This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
DNA diagnostics for reliable and universal identification of Helicobacter pylori
Pavol Sulo, Barbora Šipková
Pavol Sulo, Barbora Šipková, Department of Biochemistry, Comenius University, Bratislava 842 15, Slovakia
Author contributions: Sulo P designed the outline and wrote most of the manuscript; Šipková B summarized the data, prepared the figures and tables, and wrote some of the manuscript; both authors read and approved the final manuscript.
Supported bySlovak Research and Development Agency, No. PP-COVID-20-0051.
Conflict-of-interest statement: The authors declare that they have no conflict of interest.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non-Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Pavol Sulo, PhD, Research Associate, Department of Biochemistry, Comenius University, Ilkovicova 6, Bratislava 842 15, Slovakia. firstname.lastname@example.org
Received: March 20, 2021 Peer-review started: March 20, 2021 First decision: July 3, 2021 Revised: July 11, 2021 Accepted: September 19, 2021 Article in press: September 19, 2021 Published online: November 7, 2021
Reliable diagnostics are a major challenge for the detection and treatment of Helicobacter pylori (H. pylori) infection. Currently at the forefront are non-invasive urea breath test (UBT) and stool antigen test (SAT). Polymerase chain reaction (PCR) is not endorsed due to nonspecific primers and the threat of false-positives. The specificity of DNA amplification can be achieved by nested PCR (NPCR), which involves two rounds of PCR. If the primers are properly designed for the variable regions of the 16S rRNA gene, it is not difficult to develop an NPCR assay for the unambiguous identification of H. pylori. Elaborate NPCR for a 454 bp amplicon was validated on 81 clinical biopsy, stool, and saliva samples, each from the same individuals, and compared with available H. pylori assays, namely histology, rapid urease test, SAT, and 13C-UBT. The assay was much more sensitive than simple PCR, and it was equally sensitive in biopsy samples as the 13C-UBT test, which is considered the gold standard. In addition, it is sufficiently specific because sequencing of the PCR products exclusively confirmed the presence of H. pylori-specific DNA. However, due to the threshold and lower abundance, the sensitivity was much lower in amplifications from stool or saliva. Reliable detection in saliva also complicates the ability of H. pylori to survive in the oral cavity aside from and independent of the stomach. The reason for the lower sensitivity in stool is DNA degradation; therefore, a new NPCR assay was developed to obtain a shorter 148 bp 16S rRNA amplicon. The assay was validated on stool samples from 208 gastroenterological patients and compared to SAT results. Surprisingly, this NPCR revealed the presence of H. pylori in twice the number of samples as SAT, indicating that many patients are misdiagnosed, not treated by antibiotics, and their problems are interpreted as chronic. Thus, it is unclear how to properly diagnose H. pylori in practice. In the first approach, SAT or UBT is sufficient. If samples are negative, the 148 bp amplicon NPCR assay should be performed. If problems persist, patients should not be considered negative, but due to threshold H. pylori abundance, they should be periodically tested. The advantage of NPCR over UBT is that it can be used universally, including questionable samples taken from patients with achlorhydria, receiving proton pump inhibitors, antibiotics, bismuth compound, intestinal metaplasia, or gastric ulcer bleeding.
Core Tip: Polymerase chain reaction (PCR) is not endorsed for Helicobacter pylori (H. pylori) diagnostics due to nonspecific primers and the threat of false-positives. However, a nested PCR that is as specific and equally sensitive in biopsy samples as the 13C-urea breath test was developed. Due to the threshold of H. pylori abundance and the ability to survive in the oral cavity, it is not suitable for saliva samples. Despite DNA degradation in stool samples, nested PCR for a shorter 148 bp amplicon identified twice the number of positive samples as stool antigen test, indicating that many patients are misdiagnosed, not treated by antibiotics, explaining why their problems are interpreted as chronic.