Initial Reverse Transcription is Essential for Reliable Helicobacter pylori 23S rDNA Real-Time PCR Diagnostics from Fecal Samples.

Abstract

A sensitive, non-invasive molecular test for identifying Helicobacter pylori (H. pylori) in stool samples is important in the context of a "test-and-treat strategy". The goal of this study was to elucidate the benefit of an initial reverse transcription (RT) step for the sensitivity of H. pylori 23S rDNA Real-time PCR in stool samples. We compared a LightMix® Modular Helicobacter 23S rDNA PCR assay without (LightMix® PCR) and with an initial reverse transcription step (LightMix® RT-PCR) assay for rapid detection of Helicobacter pylori in fecal samples. For maximum sensitivity, all Lightmix® RT-PCR assays were performed in triplicate for each sample. Additionally, the LightMix® RT-PCR was compared to H. pylori AG ELISA. Direct detection of H. pylori in feces by Lightmix® PCR was less sensitive than LightMix® RT-PCR. Only 2 out of 44 stool samples (4.5%) from patients with diarrhea were positive for H. pylori in the LightMix® PCR assay. In contrast, a reverse transcription step prior Lightmix® PCR increased the assay sensitivity markedly (19 out of 44 positives, 43.2%). When re-testing 65 samples initially analyzed with H. pylori AG ELISA with LightMix® RT-PCR, the detection rate for H. pylori was similarly increased from 23.1% (15/65) with H. pylori AG ELISA to 43.1% (28/65) with H. pylori LightMix® RT-PCR. 21.5% of the 28 H. pylori positive samples were positive in 1/3, 32% in 2/3, and 46.5% in 3/3 triple RT-PCR approaches. When re-testing the 28 LightMix® RT-PCR positive samples by LightMix® PCR, only 17.9% (5/28) RT-PCR positive samples were positive in PCR. LightMix® RT-PCR is much more sensitive than Lightmix® PCR. In comparison to H. pylori AG ELISA, the LightMix® RT-PCR shows a markedly higher sensitivity. An initial reverse transcription step is crucial for reliable Helicobacter pylori 23S rDNA Real-time PCR diagnostics. The triple RT-PCR approach can additionally improve the detection of H. pylori in fecal samples. Thus, the method presented provides a highly sensitive, noninvasive assay to detect H. pylori in fecal samples with potential advantages compared to other H. pylori detection methods currently used in routine diagnostics.