Comparison of diet, lactulose, and metronidazole combinations in the control of pre‐surgical clinical signs in dogs with congenital extrahepatic portosystemic shunts

Abstract Background Hepatic supportive diet (HSD), lactulose, and antimicrobials are medical treatments for dogs with congenital extrahepatic portosystemic shunts (cEHPSS). The relative contribution of these treatment components is currently unknown. Objectives To determine which treatment combinations are most efficacious in pre‐surgical control of clinical signs of cEHPSS in dogs. Animals Thirty‐six dogs with untreated cEHPSS. Methods Three‐arm randomized clinical trial. At inclusion (T0), dogs were divided into 3 groups: HSD (n = 12), HSD + lactulose (n = 12), or HSD + metronidazole (n = 12) and received the randomized treatment for 4 weeks (T1) followed by combined treatment of HSD + lactulose + metronidazole for 2 weeks or until cEHPSS attenuation (T2). Clinical score as well as fasting ammonia (FA) and C‐reactive protein (CRP) concentrations were compared among groups and time points. Results Thirty‐four dogs were evaluated. Thirty‐four dogs reached T1 and 29 dogs T2. At T1, clinical scores decreased in the HSD + lactulose (n = 11; P = .001), but not in the HSD (n = 8; P = .96) and HSD + metronidazole (n = 10; P = .06) groups. Adding metronidazole to HSD + lactulose (n = 11) did not result in further clinical score improvement (T2; P = 1.000). Moderate and weak correlation between clinical score and FA and clinical score and CRP was present (ρ = .35, P < .001; ρ = .27, P = .01, respectively) with FA decreasing over time on medical treatment (P = .001). Conclusions and Clinical Importance Combined HSD + lactulose seems sufficient for pre‐surgical cEHPSS stabilization unlike sole HSD or HSD + metronidazole. Medical treatment of cEHPSS clinical signs decreases FA.

communication allows for toxin-rich blood to bypass the liver, entering the systemic circulation directly and causing a myriad of clinical signs. Hepatic encephalopathy (HE) is usually the most common clinical sign at presentation, although gastrointestinal and urinary tract signs are also frequently reported. 2 Several factors are implicated in the HE pathogenesis with ammonia playing a central role, whereas inflammation has been suggested as an important contributing factor. 3,4 Furthermore, after surgical attenuation of cPSS, C-reactive protein (CRP) concentration was observed to decrease significantly. 5 Medical treatment, consisting of a combination of hepatic supportive diet (HSD), lactulose, antimicrobial drugs, or some combination of these, and attenuation by surgery or interventional radiologic procedure have been described as valid treatments for cPSS. 6 Long-term survival is reported to be superior in dogs that undergo surgery compared to dogs treated medically. 7 Notwithstanding its inferiority as long-term management, medical treatment is an important step in the pre-surgical stabilization of surgical candidates and in the post-surgical period when gradual occlusion techniques are used and the cPSS is still patent. This preoperative and postoperative medical management is thought to lead to a reduction of clinical signs, anesthetic risk and perioperative complications. 8 As mentioned above, medical management is a combination of different treatments, each with a different mechanism of action. Lactulose, a non-absorbable disaccharide, decreases ammonia production and absorption in the colon by decreasing colonic pH, causing a shift from ammonia to ammonium, with the latter being poorly absorbed by the gastrointestinal mucosa. It also decreases gastrointestinal transit time and inhibits glutaminase activity, decreasing intestinal uptake of glutamine and its metabolism to ammonia. 9 Antimicrobials such as metronidazole and ampicillin, which have been widely used in veterinary medicine, have their main therapeutic effect in controlling cPSS clinical signs by eliminating urease-producing bacteria, subsequently decreasing ammonia production and absorption. 10,11 Hepatic supportive diets, another component of medical management, are traditionally lower in protein, while incorporating highly digestive protein, leaving less protein substrate to be metabolized to ammonia by gastrointestinal bacteria. 12 Medically treated dogs with cPSS receiving HSD vs HSD and lactulose had no difference in median survival time when followed for 3.2 years. 13 Additionally, a meta-analysis in people with HE found that rifaximin, a poorly absorbed antimicrobial, is at least as effective as nonabsorbable disaccharides in controlling clinical signs of HE. 11 These findings in people and dogs prompt the question of which part of medical management is effective for control of clinical signs in cPSS dogs. Little information is available in the veterinary literature to compare efficacy of different medical treatment components. 6 To investigate which components of medical management are more effective in pre-surgical stabilization, a 3-arm randomized clinical trial was designed, including dogs recently diagnosed with a congenital extrahepatic portosystemic shunt (cEHPSS) that were not receiving any medication or a decreased protein diet. We aimed to compare the effect of HSD, HSD + lactulose and HSD + metronidazole on clinical signs as well as fasting ammonia (FA) and CRP concentrations. A secondary aim was to investigate whether combination treatment (HSD + lactulose + metronidazole) would have superior effects to these 3 treatments on the same variables.

| Animals
The study was approved by the local ethical and deontological committee (EC2017/49 and DC2017N06), and written consent was obtained from each dog owner before study inclusion. In total, 36 client-owned dogs with cEHPSS referred to Small Animal Hospital-Ghent University were enrolled. The dogs had a definitive diagnosis of cEHPSS, confirmed either by abdominal doppler ultrasonography, transsplenic portal scintigraphy (TSPS), computed tomography angiography, or some combination of these procedures. Dogs were not included in the study if they had received antimicrobial drugs, lactulose, HSD or probiotics in the 5 days before study inclusion or if they had clinically relevant concurrent disease potentially contributing to the clinical score.

| Clinical score
Clinical score was calculated from standardized questionnaires that were filled out by the owners at the different time points (T0, T1, T2), as published before (Table 1). 14,15 Briefly, any clinical sign occurring often was allocated 2 points and those occurring occasionally were allocated 1 point. The points for gastrointestinal and urinary signs were not multiplied, but those for seizure and coma were multiplied by 3 and all other neurologic signs multiplied by 2, resulting in a maximal clinical sign score of 62. The higher the score, the worse the clinical status.

| Blood sampling
Venous blood samples were collected at T0, T1, and T2 for FA and CRP measurements. Venous FA concentration was immediately determined using a commercially available and validated hand-held ammonia analyzer (PocketChem BA, A. Menarini Diagnostics; upper limit, 45 μmol/L; detection range, 8-285 μmol/L). 16 Once blood in the serum tubes was clotted, samples were centrifuged at 3500g for 5 minutes and serum was immediately stored at À80 C for later CRP measurement. C-reactive protein concentrations were measured in batch using a validated commercial kit (Gentian canine CRP reagent kit; upper limit, 10 mg/L; detection range, 10-1000 mg/L). 17

| Statistical analysis
Statistical analyses were performed using SPSS Statistics 26 (IBM, Armonk). Kruskal-Wallis tests were performed to assess differences in age, body weight, times receiving the different treatments, clinical score, FA and CRP concentrations, and number of dogs with FA concentrations within reference interval at T0 vs T2. In case of significant differences, Bonferroni corrections were used to take multiple comparisons into account. To assess differences in groups' clinical scores over time, Friedman's two-way analysis was performed with Bonferroni corrections for multiple comparisons in case of significant differences. A Wilcoxon signed rank test was performed to assess the difference of FA and CRP concentrations at diagnosis vs T2. Subsequently, a Mann-Whitney U-test was used to assess FA and CRP concentrations in dogs receiving combined treatment during <15 days vs those that received treatment for >25 days. Finally, Spearman correlation tests were used to assess correlations between clinical score on the one hand, and FA and CRP on the other hand, and between FA and CRP. Results were considered significant if P ≤ .05.

| Study population at baseline (time point T0)
Between October 2017 and March 2020, 36 dogs were enrolled.
Twelve dogs were originally included at T0 in the HSD group, 12 in the HSD + lactulose diet group and 12 in the HSD + metronidazole group. Between T0 and T1, 2 dogs had to be excluded, 1 in the HSD + metronidazole group and 1 in the HSD + lactulose group.
The former was euthanized related to paralysis caused by a subarachnoid cyst and the latter was excluded because of uncontrollable central nervous signs present at study inclusion and that persisted almost 3 years after cEHPSS closure was confirmed by imaging. The Median age and body weight at T0 are presented in Table 2. No differences in median age or weight were present at T0 (P = .36 and .17, respectively).
T A B L E 1 Clinical scoring system in dogs with an extrahepatic portosystemic shunt 14,15 Frequency Multiplication factor Maximal score often-occasionally-never  Table 3. At T0, no differences in clinical signs score were present among groups (P = .8).

| End of initial treatment (time point T1)
Dogs presented at T1 a median of 29 days (range, 3-54) after T0. Two dogs in group HSD and 1 in group HSD + metronidazole had to be started earlier on combined treatment because of unsatisfactory con-

| Influence of medical treatments on clinical score over time points
The HSD group had a clinical score decrease at T2 compared to T0 both when all dogs were included (P = .01) and when treatment failure dogs were excluded (P = .01). The HSD group did not have clinical score changes between T0 and T1 nor between T2 and T1. The  Table 4. Figure 1 shows the changes in clinical score when all dogs were included.   No correlation between FA and CRP concentration was present (P = .14). shown to be as effective as metronidazole in the management of acute overt type C HE (HE resulting from chronic liver disease) in people. 22 Furthermore, a recent meta-analysis in people with type C HE identified a clinical benefit when rifaximin and lactulose were combined compared with lactulose in decreasing mortality and clinical signs. 23 Type C HE has a different underlying pathology than PSSrelated HE and conclusions regarding antimicrobial use might therefore not translate to type B HE (the type of HE in cPSS, in which no underlying liver parenchymal disease is present). Although it would be interesting and pertinent to include a group receiving rifaximin, its current price precludes routine use in veterinary medicine. Furthermore, from the data obtained in our study, HSD + lactulose without the addition of metronidazole seems to be an acceptable treatment protocol for cEHPSS dogs, avoiding the potential adverse effects and additional costs associated with metronidazole. More importantly, decreased antimicrobial usage contributes to decreased overall antimicrobial resistance of bacteria in animals as well as people. 24 From the 11 dogs receiving HSD + lactulose that reached T1, all but 1 had noticeably decreased clinical signs, and adding metronidazole to the initial treatment protocol of the only dog described as having a higher clinical score at T1 also failed to improve its clinical score ( Figure 1).

| DISCUSSION
Medical treatment for cPSS was shown to decrease the severity of clinical signs, as has been described before, 12  and might wish to start combined treatment earlier or might be content to wait until T1 is completed. Nevertheless, all dogs that reached T1 earlier than expected did so because of uncontrollable clinical signs that were not expected to subside, even with continuation of the allocated treatment for a longer period. These dogs were considered treatment failures and, in order to represent treatment failure, a "worst observation carried forward" approach was chosen, meaning that a maximum possible score was assigned for the failed time point.
We also presented the data without including the dogs considered treatment failures because doing so allows clinicians to assess and decide which treatment combinations are safest. The exclusion of treatment failures from statistical analysis however induces a significant bias, and this data that excludes treatment failures must be interpreted carefully. Fourth, T2 was variable in the dogs included, although no difference in follow-up time among groups was detected.
If all dogs included at T2 had similar follow-up time, conclusions regarding the effect of combined treatment would be stronger. We tried to standardized T2 time, but because of the dogs being privately owned and as a result of clinical practice organization (eg, availability of surgery) a wider dispersion of time at T2 occurred. Fifth, the 5-day period without having received HSD, lactulose, antimicrobials, or probiotics before study inclusion might have been too short. Recent data indicate that antimicrobials might alter the gastrointestinal microbiota several weeks after discontinuation. 26 This finding means that the amount of ammonia produced by intestinal bacteria and associated clinical signs of HE may be lower several weeks after antimicrobial discontinuation. Ideally, all included dogs should not have received any medications before referral, but doing so would limit study size too much. Although the majority of dogs presented had high clinical scores at T0, decreases in clinical score before and after treatment might have been underestimated by this study limitation.
In conclusion, the combination of HSD with lactulose provided appropriate control of clinical signs in the studied population and it seems reasonable to suggest that metronidazole can be omitted from the treatment protocol for dogs with cEHPSS awaiting surgical cEHPSS attenuation. However, further work is needed to allow more specific conclusions about the preferred pre-surgical medical management of dogs with cEHPSS.

ACKNOWLEDGMENT
No funding was received for this study.