Absence of Helicobacter species in biliary atresia.

Abstract

To The Editors: Since the discovery of nongastric species of Helicobacter, ∼30 species have been described, some of which are able to grow in bile (Helicobacter pullorum, Helicobacter hepaticus, Helicobacter bilis and Flexispira rappini). It has been suggested that these bile-tolerant species of Helicobacter could be involved in inflammatory lesions of the biliary tract or liver and might induce chronic infection. Chronic hepatic and pancreatic disorders caused by Helicobacter species have been reported in animal models, and the question of a possible role of Helicobacter species in human liver pathology has been raised. 1 In infants biliary atresia is the primary cause of chronic cholestasis and is the main indication for liver transplantation. This acquired, infantile, obstructive cholangiopathy results from as yet unexplained sclerosis and a progressive inflammatory process initiated in the bile duct epithelium. Liver biopsies reveal the following histologic features: an enlarged portal tract with numerous bile ducts and cholestasis; increased fibrosis; and general infiltration with polymorphonuclear cells. The etiology of this disorder is unexplained. Several infectious hypotheses (e.g. cytomegalovirus, Epstein-Barr virus, rubella virus or reovirus) have been evoked, but none has been retained. 2 More recently the association with gastric Helicobacter pylori infection was studied in children with biliary atresia. The H. pylori infection rate was no higher than in the general population; thus the authors concluded that colonization was benign. 3 At present the etiology and pathogenesis of the progressive inflammation process observed in biliary atresia are unclear, and the disorder is considered to be a “primary” disease. However, the recent emergence of enterohepatic Helicobacter species as potential human biliary pathogens prompted us to revisit the infection hypothesis. The aim of our study was to determine whether inflammation in primary biliary atresia is associated with the presence of enterohepatic Helicobacter species. Ten sequential cases of primary biliary atresia were studied (5 boys and 5 girls, ages ranging from 19 to 92 days; median age, 50 days). To control for possible natural colonization of the infants’ biliary tracts by Helicobacter species, 10 children presenting with a range of other hepatic diseases of infancy were also examined (6 boys and 4 girls, ages ranging from 43 to 514 days; median age, 80 days). The controls presented with arteriohepatic dysplasia (n = 2); nonsyndromic, intrahepatic biliary hypoplasia (n = 2); progressive, fibrosing, intrahepatic cholestasis (n = 2); Niemann-Pick disease (n = 1); Gaucher disease (n = 1); inspissated bile syndrome (n = 1); and parenteral nutrition-associated cholestasis (n = 1). Percutaneous liver biopsies were performed with a 16-gauge Tru-cut needle. A large biopsy sample (weighing at least 10 mg) was immediately frozen and stored at –80°C. The presence or absence of Helicobacter species was tested using 16S ribosomal DNA PCR, which has previously been described as the best way to study new bacterial species with unknown growth requirements. The primers used amplify a 399-bp sequence that is 100% specific and sensitive for the Helicobacter genus: 5′-AAC GAT GAA GCT TCT AGC TTG CTA G-3′ and 5′-GTG CTT ATT CST NAG ATA CCG TCA T-3′. 4 DNA was extracted with sodium dodecyl sulfate and proteinase K. 4 The potential presence of polymerase inhibitors in hepatic tissue was tested by amplification of control aliquot of tissue extract to which a dilute suspension of H. pylori had been added. Inhibitors were detected and then successfully removed, according to a previously described method. 5 Briefly tissue extracts were embedded in low melting point agarose blocks, immersed in lysis solution and then washed; the cycle was repeated several times. DNA was then extracted from the agarose blocks using SpinX columns (Costar) and precipitated with ethanol. H. pylori controls showed good amplification. Helicobacter species was not found either in primary biliary atresia or in control biopsy samples by PCR. Consequently in spite of the low number of cases studied, we conclude that none of these species seems to be involved in pathogenesis of biliary atresia and that Helicobacter species cannot be incriminated as the missing etiologic agent of this disorder. Moreover it appears that none of the presently known Helicobacter species (whether bile-tolerant or not) can be considered to be colonizers of infants with cholestasis. This suggests that enterohepatic Helicobacter species do not, as a rule, colonize the intestine during the first months of life. Pascal Vincent, M.D. Karinne Mention, M.D. Emmanuelle Leteurtre, M.D. Sylvie Armand, M.S. Frédéric Gottrand, M.D.