Significantly elevated Helicobacter pylori density and different genotype distribution in erosions as compared with normal gastric biopsy specimen detected by quantitative real-time PCR.
OBJECTIVE
Determination of the local densities of Helicobacter pylori and its genotypic variations in gastric biopsy specimens by using novel real-time PCR-based methods could support the precise diagnosis and understanding of H. pylori infections.


METHODS
Serial dilutions of H. pylori (0.016-16 microg/microl), control, bacterial, and human DNA samples were prepared. Fresh-frozen gastric biopsy specimens were taken from 103 patients, and the DNA was isolated. Quantitative determination of the ureaseA gene using hybridization probes with parallel evaluation of an internal human control gene (beta-globin) was performed by real-time PCR. CagA and VacA s1 genotypic characterizations were also performed. The data were compared with urea breath test (UBT), histology, and serological testing.


RESULTS
The presence of H. pylori could be detected by ureaseA-fluorescence energy transfer (53%), UBT (51%), serological testing (48%), and histology (52%) when compared with the gold standard (54%). A significant correlation was found between the quantitative real-time ureaseA/beta-globin ratio-based H. pylori frequency and the UBT results (P<0.01). Significantly increased bacterial density was found in the erosions when compared with the healthy part of the antrum and corpus (P<0.01). Real-time PCR VacA s1 results were in significant correlation (P<0.01) with those of serological tests, but CagA results were not. The genomic profiles (VAC/GAC) were different in 13.7% of the cases, which involved three different locations in the stomach.


CONCLUSIONS
Real-time PCR was the most reliable method for H. pylori diagnosis. Furthermore, quantification and genotyping could also be performed using this technique. The density of H. pylori was significantly increased in macroscopic erosions.
Molnar
Bela
B
Clinical Research Unit, Hungarian Academy of Sciences, Budapest, Hungary. mb@bel2.sote.hu
Szoke
Dominika
D
Ruzsovics
Agnes
A
Tulassay
Zsolt
Z
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001706:Biopsy; D001944:Breath Tests; D005260:Female; D005756:Gastritis / Q000382:microbiology*; D005838:Genotype; D016481:Helicobacter Infections / Q000235:genetics / Q000378:metabolism*; D016480:Helicobacter pylori / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013270:Stomach / Q000473:pathology*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C080866:cagA protein, Helicobacter pylori