Three Tests Used to Identify Non-Culturable Form of Helicobacter pylori in Water Samples.

Abstract

Background:Helicobacter pylori, causing the most common chronic bacterial infection, exist in two forms; bacilli and coccoid. The coccoid form is identified as viable but non-culturable bacteria.Objectives:The current study aimed to conduct culture, polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP) tests to identify coccoid forms of H. pylori.Materials and Methods:The PCR and LAMP tests were optimized using specific primers for glmM gene. The sensitivity and specificity of the tests were determined. The current experimental study was conducted on 10 different strains isolated from clinical cases (H1-H10). The isolates were added to tap water and incubated at three different temperatures for one and two months intervals. After pure-culturing of the bacteria, DNAs were extracted and PCR and LAMP were performed.Results:Ten copies of targeted DNA were required for PCR detection whereas only five copies gave a positive reaction by LAMP assay, with 100% specificity. Of the 10 isolates inoculated in water for one and two months at three different temperatures 4, 22, and 37°C, only three cases (5%) were found positive in the first month; 13 (21.6%) and 29 cases (48.3%) were also positive by PCR and LAMP tests in the first and second months.Conclusions:Results of the current study confirmed that molecular methods such as PCR and LAMP were much more sensitive, rapid, and specific than culturing to identify non-culturable coccoid forms of H. pylori in water.