Peculiarities of Diagnostic Reliability-Nested PCR Versus SAT in the Identification of Helicobacter pylori

Abstract

H. pylori detection via the stool antigen test (SAT) requires 100 times more cells than nested PCR (NPCR) for a 454 bp amplicon, but is significantly more sensitive in identifying positive stool samples. To understand this contradiction, we developed an NPCR assay to amplify a shorter 148 bp segment of the 16S rRNA gene. The assay was extremely sensitive and reliable when adhering to particular rules commonly used in forensic laboratories. The SAT and NPCR for long and short amplicons were compared using stool samples from 208 gastroenterological patients, of which 27.9% were identified as positive according to the SAT and only 6.25% according to the 454 bp NPCR amplicon, but 51.0% in the short 148 bp NPCR. Among 100 asymptomatic volunteers, the prevalence was 35% in the SAT assay and 22% in the long NPCR, but as much as 66.6% of positives were determined in the short 148 bp NPCR. The specificity of the PCR product was determined via DNA sequencing, which confirmed H. pylori's origin in all NPCR-positive samples. Apparently, the stool contains mostly short fragments of H. pylori DNA, and the most plausible explanation for the SAT/NPCR paradox is the degradation of H. pylori DNA in the digestive system.

Keywords: H. pylori; diagnostic reliability; nested PCR; stool antigen test.

Figure 1

Representative data of the 148 bp amplicon nested PCR assay for H. pylori detection in stool samples. Lanes: λ λ/PstI size marker; 1, 4, 7, 10, and 13 samples; 16 positive control; 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, and 18 negative controls (no DNA). (A) Amplified by external primers HeliS-trim/Satout (192 bp). (B) PCR product amplified by internal primers Hpup/Satin (148 bp), 2% agarose gel electrophoresis.

Figure 2

Circular cladogram showing the relatedness of H. pylori strains identified in stool samples. The cladogram was calculated from the comparison of the 94 bp 16S rDNA fragment (neighbor-joining algorithm) of 96 stool samples. Red highlights SAT-positive samples, blue highlights SAT-negative samples.