Identification of Helicobacter pylori and other Helicobacter species by PCR, hybridization, and partial DNA sequencing in human liver samples from patients with primary sclerosing cholangitis or primary biliary cirrhosis

Abstract

Helicobacter pylori was identified in human liver tissue by PCR, hybridization, and partial DNA sequencing. Liver biopsies were obtained from patients with primary sclerosing cholangitis (n = 12), primary biliary cirrhosis (n = 12), and noncholestatic liver cirrhosis (n = 13) and (as controls) normal livers (n = 10). PCR analyses were carried out using primers for the Helicobacter genus, Helicobacter pylori (the gene encoding a species-specific 26-kDa protein and the 16S rRNA), Helicobacter bilis, Helicobacter pullorum, and Helicobacter hepaticus. Samples from patients with primary biliary cirrhosis and primary sclerosing cholangitis (11 and 9 samples, respectively) were positive by PCR with Helicobacter genus-specific primers. Of these 20 samples, 8 were positive with the 16S rRNA primer and 9 were positive with the 26-kDa protein primer of H. pylori. These nine latter samples were also positive by Southern blot hybridization for the amplified 26-kDa fragment, and four of those were verified to be H. pylori by partial 16S rDNA sequencing. None of the samples reacted with primers for H. bilis, H. pullorum, or H. hepaticus. None of the normal livers had positive results in the Helicobacter genus PCR assay, and only one patient in the noncholestatic liver cirrhosis group, a young boy who at reexamination showed histological features suggesting primary sclerosing cholangitis, had a positive result in the same assay. Helicobacter positivity was thus significantly more common in patients with cholestatic diseases (20 of 24) than in patients with noncholestatic diseases and normal controls (1 of 23) (P = <0.00001). Patients positive for Helicobacter genus had significantly higher values of alkaline phosphatases and prothrombin complex than Helicobacter-negative patients (P = 0.0001 and P = 0.0003, respectively). Among primary sclerosing cholangitis patients, Helicobacter genus PCR positivity was weakly associated with ulcerative colitis (P = 0.05). Significant differences related to blood group or HLA status were not found.

FIG. 1

Analysis of Helicobacter genus-specific PCR products from liver samples of patients with PSC and PBC. The 400-bp fragments were analyzed by 2% agarose gel electrophoresis. Lanes 1 to 5, positive samples from PSC patients; lanes 6 to 11, positive samples from PBC patients; lane 12, negative control (double-distilled water); lane 13, H. pylori DNA; lane 14, H. bilis DNA; lane M, 100-bp DNA ladder size markers.

FIG. 2

Southern blot hybridization of PCR products generated by primers based on the gene encoding a 26-kDa surface protein of H. pylori. Hybridization was performed using a digoxygenin-labeled probe generated by amplification of DNA from H. pylori strain 17874 with the species-specific D primers. Lane 1, positive control (H. pylori DNA); lane 2, negative control (double-distilled water); lanes 3 and 4, positive samples from PSC patients; lanes 5 to 7, positive samples from PBC patients; lanes 8 and 9, Helicobacter-positive but H. pylori-negative samples from PSC patients; lane 10, Helicobacter-positive but H. pylori-negative sample from a PBC patient.