Culture of Helicobacter pylori from stool samples in children.(Clinical report)

Abstract: We evaluated two protocols for isolation of Helicobacter pylori in stool from biopsied and nonbiopsied children. Twenty-three child patients whose presumptive positivity or negativity was diagnosed by endoscopy and a rapid urease test at site were used to compare biopsy-based tests with stool-based tests (H. pylori stool antigen test and stool culture). Their gastric activity and bacterial density were graded by the updated Sydney system. Biopsy and stool specimens were cultured on Campy-blood and Belo horizonte agar plates after enrichment in selective Campy-Thio medium. To compare two stool culture protocols, stools from 20 nonbiopsied children were tested by the HpSA test and cultured either as above or after treatment with cholestyramine. Grown colonies were screened by Gram staining, slide agglutination using anti-H. pylori monoclonal IgG; positive isolates were tested by biochemical tests and polymerase chain reaction for H. pylorispecific ureA gene. Coccoid H. pylori was isolated in stool samples from the biopsied patients whose bacterial density was two to four in histology. Their oxidase was slightly positive but became positive after two subcultures, while additional biochemical tests confirmed the isolation of H. pylori. Similar coccoid but oxidase positive H. pylori was isolated from three nonbiopsied children with the protocol of cholestyramine treatment only. The density of bacteria in the stomach may influence the recovery of H. pylori from stool; inactivation of bile with cholestyramine improves the yield in culture and favors isolation of an enhanced metabolic

form of bacteria.

Key words: Helicobacter pylori, HpSA, stool culture, cholestyramine, children, Iran.

Resume: Nous avons evalue deux protocoles d'isolement de Helicobacter pylori a partir des selles d'enfants ayant subi ou non une biopsie. Vingt-deux enfants, dont la positivite ou la negativite presumee a ete diagnostiquee par endoscopie et par un test rapide a l'urease, ont ete recrutes pour comparer les tests bases sur la biopsie et les tests bases sur les selles (test de detection de l'antigene fecal de H. pylori HpSA et culture des selles). La gradation de leur activite gastrique et de la densite bacterienne a ete realisee par le systeme de Sydney revise. Les specimens de biopsies et de selles ont ete cultive s sur des plaques d'agar Campy-blood et Belo horizonte apres une periode d'enrichissement sur milieu selectif Campythio. Afin de comparer deux protocoles de culture des selles, les selles de 20 enfants n'ayant pas subi de biopsie ont ete testees par la detection du HpSA et cultivees comme decrit precedemment, ou apres traitement a la cholestyramine. Les colonies obtenues ont ete criblees par coloration Gram, l'agglutination sur lame a l'aide d'un IgG monoclonal anti H. pylori; les isolats positifs ont ete soumis a des tests biochimiques et a une reaction en chaine de la polymerase visant a detecter le gene ureA specifique de H. pylori. Helicobacter pylori sous forme coccoide a ete isole des selles de patients ayant subi une biopsie dont la densite bacterienne etait de deux a quatre a l'histologie. L'activite oxydase etait legerement detectable, mais est devenue positive apres deux sous-cultures, alors que des tests biochimiques additionnels ont confirme l'isolement de H. pylori. Des formes coccoides similaires mais negatives a l'oxydase ont ete isolees de trois enfants n'ayant pas subi de biopsies, lors du protocole utilisant la cholestyramine seulement. La densite bacterienne de l'estomac peut influencer la recuperation de H. pylori dans les selles; l'inactivation de la bile avec la cholestyramine ameliore le rendement de la culture et favorise l'isolement de formes metaboliquement plus actives de la bacterie.

Mots-cles : Helicobacter pylori, HpSA, culture de selles, cholestyramine, enfants, Iran.

[Traduit par la Redaction]

Introduction

Helicobacter pylori colonizes the human stomach and causes chronic active gastritis, peptic ulcer disease, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma (Fischbach et al. 2005; Wundisch et al. 2005). Colonization of the stomach by H. pylori begins mainly in early childhood and persists during later life. Presence of H. pylori in the stomach can be detected by biopsy-based methods including culture, histology, rapid urease test (RUT), and noninvasive methods, such as the urea breath test (UBT), serology, and stool tests (Krogfelt et al. 2005). Among the noninvasive methods, fecal testing is particularly appropriate for children since feces can be …