Unreliability of Results of PCR Detection of Helicobacter pylori in Clinical or Environmental Samples

Abstract

The aim of this study was to compare published Helicobacter pylori primer pairs for their ability to reliably detect H. pylori in gastric biopsy specimens and salivary samples. Detection limits of the 26 PCR primer pairs previously described for detection of H. pylori DNA in clinical samples were determined. Sensitivity and specificity were determined using primers with detection limits of <100 CFU/ml using 50 H. pylori-positive and -negative (by concordance by culture and histology) coded gastric biopsy specimens. These results were then confirmed with gastric biopsy specimens and saliva from patients with confirmed H. pylori status. Five of the twenty-six previously reported primer pairs (HP64-f/HP64-r, HP1/HP2, EHC-U/EHC-L, VAG-F/VAG-R, and ICT37/ICT38) had detection limits of <100 CFU/ml in the presence of gastric tissue. None had 100% specificity or sensitivity; all produced false-positive results. The HP64-f/HP64-r for ureA and HP1/HP2 for 16S rRNA individually had sensitivities and specificities of >90% with gastric biopsy specimens. No combinations of primer pairs improved the results. Using these five primer pairs, 54% of the positive saliva samples were determined to be false positive; both the HP64-f/HP64-r and the HP1/HP2 sets produced false positives with saliva. We conclude that clinicians should not rely on results using current PCR primers alone to decide the H. pylori status of an individual patient or as a basis for treatment decisions. The results of studies based on PCR identification of H. pylori in environmental samples should be viewed with caution. Possibly, specific primers sets can be identified based on the presence of multiple putative virulence factor genes.

An evaluation of selected oral health indicators and cariogenic bacteria titer in patients with Helicobacter pylori

“…Other species of bacteria that secrete urease in the plaque, such as Streptococci, Actinomyces or Haemophilus spp, can cause false positives. 18,19 The research team of Silva et al demonstrated a correlation between the colonization of supragingival plaque and oral health parameters -specifically, between the percentage of the surface covered with dental plaque and the bleeding rates of periodontal pockets. 16 Patients with higher values of these 2 indicators were likely to report the presence of H. pylori in their oral cavities.…”

“…Dental plaque has been called "a reservoir for genotypes", where horizontal gene transfer can take place between the bacteria that reside in the biofilm. 17,18 The genotypic variability of this bacteria manifests itself in the presence of point mutations and changes in the gene sequence. This may lead to both false positive and false negative results if the PCR test is limited to determining a single gene.…”

“…Recognition of a combination of at least 2 different, simultaneously occurring genes was regarded as a positive result. 18 Individually marked genes were analyzed for the sensitivity and specificity of the primer. A 16S rRNA region that amplifies the 106bp fragment and the region from human leukocytes presents low specificity.…”

Biomarkers and diagnostic tools for detection of Helicobacter pylori

“…This type of test not only diagnoses the amount of damage in the stomach but also allows the clinician to directly sample the tissue to test it for the presence of the H. pylori organism. Noninvasive tests can identify H. pylori in a sample, but do not reveal the amount of tissue damage (Fleming et al 2009;Sugimoto et al 2009). …”

“…Reprinted with permission from Deretsky (2015) et al 2000; Monteiro et al 2001b). However, the specificity and sensitivity of the diagnosis varies, according to different sample preparations and various laboratory conditions (Sugimoto et al 2009). Monoplex, multiplex, and nested PCR methods are useful for specially identifying H. pylori in biological samples.…”