Helicobacter

Test codes:

B0021 - Ultrasensitive qualitative detection of Helicobacter pylori by real time polymerase chain reaction

B0023 - Ultrasensitive qualitative detection of Helicobacter heilmannii by real time polymerase chain reaction

B0097 - Ultrasensitive qualitative detection of Helicobacter mustelae by real time polymerase chain reaction

P0010 - Ultrasensitive Helicobacter species screen by real time polymerase chain reaction.  This screen detects but does not differentiate H. pylori, H. heilmannii, H. bilis, H. hepaticus, H. rappini, H. felis, H. salomonis and other Helicobacter species.

P0011 - Ultrasensitive Helicobacter species identification by real time polymerase chain reaction and PCR product sequence analysis.  This 2-stage assay detects and differentiates H. pylori, H. heilmannii, H. bilis, H. hepaticus, H. rappini, H. felis, H. salomonis and other Helicobacter species.

Many species of the genus Helicobacter have been identified in mammals and their pathogenicity varies. Some species can induce significant disease while others appear to merely colonize the gastrointestinal tract.

Helicobacter pylori
Helicobacter pylori is a gram-negative spiral bacterium found in gastric mucosa and associated with active and chronic gastritis. H. pylori can establish a chronic, persistent infection, which may lead to gastric or duodenual ulcers, gastric cancer and gastric lymphomas. Studies have revealed that approximately 50% of the world’s human population is infected with H. pylori.

Biochemically, the bacterium produces catalase, oxidase and urease enzymes. The urease enzyme permits the bacterium to metabolize urea present in the gastric mucosa and establish a microenvironment favorable to the organism. H. pylori is a highly motile organism with multiple unipolar flagella. Both the urease enzyme and the flagella are considered to be important virulence factors.

Diagnosis of Helicobacter pylori infection in humans relies on upper endoscopy or the 13C-urea breath test (see review by Nakamura, 2001). Although the endoscopy procedure permits culture of the bacterium from biopsy specimens (the gold standard for diagnosis), demonstration of urease activity and histological detection of the germ, the procedure is expensive and invasive. The 13C-urea breath test is a well-established, relatively sensitive, specific and noninvasive method. Molecular tests, such as PCR, can also offer precise diagnosis of H. pylori infections. In fact, molecular testing by PCR can complement other diagnostic tests because it can be applied to archival fixed tissue, environmental samples, gastric fluid, oral secretions, and stool samples, in which traditional diagnostic tests do not have sensitivity and perform poorly. Studies have shown than PCR detection of H. pylori in gastric fluid specimens can reach a sensitivity of 96% and a specificity of 100% (Westblom et al., 1993; Yoshida et al., 1999). This capability is especially useful in monitoring active H. pylori infection in primates and other animals, as the breath test is difficult to conduct for these animals.

Helicobacter heilmannii
Helicobacter heilmannii (previously known as Gastrospirillum hominis) is a 4-10 µm long, spiral-shaped, motile bacterium with three to eight coils, a wavelength of about 1 µm, up to 14 uni- or bipolar flagella, and no periplasmic filaments. In humans, gastric infection with H. heilmannii is associated with the development of chronic gastritis (found in the stomachs of 0.2 to 4% of patients with gastritis) and low-grade mucosa-associated lymphoid tissue lymphoma in humans. Eradication of H. heilmannii by antibiotic treatment of patients can result in complete remission of MALT lymphoma, indicating a causal relationship between H. heilmannii infection and MALT lymphoma. Unlike H. pylori infections, gastric infections with H. heilmannii or Gastrospirillum-like organisms are not restricted to humans. A broad range of animals, including dogs, cats, pigs, and cattle, are naturally infected, with frequencies ranging from 80% to 100%. It has been suggested that H. heilmannii infection in humans may be a zoonosis and that animals may serve as a reservoir for transmission to humans.

Definitive culture of H. heilmannii has not been achieved to date (Anderson et al., 1996) and diagnosis of H. heilmannii infection is usually made on the basis of its distinct spiral morphology, compared with H. pylori, on silver- stained tissue sections. However, there are a number of large, gastric, spiral organisms such as H. felis, H. salomonis, and H. bizzozeronii which are indistinguishable from H. heilmannii on routine light microscopy, and H. pylori grown in a broth culture can also adopt a morphology identical to that of H. heilmannii (Fawcett et al., 1999). Molecular detection methods such as PCR are required for more definitive identification (Trebesius et al., 2001).

Helicobacter mustelae
The genus Helicobacter contains more than 50 species which have been isolated from a wide range of vertebrate hosts. Among these species, Helicobacter mustelae is especially interesting because it is the only Helicobacter species other than H. pylori that is known to cause gastritis, ulcers and gastric cancer, though these only occur in ferrets and other mustelids.

When ferrets are infected with H. mustelae, the bacteria stimulate gastric epithelial cells to proliferate and it has been suggested that this stimulation results in gastric adenocarcinoma and MALT lymphoma formation. Ulcers are also common in infected ferrets. In clinical research, H. mustelae infection of ferrets is the only natural model of Helicobacter-associated ulcer disease.

It has been suggested that nearly 100% of ferrets may be infected with Helicobacter mustelae by weaning. However, only a small percentage of these ferrets show clinical symptoms. Disease is seen most commonly in ferrets that have been stressed or have other concurrent diseases. Clinical signs of infection include lack of appetite, diarrhea, black tarry stools, vomiting and abdominal pain (as manifested by grinding teeth, hunched posture, and reluctance to move). Signs of nausea such as increased salivation and pawing at the mouth may be seen. With chronic infection, weight loss, weakness, and loss of muscle mass are common symptoms.

Culture detection of H. mustelae is difficult because of its fastidious growth requirements. Molecular detection by polymerase chain reaction (PCR) is rapid, sensitive and specific, and is a good alternative to the traditional approach (Forester et al., 2003.

Utilities:

• Help confirm the disease causing agent
• Shorten the time required to confirm a clinical diagnosis of Helicobacter infection
• Help ensure that animal facilities are free of Helicobacter
• Early prevention of spread of these bacteria among a group of animals
• Minimize personnel exposure to these bacteria
• Safety monitoring of biological products and vaccines that derive from susceptible animals

References:
Nakamura, R.M. (2001) Laboratory tests for the evaluation of Helicobacter pylori infections. J. Clin. Lab. Analysis 15: 301-307.
Westblom, T.U., Phadmis, S., Yang, P. and Czinn, S.J. (1993) Diagnosis of Helicobacter pylori infection by means of a polymerase chain reaction for gastric juice aspirates. Clin. Infect. Dis. 16: 367-371.
Yoshida, H., Maeda, S. and Ogura, K. (1999) PCR-monitoring of gastric juice obtained with the capsulated string for evaluation of H. pylori infection. Nippon Rhinsho 57: 107-110.
Andersen, L.P., Norgaard, A., Holck, S., Blom, J. and Elsborg, L. (1996) Isolation of a "Helicobacter heilmannii"-like organism from the human stomach. Eur. J. Clin. Microbiol. Infect. Dis. 15:95-96.
Fawcett, P.T., Gibney, K.M. and Vinette, K.M. (1999) Helicobacter pylori can be induced to assume the morphology of Helicobacter heilmannii. J. Clin. Microbiol. 37:1045-1048.
Trebesius, K., Adler, K., Vieth, M., Stolte, M. and Haas, R. (2001) Specific detection and prevalence of Helicobacter heilmannii-like organisms in the human gastric mucosa by fluorescent in situ hybridization and partial 16S ribosomal DNA sequencing. J. Clin. Microbiol. 39:1510-1516.
Forester, N.T., Lumsden, J.S., Parton, K., Cowan, P.E. and O'Toole, P.W. (2003) Detection and isolation of Helicobacter mustelae from stoats in New Zealand. New Zealand Vet. J. 51:142-145.

Specimen requirement:
B0021, B0023, B0097 - 0.2 ml gastric lavage, or rectal swab or 0.2ml feces, or 0.2 ml fresh, frozen or fixed tissue, or 0.2 ml cell culture
P0010, P0011 - 0.2 ml cell culture

Contact Zoologix if advice is needed to determine an appropriate specimen type for a specific diagnostic application. For specimen types not listed here, please contact Zoologix to confirm specimen acceptability and shipping instructions.

For all specimen types, if there will be a delay in shipping, or during very warm weather, refrigerate specimens until shipped and ship with a cold pack unless more stringent shipping requirements are specified. Frozen specimens should be shipped so as to remain frozen in transit. See shipping instructions for more information.

Turnaround time: 2 business days (1 week for P0011)

Methodologies:
B0021, B0023, B0097, P0010:  Qualitative real time PCR
P0011:  Qualitative real time PCR and PCR product sequence analysis

Normal range: Nondetected