Detection of Helicobacter pylori by Real-Time PCR for 16s rRNA in Stools of NonInfected Healthy Children, Using ELISA Antigen Stool Test as the Gold Standard

SUMMARY
The importance of Helicobacter pylori (H. pylori) infection in gastroduodenal disease is well established. However, most infected individuals never experience clinically relevant signs and/or symptoms. Reliable noninvasive diagnostic methods have been developed for H. pylori detection. Antibody-based enzyme-linked immunosorbent assay (ELISA) for H. pylori detection in stools has shown high sensitivity and specificity. Real-time PCR (rt-PCR), meanwhile, is not widely used for stool detection of H. pylori, and results have been variable. Infection status in asymptomatic children is currently categorized into two groups: transient infections or persistent infections.
Data of a previous study showed that  33% of ELISA-negative samples were rtPCR positive. So, the authors aimed to determine whether persistent infections could be accurately detected by rtPCR, despite the possibility of false ELISA negative results. For the purpose, ELISA antigen stool tests were used as “gold standard”, and rtPCR for 16s rRNA as the test method. The study was based on two previous prospective cohort studies of Chilean children: the authors selected 36 subjects with an ELISA-negative/rtPCR-positive stool sample. 25 of these children had never been infected with H. pylori and 11 had a transient infection.
To determine the proportion of these children with a possible persistent infection detectable only by rtPCR, two to three ELISA-negative stool samples from each child were tested by rtPCR for 16s rRNA. A total of 78 samples were analyzed. To confirm the reproducibility of the rtPCR protocol, five samples from infected children were reanalyzed by rtPCR: as expected, rtPCR for 16s rRNA was consistently positive. The authors performed 156 rtPCR on ELISA-negative stool samples, of which 14 (18%) were rtPCR positive, but only 4 of 78 (5.1%) samples were consistently positive by rtPCR for 16S rRNA, from a total of 3/36 (8.3%) children who had an initial rtPCR-positive/ELISA-negative sample. Only 4% of noninfected children had one positive sample, in both duplicates, by rtPCR. Two of the eleven children with a transient infection by ELISA (18.1%) had one or two additional rtPCR-positive samples in both duplicates. Overall, rtPCR testing for 16s rRNA was able to detect a possible persistent infection not detected by ELISA in one of 36 (2.7%) children.
The authors highlighted that both noninvasive tests, ELISA and rtPCR run in duplicate, are highly concordant when defining the infection status of children, not only for persistently infected children, but also for transient or noninfected children. Although scarce, ELISA-negative/rtPCR-positive samples did occur and their significance remains to be elucidated. Importantly, the majority of positive results occurred in only one of the two duplicates, strongly suggesting that these were false positives. The relatively higher 16S rRNA detection rate in stools from children with a transient infection compared to those who never had an ELISA-positive sample, suggests that the intermittency of bacterial/antigen load in the stools of these children may be playing a role in determining ELISA versus rtPCR positivity. The authors concluded that, despite the need of further research, stool ELISA is a reliable method in characterizing H. pylori dynamics in apparently healthy children.

AUTHORS: George S, Mamani N, Lucero Y, Torres JP, Farfán M, Lagomarcino AJ, Orellana A, O'Ryan M.

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