Non-invasive detection and successful treatment of a Helicobacter pylori infection in a captive rhesus macaque

Abstract

Gastritis is a commonly diagnosed condition in non-human primates used in biomedical research. As in humans, Helicobacter pylori infection may cause gastritis. The following report presents a method of non-invasive detection and a successful treatment protocol for this common pathogen.

Gastritis and gastric ulcerations in non-human primates frequently present as an incidental finding at necropsy and are normally not associated with major clinical signs. ¹ In cases of severe gastritis, animals may show anorexia, weight loss, abdominal pain, bloating, vomiting or regurgitation, melena, hematemesis, ascites, dehydration, tachypnea, tachycardia, pale mucous membranes and/or lethargy.^1,2 As in humans, gastric disease can be associated with Helicobacter pylori,^1,2 and due to a similar anatomy and pathophysiology rhesus macaques may represent a valuable animal model. ³

In vivo diagnostic tests for Helicobacter spp. in non-human primates typically involve gastric endoscopy and biopsy. The biopsy specimens are then submitted for histology, rapid urease testing, polymerase chain reaction or culture.^1,2 For humans, non-invasive diagnostic tests such as quantitative and qualitative antibody testing, urea breath test and fecal antigen tests (FATs) are available.^4,5 Amongst these tests, FATs are considered to be suitable for children and handicapped individuals since they do not require the patient’s cooperation, and the use of monoclonal FATs is clinically accepted. ⁵

Here we report a case of an 8-year-old, male rhesus monkey showing weight loss, paleness, persistent alopecia, deteriorated general appearance and a distinct sour foetor ex ore. Apart from severe hypophosphatemia (0.291 mmol/L [0.872–1.453 mmol/L] ⁶ ), hematology and serum chemistry values (white blood count [WBC], red blood count [RBC], hemoglobin [HGB], hematocrit [HCT], platelet [PLT], mean corpuscular volume [MCV], mean corpuscular haemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC], albumin [ALB], alkaline phosphatase [ALKP], alanine aminotransferase [ALT], amylase [AMYL], urea nitrogen [BUN], cholesterol [CHOL], creatinine [CREA], globulin [GLOB], glucose [GLU], total bilirubin [TBIL], total protein [TP], calcium [Ca], chloride [Cl], potassium [K], magnesium [Mg], sodium [Na]) were normal.

Based on the clinical symptoms, a case of severe gastritis was suspected. Hence a proton pump inhibitor (esomeprazole [Nexium™10 mg; AstraZeneca, Wedel, Germany], 0.4 mg/kg twice a day) was used for symptomatic treatment, and a monoclonal FAT (RIDASCREEN™ FemtoLab H. Pylori; R-Biopharm AG, Darmstadt, Germany) was performed. The enzyme-linked immunoassay was done in an accredited laboratory (Institute of Medical Microbiology and Hygiene, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany) according to the manufacturer’s instructions. ⁷ Upon receipt of a positive test result, the treatment was extended to a 10-day oral clarithromycin-based triple therapy (esomeprazole, amoxicillin and clarithromycin [the latter two from Hexal AG, Holzkirchen, Germany], 0.4, 10 and 10 mg/kg respectively, twice a day). After the 10-day treatment and in the subsequent weeks, the monkey’s average body weight increased by more than 15% (30 weeks before treatment: $\overline{\text{x}}$  = 7.5 kg; 30 weeks after treatment: $\overline{\text{x}}$  = 8.6 kg) (Figure 1), the phosphate value returned to a normal level (1.033 mmol/L), the fur started to regrow, and paleness and foetor ex ore disappeared.

Figure 1.

Weight change before and after triple therapy and results of consecutive fecal antigen tests (FATs).

To examine the effectiveness of the treatment, fecal samples were collected and tested at four weeks ⁵ and again at 8, 12, 16, 20 and 24 weeks after the end of the treatment. Four weeks after the end of the antibiotic treatment, the animal was diagnosed to be negative for H. pylori by FAT and remained negative for the subsequent 20 weeks. At 24 weeks the FAT was again positive and the result confirmed by retesting one and three weeks later (Figure 1). However, the animal’s body weight remained stable and none of the clinical signs observed before treatment developed again. To monitor the status of other animals in the facility, more fecal samples were tested. An animal was considered to be positive for H. pylori if the result was positive twice ⁵ at a weekly interval, and negative if the result was negative three times at a weekly interval, since intermittent shedding, low amounts of antigen and inhomogeneous dispersal of the antigen in the sample could occur. All animals of the same origin, that arrived together as a group (n = 3), and two out of the five animals which were temporarily in direct or continuously in indirect (alternate group cage sharing) contact with the monkey were tested positive. As none of the monkeys tested positive displayed clinical signs of disease no antibiotic treatment was administered.

The case presented demonstrates that a clarithromycin-based triple therapy, at the dosage and duration used, can control H. pylori infection in a naturally infected rhesus macaque. Clarithromycin was not used in the facility prior to the present treatment, which may explain the efficiency of the drug combination, although the emergence of clarithromycin-resistant strains is of considerable concern in human medicine. ⁸ Neither of the symptoms observed before treatment nor any other clinical signs of H. pylori-associated gastritis^1,2 appeared again. Whether a reinfection or a re-emergence of H. pylori had occurred remained unclear.

Notably, this report presents the possibility of a non-invasive detection of H. pylori in captive macaques. It has to be pointed out that a FAT is solely a qualitative test, which cannot provide a quantitative statement or prediction of the severity of disease or clinical outcome. Therefore all results have to be interpreted in conjunction with clinical data and/or other direct test procedures, such as histology, rapid urease testing, polymerase chain reaction or culture of a biopsy specimen. However, all direct methods are more costly and require the use of invasive endoscopy, as well as a certain setting such as trained personnel and a specialized laboratory (Table 1). Due to these technical difficulties and the varying prevalence and density of H. pylori in the stomach, the reliability of the results could be significantly affected, particularly during the treatment period when the amount of H. pylori is changing. Comparing FAT to other non-invasive diagnostic methods (Table 1), the lack of cooperation from the animal and the technical equipment necessary will be the limiting factors in performing a urea breath test. Serological testing, which needs a blood sample, can be regarded as a ‘semi-invasive’ method. Reported values for sensitivity and specificity of a recently established serological test lie at 93% and 100%, respectively. ⁹ The monoclonal FAT used here detects H. pylori antigen with a sensitivity of 98.6 to 100% and a specificity of 97.1 to 99.4% in a human fecal sample, and cross reactions with a large number of common pathogenic microorganisms are excluded. ⁷

Table 1.

Comparison of diagnostic tests for Helicobacter pylori in non-human primates.

	Endoscopic methods				Non-endoscopic methods
	Culture	Histology	RUT	PCR	Serology	UBT	mFAT
Invasiveness	+	+	+	+	(+)	–	–
Need for anesthesia	+	+	+	+	–/+*	–	–
Need for patient cooperation	na	na	na	na	+/na*	+	–
Need for trained personnel and/or equipment	+	+	+	+	–	+	–
Sensitivity (%)	70–905	80–984,5	90–>954,5	90–955	939	85–>955	98.6–1007
Specificity (%)	1005	90–984,5	90–>954,5	90–955	1009	85–>955	97.1–99.47
Expense	Expensive	Expensive	Inexpensive	Expensive	Inexpensive	Expensive	Inexpensive
Else	Allows determination of antibiotic sensitivities4,5	Considered by some to be the gold standard 4	Sensitivity reduced in post-treatment setting4,5	Allows determination of antibiotic sensitivities4,5	Not recommended after therapy4,5	Identifies active infection; useful before and after therapy4,5	Identifies active infection; useful before and after therapy4,5

RUT: rapid urease testing; PCR: polymerase chain reaction; UBT: urea breath test; mFAT: monoclonal fecal antigen test; na: not applicable; *in untrained animals.

To apply the monoclonal FAT for routine Helicobacter diagnostic, further studies are required to test sensitivity and specificity as well as potential cross reactions with other new and old world monkey gastric and enterohepatic Helicobacter sp^6,10. However, monoclonal FAT presents a promising, non-invasive, inexpensive and simple, though accurate, method for the veterinary clinician to monitor the infection status of H. pylori in primates in captivity.