Chronic vomiting in dogs is a common problem that

Transcription

1 J Vet Intern Med 2007;21: Triple Antimicrobial Therapy and Acid Suppression in Dogs with Chronic Vomiting and Gastric Helicobacter spp Michael S. Leib, Robert B. Duncan,{ and Daniel L. Ward Background: Helicobacter pylori is a common cause of gastritis and peptic ulcers in humans. Many dogs, including those with gastritis and chronic vomiting, are infected with Helicobacter spp. Hypothesis: Triple antimicrobial therapy will eradicate Helicobacter infection, improve gastritis, and reduce clinical signs. The addition of acid suppression medication will not improve results. Animals: Twenty-four pet dogs with chronic vomiting and gastric Helicobacter spp. Methods: Dogs were randomly assigned to triple antimicrobial therapy with or without famotidine. Gastroduodenoscopy was performed 4 weeks and 6 months after therapy. Helicobacter spp status was determined by histologic assessment of gastric mucosal biopsy specimens. Results: Eradication rates for each treatment were not significantly different and combined were 75 and 42.9% at 4 weeks and 6 months, respectively. A greater improvement in gastritis scores occurred in dogs that became Helicobacter spp negative. Overall, the frequency of vomiting was reduced by 86.4%, but there were no differences between treatments. Conclusions and Clinical Importance: Eradication rates of Helicobacter spp with both treatments were not significantly different. Eradication rates at 6 months were modest, and more effective treatments should be developed. Acid suppression is not a necessary component of treatment protocols for dogs. Eradication of gastric Helicobacter spp was associated with improvement in gastritis scores. Dramatic reduction of the vomiting frequency occurred with both treatment protocols. Gastric Helicobacter spp may cause or contribute to chronic vomiting and gastritis in some dogs. Key words: Endoscopy; Famotidine; Gastritis. Chronic vomiting in dogs is a common problem that has many causes. Chronic gastritis is a very frequent cause of chronic vomiting. Some of the causes of chronic gastritis in dogs are dietary indiscretion and intolerance, foreign body ingestion, drug administration (eg, nonsteroidal anti-inflammatory drugs), toxin ingestion, renal failure, hepatobiliary diseases, inflammatory bowel disease (IBD), and potentially Helicobacter spp. 1,2 In humans, Helicobacter pylori has been identified as one of the most common causes of gastritis and peptic ulcers; it is also known to increase the risk for development of gastric lymphoma and adenocarcinoma. 3 6 Eradication of H pylori usually results in healing of gastric and duodenal ulcers. 4,7 The most effective treatment regimens contain 2 or 3 antimicrobials combined with a proton pump antagonist, given for 1 2 weeks. 4,7 9 Infection rates in humans can approach 100% in developing countries and 25 60% in developed countries. 10 Infection usually is acquired in childhood and most often persists for life. Most infected humans remain asymptomatic. 11 Studies in dogs and cats have demonstrated that gastric Helicobacter spp are commonly found in clinically normal animals, as well as in those with signs of gastrointestinal disease Gastric Helicobacter spp From the Virginia Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA (Leib, Duncan, Ward). Previously presented in part at the Comparative Gastroenterology Society GUTSKI, Winter Park, CO, February 2002, and the 21st Annual Veterinary Medical Forum, American College of Veterinary Internal Medicine, Charlotte, NC, June {Deceased. Reprint requests: Michael S. Leib DVM, MS, Diplomate ACVIM, C.R. Roberts Professor of Small Animal Medicine, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061; mleib@vt.edu Submitted June 5, 2006; Revised April 13, 2007; Accepted May 18, Copyright E 2007 by the American College of Veterinary Internal Medicine /07/ /$3.00/0 that commonly reside in dogs and cats are larger than H pylori. 20 These large spiral bacteria include Helicobacter heilmannii, H felis, H bizzozeronii, and H Salomonis. 1,21 Large spiral bacteria are indistinguishable by routine light microscopy. 22,23 Multiple species can be present within an individual animal. 24 Despite their prevalence in dogs and cats, a direct causal relationship between Helicobacter spp, gastritis, and clinical signs has not been established in dogs or cats. Based on the authors clinical experience, gastric ulcers have been observed infrequently during gastroduodenoscopy in pet dogs with chronic vomiting, gastritis, and Helicobacter spp. In addition, abnormalities in the gastric acid secretory axis (hypergastrinemia and increased acid secretion), which commonly occur in humans with H pylori, have not been documented in dogs. 25,26 For these reasons, the authors evaluated the effects of acid suppression in the treatment of gastric Helicobacter spp in dogs. If acid suppression is not necessary, fewer medications could be administered to treat dogs with gastric Helicobacter spp, and thus treatment compliance might improve. The quadruple therapy used in this study is similar to the gold standard treatment protocol used in humans infected with H pylori. 3,7,9 The purposes of this study were to compare the effects of triple antimicrobial therapy, with and without acid suppression, in dogs with chronic vomiting, regarding: (1) the eradication rates of Helicobacter spp at 4 weeks and 6 months after therapy, (2) the frequency of vomiting during the 6 months after therapy, and (3) the gastritis histologic scores 4 weeks and 6 months after therapy. Materials and Methods Dogs evaluated at the Veterinary Teaching Hospital (VTH) of the Virginia Maryland Regional College of Veterinary Medicine and that vomited chronically were eligible for inclusion in this study based on the following criteria: (1) vomiting of at least 2 weeks duration; (2) vomiting episodes occurring for a minimum

2 1186 Leib, Duncan, and Ward of 6 days for dogs with only a 2- to 4-week duration of vomiting; (3) owner s willingness to comply with all study requirements and informed consent; (4) histologic identification of spiral bacteria in gastric mucosal biopsy samples; (5) histologic evidence of a normal stomach, gastritis, or gastritis and duodenitis; (6) absence of systemic diseases that commonly cause vomiting; (7) lack of serious concurrent diseases that required additional diagnostic testing, treatment, or both; (8) absence of other gastrointestinal diseases that commonly cause vomiting; and (9) availability of the primary author (MSL) to manage the case within the VTH. The diagnostic evaluation included CBC, serum biochemical profile, urinalysis, fecal flotation by zinc sulfate, duodenal aspiration for Giardia, abdominal ultrasonography, and upper gastrointestinal (GI) endoscopy. If it was not possible to historically distinguish between vomiting and regurgitation, a barium contrast esophagram was performed. A standard upper GI endoscopic examination and duodenal aspiration for Giardia trophozoites, with a flexible video gastroscope, was performed with the dog under general anesthesia, as described in the literature. Two mucosal samples were collected with flexible biopsy forceps from each of the following 4 gastric locations: pylorus, angularis incisura, cardia, and body along the greater curvature. A minimum of 4 mucosal samples were obtained from the orad duodenum. Mucosal samples were fixed for at least 24 hours in 10% buffered neutral formalin, routinely processed, embedded, sectioned (5-mm thick), and stained with hematoxylin and eosin (H&E). Sections were evaluated histologically. Duodenal sections were designated as normal or duodenitis, based on the number and distribution of inflammatory cells within the lamina propria by applying criteria previously published for the colon. 27 Gastric mucosal sections were designated as normal or minimally, mildly, modestly, moderately, or severely inflamed. Inflammatory cells were identified morphologically. Absence of inflammatory cells within the lamina propria was designated as normal. The presence of rare, widely scattered inflammatory cells in the lamina propria was scored as minimal inflammation. The presence of low numbers of inflammatory cells evenly distributed in the superficial lamina propria was scored as mild inflammation. The presence of large numbers of cells extending deeper into the lamina propria and occasional lymphoid aggregates encountered among the sections was scored as modest inflammation. Additional increases in inflammatory cells with separation of glandular elements, increasing numbers of lymphoid aggregates, and occasional intraepithelial lymphocytes was scored as moderate inflammation. Dense infiltration of the lamina propria with inflammatory cells extending from the superficial to deep lamina propria, frequent lymphoid aggregates (often more than 2 per tissue section), and frequent intraepithelial lymphocytes were scored as severe inflammation. Although each of the 4 areas of the stomach was examined separately, an overall gastric diagnosis was assigned by taking into account the combined histopathologic changes. The overall histologic diagnosis of the stomach then was assigned a gastritis score as follows: 0-normal, 1-minimal, 2-mild, 3-modest, 4-moderate, or 5-severe inflammation. Changes in gastritis scores were calculated at 4 weeks and 6 months by comparison with initial gastritis scores. Spiral bacteria were determined to be present or absent based on H&E-stained sections. If spiral bacteria were not identified in H&E-stained sections, duplicate sections were stained with modified Steiner s silver impregnation method, and the presence or absence of spiral bacteria was again determined. Dogs were randomly assigned (by a coin toss performed before the initiation of the study) to receive 1 of 2 treatment protocols. Triple therapy consisted of amoxicillin 15 mg/kg, metronidazole 10 mg/kg, and 262-mg bismuth subsalicylate tablets a on a scale based on body weight (,5 kg, 0.25 tablet; kg, 0.5 tablet; kg, 1.0 tablet; and.25 kg, 2.0 tablets). Quadruple therapy consisted of famotidine, 0.5 mg/kg, added to triple therapy. All medications were given PO q12h for 2 weeks. Within each treatment group, dogs were stratified based on histologic findings: normal stomach (category 1), gastritis (category 2), and gastritis and duodenitis (category 3). Owners maintained a daily record of medications administered, frequency of vomiting, and appetite. Owners were contacted after completing 1 week of therapy to identify and correct treatment problems and further document compliance, and they were contacted again after completion of therapy to assure compliance. The dog s current diet was continued. The use of other antimicrobials and anti-inflammatory drugs was discouraged for the 6 months after treatment. Upper GI endoscopy was repeated 4 weeks and 6 months after treatment was completed. The histologic presence of gastric spiral bacteria and the gastritis score was determined at each visit, as previously described. From the owners daily records, the number of vomiting episodes was tabulated for each of six 4-week periods after therapy. The 1st 4-week period started after completion of treatment and continued until the 1st endoscopic reevaluation. The last 4-week period commenced after completion of the 5th 4-week period and continued until the final endoscopic examination. Because the length of these 2 periods could be longer than 28 days in some dogs, the number of vomiting episodes per 28 days was calculated. The percentage reduction in the number of vomiting episodes was determined for each 4-week period by comparing the number of recorded vomiting episodes to the number of expected episodes from the dog s history. The historical vomiting frequency, provided by the owner, was the average number of vomiting episodes per week for the entire time that the owner observed vomiting and did not represent the intervals with the highest frequencies or the most recent period before evaluation. The Animal Care Committee of Virginia Tech approved this study. Statistical Evaluation For categorical responses (eg, presence of spiral bacteria 4 weeks and 6 months after therapy), a logistic regression model (LOGISTIC procedure of SAS b ) with histologic category and therapy, and their interaction, was fit to the data to provide exact (ie, not asymptotic approximations) hypothesis tests. The reported change in vomiting response was analyzed by SAS s MIXED procedure to perform a mixed effects repeated measures analysis of variance testing for the main effects of category, therapy, and period, as well as all of their 2- and 3-way interactions. Significant interactions were further investigated with the SLICE option to test for the simple main effects of therapy within category and period combinations. Model adequacy was assessed by standardized residual plots. Characteristics of the 2 therapy groups and histologic categories (age, duration of vomiting, and frequency of vomiting) were evaluated with a 2-factor analysis of variance. Mann-Whitney U-tests were used to evaluate the relationships between the initial gastritis scores and treatment groups and the relationships between changes in gastritis scores and Helicobacter spp status. For those relationships between gastritis scores and other variables, Spearman s correlation coefficient was used, for all other relationships Pearson s correlation coefficient was used. The FREQ procedure of SAS was used to perform McNemar s exact test to evaluate the association between the presence of spiral bacteria at 4 weeks and at 6 months. Significance for all tests was determined at P,.05. Results Fourteen females and 10 males evaluated between April 1999 and July 2002 were included in the study. There were 2 Boxers, 2 Rottweilers, 2 Standard Poodles, 1 each of 9 other breeds, and 9 mixed breed dogs. The

3 Treatment of Helicobacter 1187 Table 1. Treatment group characteristics. Treatment Group Male/ Female Median Age in Years (range) Median Vomiting Frequency/ Wk (range) Median Duration in weeks of Vomiting (range) Histologic Category 2/3 Median Gastritis Score Median Days (range) from End of Treatment to Endoscopy at 4 Wk/6 Mo Triple (10 dogs) 5/5 2.4 (1 11) 4 ( ) 23.5 (2 104) 6/ (24 36)/ 188 ( ) Quadruple (14 dogs) 5/9 3.5 ( ) 3 ( ) 18.5 (2 364) 7/ (25 38)/ ( ) median age was 3 years (range, 1 12 years). The number of dogs in each histologic category was as follows: normal stomach, 1; gastritis only, 13; and duodenitis, 10. Because there was only 1 dog within histologic category 1, results from this dog were excluded from all statistical evaluations in which category was examined. The median vomiting frequency was 3.5 episodes per week (range, ). The median duration of vomiting was 19 weeks (range, weeks). Ten dogs received triple therapy and 14 received quadruple therapy. There were no significant differences between treatment groups or histologic categories 2 and 3 with regard to age, historical vomiting frequency, historical vomiting duration, initial gastritis score, or days between completion of treatment and both endoscopic examinations (Table 1). There was no relationship between initial gastritis scores and vomiting frequency or duration. Increasing age was associated with initial gastritis scores (P , r ). Two dogs, in which regurgitation could not be historically distinguished from vomiting, had normal esophageal function demonstrated with barium contrast esophagrams. Fecal flotation was negative in 23/24 dogs; whipworms were detected in 1 sample. Duodenal aspiration for identification of Giardia trophozoites was negative in all 22 dogs in which it was performed. CBC and serum biochemical profiles were performed in all dogs, and urinalyses were performed in 23 dogs. Abdominal ultrasound examination was performed in 18 dogs. Spiral bacteria were not detected during histologic evaluation of gastric mucosal samples 4 weeks after treatment in 7/10 (70%) dogs that received triple therapy and in 11/14 (78.5%) dogs that received quadruple therapy (P ). Nine of 13 (69.2%) category-2 dogs and 9/10 (90%) category-3 dogs were histologically negative for spiral bacteria (P ). Both treatments resulted in 18/24 (75%) dogs being histologically negative. There were no significant effects of age (P ), historical vomiting frequency (P ), or duration of vomiting (P ) on the presence of spiral bacteria 4 weeks after therapy. There were no significant differences in the change in gastritis scores at 4 weeks between treatment groups (P ). Dogs that became Helicobacter spp negative had a median reduction of 0.5 in their gastritis scores, whereas those that remained positive had a median increase of 0.5 (P ). The median reduction in the frequency of vomiting episodes during the 1st 4-week period after therapy was 91.8% for triple therapy dogs and 72% for quadruple therapy dogs (P ). During this same period, median reduction in the frequency of vomiting episodes of dogs histologically negative was 85.2%, and for dogs histologically positive, 49.9% (P ). Category-2 dogs had a 77.8% reduction in vomiting frequency, whereas category-3 dogs had an 84.3% reduction in the frequency of vomiting episodes (P ). There was no significant relationship between the improvement in vomiting frequency and the change in gastritis scores (P , r ). Three dogs did not complete the final endoscopic examination, 2 due to lack of owner compliance and the 3rd because of continued clinical signs and administration of an additional treatment regimen for Helicobacter spp. All 3 dogs were histologic category 2; one received triple therapy, and two had a positive histologic examination at 4 weeks. Spiral bacteria were not detected during histologic evaluation of gastric mucosal samples 6 months after treatment in 4/9 (44.4%) dogs that received triple therapy and 5/12 (41.7%) that received quadruple therapy (P ). Four of 10 (40%) category-2 dogs and 5/10 (50%) category-3 dogs were histologically negative for spiral bacteria (P ). Both treatments resulted in 9/21 (42.9%) dogs being histologically negative, which was significantly less than 18/24 (75%) 4 weeks after therapy was completed (P ). There were no significant effects of age (P ), historical vomiting frequency (P ), or duration of vomiting (P ) on the presence of spiral bacteria 6 months after therapy. There were no significant differences in the changes in gastritis scores at 6 months (compared with initial scores), between treatment groups (P ). Dogs that were Helicobacter spp negative at 6 months had a median reduction of 2.0 in their gastritis scores, whereas those that remained positive had a median increase of 0.25 (P ). Of the 8 dogs that were negative at the 4-week evaluation and positive after 6 months, 3 received triple therapy, and 4 were category 2 and 4 were category 3. Nine of 17 (52.9%) dogs negative for Helicobacter spp at 4 weeks remained negative after 6 months. The median change in gastritis scores at 6 months (compared with initial scores) in the 17 dogs that were Helicobacter spp negative at 4 weeks, decreased by 1.0, whereas it increased in the 4 dogs that were positive by 1.25 (P ). The median change in gastritis scores at 6 months (compared with scores at 4 weeks) in the 8 dogs that were Helicobacter spp negative at the 4-week evaluation and positive after

4 1188 Leib, Duncan, and Ward 6 months was 0.0. However, the median change in gastritis scores in the 9 dogs that remained Helicobacter spp negative at 6 months, improved 1.0 (P ). The median reduction in the frequency of vomiting episodes for the entire 6-month period after completion of therapy was 86.4% for dogs that received triple therapy and 86.1% for those that received quadruple therapy (P ). The median reduction in the frequency of vomiting episodes for dogs histologically negative at the 6-month endoscopy was 92.4% and for dogs histologically positive was 81.5% (P ). Category-2 dogs had an 86.4% reduction, whereas category-3 dogs had an 84.4% reduction in the frequency of vomiting episodes (P ). There were no significant relationships between the frequency of vomiting during the entire 6-month study period and age (r , P ), historical vomiting frequency (r , P ), duration of vomiting (r , P ), or change in gastritis score (initial score to 6 months; r , P ). The median reduction in the frequency of vomiting episodes for each of the six 4-week periods for each treatment group is shown in Figure 1. There were no significant effects of therapy (P ), histologic category (P ), or 4-week period (P ). Five dogs received additional medications between the 4-week and 6-month endoscopic examinations that could have altered results. Two dogs received antibiotics for pyoderma (during the 4th and last 4-week periods): 1 for acute diarrhea (during the last 4-week period), and 1 for both acute diarrhea (during the 2nd 4-week period) and pyoderma (during the 2nd, 5th, and 6th 4-week periods). Spiral bacteria were not seen at the 6-month endoscopy in all 4 of these dogs. Vomiting frequency after receiving antibiotics appeared similar to the periods before therapy in 3 of these dogs and was improved in the 4th. One dog received an anti-inflammatory dose of prednisone for cutaneous allergy (for 9 days during the 2nd 4-week period). Spiral bacteria were not seen in this dog at the 6-month endoscopy. Vomiting frequency for this dog did not change dramatically throughout the observation period. The dog treated for both diarrhea and pyoderma also received a daily physiologic dose of prednisone throughout most of the study, despite having a normal corticotropin (ACTH) response test. One of the dogs that received antibiotics for pyoderma also was fed a protein hydrolysate diet, starting during the 2nd 4- week period. Vomiting frequency appeared to improve in this dog after the diet change. In 1 dog, a single Physaloptera spp worm was endoscopically removed from the duodenum at the 4-week posttreatment endoscopy. The vomiting frequency improved in this dog. Discussion In this study, the eradication rate of gastric Helicobacter spp was similar in both treatment groups, at 4 weeks and 6 months after completion of therapy. These results indicate that the addition of famotidine, for acid suppression, was not more effective than triple Fig 1. Median percentage decrease in the vomiting response for each 4-week period. The median percentage decrease in the vomiting frequency after therapy, compared with the vomiting frequency prior to therapy, is shown on the Y-axis for the triple therapy group (T; squares and closed line) and the quadruple therapy group (Q; diamonds and dashed line). Error bars indicate the standard deviation, the T group is deflected positively and the Q group deflected negatively. The six 4-week periods are represented on the X-axis. There were no significant differences between treatment groups during any of the six 4-week periods. therapy alone. In humans, acid suppression combined with triple antimicrobial therapy is more effective at eradicating H pylori than triple therapy alone. 5,28,29 The use of treatment protocols requiring fewer medications to be administered could lead to increased treatment compliance, and thereafter to a better response to therapy. When results of both treatment protocols are combined, a 75% eradication rate for Helicobacter spp was achieved 4 weeks after completion of therapy. In humans with H pylori, treatment protocols with eradication rates (determined 4 weeks after completion of therapy) greater than 80 90% are considered clinically effective. 4,7,9,29,30 The results of this study indicate that potentially more effective treatment protocols need to be investigated in dogs with Helicobacter spp. The high percentage (47.1%) of dogs that were negative for Helicobacter spp 4 weeks after therapy and positive after 6 months was disappointing. Infection at 6 months could be due to reinfection or recrudescence of infection. In humans living in industrialized countries, H pylori reappearance rates, after eradication, of approximately 1% have been demonstrated, with most of these cases thought to be due to recrudescence In the present study, antibiotic therapy could have suppressed the numbers of gastric Helicobacter spp below detection limits of light microscopy but could not have completely eliminated infection This could allow bacterial growth and reestablishment of a pathogenic infection after cessation of antimicrobial therapy. In addition, the use of a single method of detection, as used in this study, has been shown to lead to falsely high eradication rates and then to falsely high reappearance

5 Treatment of Helicobacter 1189 rates in humans. 30 In this study, the thorough histologic assessment of 2 mucosal samples from 4 gastric locations reduced this possibility. 32 However, it is possible that use of potentially more sensitive tests (eg, polymerase chain reaction [PCR]) 26,35 may have resulted in more dogs testing positive. In addition, poor owner treatment compliance 36 could have caused suppression, but not elimination, of bacteria. Treatment compliance was maximized in this study by the use of daily treatment diaries and telecommunication with owners after the 1st and final weeks of therapy. In humans, treatment compliance.60% has been shown to result in effective eradication of H pylori, 37 so it is possible that noncompliance may not have affected the results in this study. In addition, bacterial resistance to metronidazole and rarely amoxicillin also could have contributed to the failure to eliminate the infection and allow recrudescence. 4,5,9,38 Gastric Helicobacter spp have been identified in extragastric locations, such as the oral cavity (dental plaque, saliva, or gingival pockets), where they may be protected from antimicrobial therapy and could serve as a nidus for gastric recolonization and recrudescence of infection. 34,39 41 Helicobacter pylori also can revert to a coccoid form after exposure to certain antibiotics. 34 It may not be possible to identify this form by routine laboratory techniques. The coccoid form can revert back to viable H pylori and potentially reestablish infection. It is unknown whether the gastric Helicobacter spp found in dogs are capable of reverting to a coccoid form or not. In dogs, reinfection via the fecal-oral route is a likely possibility because of the social behavior of dogs and their relatively (compared with humans in industrialized countries) unsanitary living conditions. Recurrence after treatment in adult humans with H pylori is related to sanitation and population prevalence of infection. 30,31,39,42 Analysis of bacterial DNA by molecular fingerprinting techniques would be necessary to distinguish recrudescence from reinfection with a different strain 30,39,42,43 and was not done in this study. If reinfection is later found to be responsible for the high infection rates that occurred after 6 months in this study, modification of husbandry and environmental conditions could be performed to minimize reexposure to Helicobacter spp after antimicrobial treatment. As expected, all of the dogs that were positive at 4 weeks remained positive after 6 months. In humans, infection with H pylori is believed to be life-long in most individuals. However, spontaneous elimination has been documented, most often in children. 31 The authors are aware of only 1 other study that evaluated the effects of triple antimicrobial therapy on the Helicobacter spp status of pet dogs with upper gastrointestinal signs. 44 Seven of 9 dogs that received triple therapy were found to be Helicobacter spp negative after completing therapy. Four of these dogs were reevaluated a mean of 2.5 years after therapy, and all were found to be positive for Helicobacter spp. These results are similar to the results obtained at 4 weeks in the present study, but longer-term eradication rates exceeded these findings 6 months after treatment. It is possible that more Helicobacter spp positive dogs would have been identified by Happonen, Linden, and Westermarck 44 if all dogs were tested 6 months after completing therapy. Two studies have evaluated the 2-week treatment of naturally infected asymptomatic beagles with famotidine, amoxicillin, and metronidazole. 26,45 In both reports, Helicobacter spp were identified in gastric biopsy specimens 29 days after completion of therapy in all dogs. These results cannot be directly compared with the findings of the present study, because the treatment protocols differed and the experimental studies evaluated asymptomatic dogs. In the present study, eradication results after 4 weeks may have been better because of the inclusion of bismuth as a 3rd antimicrobial agent. Bismuth-containing compounds have been used to heal duodenal ulcers in humans, have topical bactericidal effects against H pylori (possibly by disrupting adhesion to epithelial cells and inhibiting secreted enzymes), and may prevent the development of bacterial resistance to imidazoles. 46 Eradication of gastric Helicobacter spp was associated with improvement in gastritis scores, especially 6 months after treatment. Reduction in the severity of gastritis likely contributed to the reduction in clinical signs in these dogs. Although changes in gastritis scores and Helicobacter status were not significantly related to changes in vomiting frequency, results approached statistical significance. Lack of significance was possibly due to extreme variation in vomiting frequency among dogs (Fig 1). This finding suggests that in some dogs, Helicobacter spp were unrelated to gastritis and the presence of clinical signs. However, it also suggests that the opposite may occur in other dogs. Regardless of Helicobacter spp status at 6 months, dogs that were negative at 4 weeks had a significant improvement in gastritis scores compared with those that were positive at 4 weeks. This improvement in gastritis associated with eradication at 4 weeks helps to explain the reduced vomiting frequency in dogs that became positive at 6 months. The degree of gastric inflammation remained unchanged in a group of naturally infected beagles 4 weeks after failed eradication therapy with amoxicillin, metronidazole, and famotidine. 26 However, the histologic results from the present study differ from those of Happonen s treatment of pet dogs with clinical signs, in which they reported no histologic changes after eradication of Helicobacter spp despite clinical improvement. 44 In that study, details of histologic findings after treatment were not presented, but gastritis did not persist in 3/6 dogs after therapy. Both treatment protocols resulted in substantial reduction of the frequency of vomiting during each of the 4-week periods but were not significantly different from each other. These results also suggest that acid suppression did not have a beneficial effect and could be eliminated from treatment protocols in dogs. Dogs negative for Helicobacter spp had a greater reduction in vomiting frequency than those that were positive 4 weeks and 6 months after completion of therapy.

6 1190 Leib, Duncan, and Ward Although this difference approached statistical significance at 4 weeks (P ), it did not at 6 months. The potential impact of variance within a group is discussed above, and the results of the present study suggest that eradication of Helicobacter spp will reduce clinical signs only in some dogs. The reduction of vomiting frequency in the dogs positive for Helicobacter spp after 6 months was unexpected. Of the 12 positive dogs after 6 months, 8 were negative at 4 weeks. In these 8 dogs, reinfection or recrudescence occurred sometime during the last 5 months of the study. Because evaluation for the presence of Helicobacter spp was performed only twice, it was not possible to determine when reinfection or recrudescence occurred. In these dogs, bacteria either were eliminated or reduced below detection limits of light microscopy at 4 weeks, decreasing their potentially pathogenic effects and the degree of gastritis, and reducing the severity of clinical signs. As reinfection or recrudescence occurred sometime during the next 5 months, bacterial numbers and the severity of gastritis increased, but possibly not to pretreatment levels. When clinical signs develop during adulthood in humans infected with H pylori, they are preceded by a long asymptomatic period, during which progressive gastric structural and functional damage occurs. 5,47,48 The frequency of vomiting in these dogs may increase as progressive gastritis develops. A longer study period is needed to address this question. The overall reduction in vomiting frequency in the 4 dogs that remained positive throughout the study was only modest (median reduction of 25.3%). Only 1 dog had a satisfactory response, a reduction in vomiting frequency by 86%. In Happonen s study, there were 2 dogs that remained positive after triple therapy and both had either clearly milder or slightly milder clinical signs after treatment. 44 Histologic category did not affect Helicobacter spp status, at either 4 weeks or 6 months, or the frequency of vomiting after treatment. This finding suggests that duodenitis did not contribute substantially to vomiting frequency or that the study treatments reduced intestinal inflammation. The assessment of intestinal inflammation has been shown to be inconsistent among pathologists 49 and uniformly accepted histologic standards for IBD in dogs do not exist. Of the 10 dogs with duodenitis, 8 were assessed as mild, whereas 2 were considered to be mild to moderate (data not shown). A different pathologist might have characterized some of these intestinal biopsy samples as normal. Antibiotic therapy can have substantial effects on the gastrointestinal flora, which is thought to play a major role in the initiation and perpetuation of IBD in humans. 50 The inhibitory effects of metronidazole, amoxicillin, or bismuth on small bowel bacteria could have resulted in reduced intestinal inflammation and clinical signs. Because metronidazole can also modulate inflammation by inhibiting cell-mediated immunity, altering neutrophil chemotaxis and motility, and affecting lymphocyte transformation, it has been recommended for the treatment of IBD in dogs. 51,52 There are several weaknesses in this clinical study that should be addressed. The lack of a placebo group makes it impossible to conclude that reductions in the vomiting frequency were necessarily due to one of the treatments, rather than normal variation in the severity of clinical signs. However, the long follow-up period after treatment (6 months) makes this explanation less plausible. In addition, use of a placebo in a clinical study of this length is not practical, as few owners would be willing to tolerate continued vomiting for 6 months. The effect of treatment on vomiting frequency was based on comparison of a daily posttreatment diary to the reported historical vomiting frequency. This approach could have introduced bias into the study based on the owner s recall of the vomiting frequency and could potentially have exaggerated the treatment response. Ideally a pretreatment diary should have been prepared before treatment to ensure accurate comparison with posttreatment results. Unfortunately, this approach is impractical in clinical practice. The additional treatments that 5 of the dogs received after the 4-week evaluation could have affected Helicobacter spp status, vomiting frequency, or both at 6 months. Most received a short course of a single antibiotic and all were Helicobacter spp negative at 6 months. However, in humans with H pylori infection, treatment with a single antibiotic resulted in,20% eradication rates. 7 Thus, it is probable that the additional antibiotic treatments did not affect Helicobacter spp status in these 5 dogs. However, if the additional antibiotic therapy resulted in more Helicobacter spp negative dogs at 6 months, it would not alter one of the study s important conclusions, that reinfection or recrudescence was common. It is also possible that additional antibiotic treatments could have reduced the frequency of vomiting. However, in only 1 dog did the vomiting frequency appear to change dramatically after administration of additional antibiotics. The anti-inflammatory dosage of prednisone used in 1 dog could have reduced mucosal inflammation with subsequent reduction in vomiting frequency. The vomiting frequency, however, did not appear to change after prednisone administration in this dog. In addition, this dog did not have duodenitis upon admission to the study. Vomiting frequency did improve in the 1 dog that had a dietary change to a protein hydrolysate diet during the 2nd 4-week period. Improvement could have been due to dietary effects or a delayed clinical response to eradication of the Helicobacter spp and reduction in gastritis score. Finally, the removal of a single Physaloptera spp from a dog that became Helicobacter spp positive at 6 months could have influenced the vomiting frequency. These factors could have had a small effect on the reduction in vomiting frequency in positive dogs (81.5%), but not enough to significantly impact the differences between positive and negative dogs (92.4%). In a clinical trial of 6.5 months duration, it is often not possible to prohibit additional treatments for concurrent disorders. As an alternative, dogs that received additional treatments could have been removed from the study s results. However, the authors chose not to

7 Treatment of Helicobacter 1191 remove these dogs because the potential impact on results would be very slight, removal would have reduced the statistical power of the study, and removal would not affect the study s main conclusions. Additionally, these treatments were administered after the 4- week endoscopic examination and could not affect the results before that time. The present study did not identify the species of the gastric Helicobacter spp because they cannot be distinguished histopathologically. 23 Antibiotic sensitivity may vary among different Helicobacter spp. In addition, concurrent infection with multiple species has been reported in dogs. 24,35 Helicobacter bizzozeronii, H felis, and H salomonis have been cultured from dogs, whereas H heilmannii has not. 14,53,54 If bacteria could be cultured, antibiotic sensitivity testing would be helpful in drug selection because metronidazole and clarithromycin, and rarely amoxicillin, resistance has been detected in strains of H pylori. 4,5,38,55 However, at the present time, culture of gastric biopsy specimens in veterinary clinical practice is not routinely available and practitioners must treat gastric Helicobacter spp without the benefit of sensitivity testing. In conclusion, both triple and quadruple therapy resulted in the eradication of Helicobacter spp in 75% of dogs 4 weeks after completion of therapy and in 42.9% of dogs after 6 months. These data suggest that reinfection or recrudescence of infection occurred in many dogs by 6 months. A greater improvement in gastritis scores occurred in dogs that became negative for Helicobacter spp. The frequency of vomiting was dramatically reduced at all points in the study in both treatment groups, regardless of Helicobacter status. Acid suppression with famotidine did not affect treatment efficacy or frequency of clinical signs. This study suggests that Helicobacter spp contributes to gastritis and vomiting in some dogs. Additional studies are necessary to define more effective treatments and further describe the potential pathogenic role of gastric Helicobacter spp in dogs. Footnotes a Pepto-Bismol tablets, Procter & Gamble, Cincinnati, OH b SAS Version 8.2, SAS Institute, Cary, NC Acknowledgments This study was supported in part by the Savannah and Barry French Poodle Memorial Fund and the Pet Memorial Fund of the Virginia Veterinary Medical Association. References 1. Lee A, Krakowka S, Fox JG, et al. Role of Helicobacter felis in chronic canine gastritis. Vet Pathol 1992;29: Radin MJ, Eaton K, Krakowka S, et al. Helicobacter pylori gastric infection in gnotobiotic beagle dogs. Inf Imm 1990;58: Pakodi F, Abdel-Salam O, Debreceni A, et al. Helicobacter pylori: One bacterium and a broad spectrum of human disease! An overview. J Physiol 2000;94: Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Med 2002;347: Graham D. Therapy of Helicobacter pylori: Current status and issues. Gastroenterology 2000;118(Suppl):S2 S8. 6. Sipponen P, Hyvarinen H. Role of Helicobacter pylori in the pathogenesis of gastritis, peptic ulcer, and gastric cancer. Scand J Gastroenterol 1993;28(Suppl): Chiba N, Rao BV, Rademaker JW, et al. Meta-analysis of the efficacy of antibiotic therapy in eradicating Helicobacter pylori. Am J Gastroenterol 1992;87: Graham D, Hoffman J, El-Zimaity H, et al. Twice a day quadruple therapy (bismuth subsalicylate, tetracycline, metronidazole plus lansoprazole) for treatment of Helicobacter pylori infection. Aliment Pharmacol Ther 1997;11: van der Hulst RW, Keller JJ, Rauws EA, et al. Treatment of Helicobacter pylori infection: A review of the world literature. Helicobacter 1996;1: Goodman K, Correa P. The transmission of Helicobacter pylori: A critical review of the evidence. Int J Epidemiol 1995;24: Brown LM. Helicobacter pylori: Epidemiology and routes of transmission. Epidemiol Rev 2000;22: Geyer C, Colbatzky F, Lechner J, et al. Occurrence of spiral-shaped bacteria in gastric biopsies of dogs and cats. Vet Rec 1993;133: Hermanns W, Kregel K, Breuer W, et al. Helicobacter-like organisms: Histopathological examination of gastric biopsies from dogs and cats. J Comp Pathol 1995;112: Happonen I, Linden J, Saari S, et al. Detection and effects of helicobacters in healthy dogs and dogs with signs of gastritis. J Am Vet Med Assoc 1998;213: Yamasaki K, Suematsu H, Takahashi T. Comparison of gastric lesions in dogs and cats with and without gastric spiral organisms. J Am Vet Med Assoc 1998;212: Neiger R, Dieterich C, Burnens A, et al. Detection and prevalence of Helicobacter infection in pet cats. J Clin Microbiol 1998;36: Papasouliotis K, Gruffydd-Jones TJ, Werrett G, et al. Occurrence of gastric Helicobacter-like organisms in cats. Vet Rec 1997;140: Eaton KA, Dewhirst FE, Paster BJ, et al. Prevalence and varieties of Helicobacter species in dogs from random sources and pet dogs: Animal and public health implications. J Clin Microbiol 1996;34: Otto G, Hazell SH, Fox JG, et al. Animal and public health implications of gastric colonization of cats by Helicobacter-like organisms. J Clin Microbiol 1994;32: Neiger R, Simpson K. Helicobacter infection in dogs and cats: Facts and fiction. J Vet Intern Med 2000;14: Lecoindre P, Chevallier M, Peyrol S, et al. Gastric helicobacters in cats. J Feline Med Surg 2000;2: Jalava K, Kaartinen M, Utriainen M, et al. Helicobacter salomonis sp. nov., a canine gastric Helicobacter sp. related to Helicobacter felis and Helicobacter bizzozeronii. Int J Syst Bacteriol 1997;47: Hanninen M, Happonen I, Saari S, et al. Culture and characteristics of Helicobacter bizzozeronii, a new canine gastric Helicobacter sp. Int J Sys Bacteriol 1996;46: Wiinberg B, Spohr A, Dietz H, et al. Quantitative analysis of inflammatory and immune responses in dogs with gastritis and

8 1192 Leib, Duncan, and Ward their relationship to Helicobacter spp. infection. J Vet Intern Med 2005;19: Simpson K, PLM, Strauss-Ayali D, et al. Helicobacter felis infection in dogs: Effect on gastric structure and function. Vet Pathol 1999;36: Simpson K, Strauss-Ayali D, McDonough P, et al. Gastric function in dogs with naturally acquired gastric Helicobacter spp. infection. J Vet Intern Med 1999;13: Roth L, Walton AM, Leib MS, et al. A grading system for lymphocytic plasmacytic colitis in dogs. J Vet Diagn Invest 1990;2: de Boer W, Driessen W, Jansz A, et al. Effect of acid suppression on efficacy of treatment for Helicobacter pylori infection. Lancet 1995;345: Korman MG, Tytgat GNJ. Helicobacter pylori and peptic ulcer. Scand J Gastroenterol 1995;210(Suppl): van der Hulst R, Rauws E, Koycu B, et al. Helicobacter pylori reinfection is virtually absent after successful eradication. J Infect Dis 1997;176: Xia HH, Talley NJ. Natural acquisition and spontaneous elimination of Helicobacter pylori infection: Clinical implications. Am J Gastroenterol 1997;92: Xia H, Gilvarry J, Beattie S, et al. Recrudescence of Helicobacter pylori infection in patients with healed duodenal ulcer after treatment with different regimens. Am J Gastroenterol 1995;90: Parsonnet J. The incidence of Helicobacter pylori infection. Aliment Pharmacol Ther 1995;9(Suppl): Perkins SE, Yan LL, Shen Z, et al. Use of PCR and culture to detect Helicobacter pylori in naturally infected cats following triple antimicrobial therapy. Antimicrob Agents Chemother 1996;40: Neiger R, Tschudi M, Burnens A, et al. Diagnosis and identification of gastric Helicobacter species by polymerase chain reaction in dogs. Microb Ecol Health Dis 1999;11: Adams V, Campbell J, Waldner C, et al. Evaluation of client compliance with short-term administration of antimicrobials to dogs. J Am Vet Med Assoc 2005;226: Graham D, Lew GM, Malaty HM, et al. Factors influencing the eradication of Helicobacter pylori with triple therapy. Gastroenterology 1992;102: Watanabe K, Tanaka A, Imase K, et al. Amoxicillin resistance in Helicobacter pylori: Studies from Tokyo, Japan from 1985 to Helicobacter 2005;10: Seo M, Okada M, Shirotani T, et al. Recurrence of Helicobacter pylori infection and the long-term outcome of peptic ulcer after successful eradication in Japan. J Clin Gastroenterol 2002;34: Pytko-Polonczyk J, Konturek S, Karczewska E, et al. Oral cavity as permanent reservoir of Helicobacter pylori and potential source of reinfection. J Physiol Pharmacol 1996;47: Recordati C, Gualdi V, Tosi S, et al. Detection of Helicobacter spp. DNA in the oral cavity of dogs. Vet Microbiol 2007;119: Hildebrand P, Bardhan P, Rossi L, et al. Recrudescence and reinfection with Helicobacter pylori after eradication therapy in Bangladeshi adults. Gastroenterology 2001;121: Bell G, Powell K, Burridge S, et al. Reinfection or recrudescence after apparently successful eradication of Helicobacter pylori infection: Implications for treatment of patients with duodenal ulcer disease. Q J Med 1993;86: Happonen I, Linden J, Westermarck E. Effect of triple therapy on eradication of canine gastric helicobacters and gastric disease. J Small Anim Pract 2000;41: Cornetta AM, Simpson KW, Strauss-Ayali D, et al. Use of [ 13 C] urea breath test for detection of gastric infection with Helicobacter spp in dogs. Am J Vet Res 1998;59: Chiba N. Effects of in vitro antibiotic resistance on treatment: Bismuth-containing regimens. Can J Gastroenterol 2000;14: Treiber G, Wittig J, Ammon S, et al. Clinical outcome and influencing factors of a new short-term quadruple therapy for Helicobacter pylori eradication: A randomized controlled trial (MACLOR study). Arch Intern Med 2002;162: Lahaie R, Farley A, Dallaire C, et al. Bismuth-based quadruple therapy with bismuth subcitrate, metronidazole, tetracycline and omeprazole in the eradication of Helicobacter pylori. Can J Gastroenterol 2001;15: Willard M, Jergens A, Duncan R, et al. Interobserver variation among histopathologic evaluations of intestinal tissues from dogs and cats. J Am Vet Med Assoc 2002;220: Linskens R, Huijsdens X, Savelkoul P, et al. The bacterial flora in inflammatory bowel disease: Current insights in pathogenesis and the influence of antibiotics and probiotics. Scand J Gastroenterol 2001;234: Marks S. Management of canine inflammatory bowel disease. Compend Contin Educ Pract Vet 1998;20: Groman R. Metronidazole. Compend Contin Educ Pract Vet 2000;22: Scanziani E, Simpson K, Monestiroli S, et al. Histological and immunohistochemical detection of different Helicobacter species in the gastric mucosa of cats. J Vet Diagn Invest 2001;13: Cattoli G, van Vugt R, Zanoni R, et al. Occurrence and characterization of gastric Helicobacter spp. in naturally infected dogs. Vet Micro 1999;70: Fallone C. Epidemiology of the antibiotic resistance of Helicobacter pylori in Canada. Can J Gastroenterol 2000;14: