Absence of Helicobacter species in the liver of patients with primary or metastatic liver cancer

Coppola et al.1 recently reported an absence of Helicobacter pylori and other Helicobacter species in patients with primary or metastatic liver cancer. However, the article failed to discuss the recent findings on this topic by Bulajic et al.2 and Fukuda et al.,3 which were consistent with previous reports demonstrating high incidence of infection with Helicobacter species in chronic liver disease and biliary tract carcinoma.4-6 The article by Coppola et al.1 raises some important questions regarding the methodology used to identify Helicobacter infection and the conclusions made based on it. The full potential of diagnostic polymerase chain reaction (PCR) is limited, in part, by the low concentration of microorganisms and the presence of PCR inhibitors that reduce or even block PCR.7 Diagnostic PCR involves 4 steps: (1) sample collection, (2) sample pretreatment, (3) nucleic acid amplification, and (4) detection of the PCR product.8 Therefore, all these factors should be taken into consideration before comparing results of independent studies. In the article by Coppola et al. the extraction and amplification methods of liver tissue were validated by amplifying DNA extracted from stomach biopsy samples and detection of hepatitis B viral (HBV) DNA in the liver of HBV-infected patients. Such validation is inadequate without checking the sensitivity of the PCR assay on Helicobacter-negative stomach and liver tissues spiked with known concentrations of Helicobacter. In addition, the validation of the Helicobacter PCR assay for liver tissue samples by detecting H. pylori DNA in the stomach of H. pylori–positive patients is incorrect, because different tissues can vary regarding PCR sensitivity due to differences in PCR inhibitor, and microorganism concentration. The detection of HBV DNA in liver samples, which may be present at a higher concentration than Helicobacter DNA, may be useful for validatation of an HBV assay, but is not suitable for entirely different microorganisms such as Helicobacters. Zanella et al. detected 105 copies HBV DNA per microgram of total DNA in liver samples of hepatitis B surface antigen (HBsAg)-positive patients (13), which is higher than the copy number of Helicobacter DNA we observed in liver tissue samples of patients with hepatocellular carcinoma (Wadström et al., unpublished data). Therefore, it is inadequate to use the HBV assay as a control for the Helicobacter assay. It has been shown before that increasing the target template concentration may overcome PCR inhibition.9 This may explain the ability of Coppola et al. to detect high concentrations of HBV DNA in liver tissue whereas Helicobacter DNA was not detectable in the singlestep PCR assay. Applying a 1or 2-step PCR is known to effect PCR detection sensitivity. Stärk et al.10 showed that nested PCR was as much as 104 times more sensitive than a 1-step PCR. Therefore, in our studies we have used the more sensitive nested PCR assay for detection of Helicobacter DNA in liver and gallbladder samples. The question is still open as to the potential role of the Helicobacter genus in the pathogenesis of hepatic and biliary tract diseases. However, the points made above must be considered to standardize the methodology and ensure comparable results from different groups. Only then can we assess the true impact of enterohepatic Helicobacters on human health.