EPIYA motif patterns among Cuban Helicobacter pylori CagA positive strains.

INTRODUCTION: It is known that polymorphisms in C-terminal region of CagA influence gastric disease development on Helicobacter pylori infection. Additionally, the geographic distribution of these polymorphisms has been associated with the appearance of more severe gastroduodenal pathologies. Objective. To determine the CagA phosphorylation motifs pattern (EPIYA pattern) in Cuban H. pylori isolates, and to study its association with patient´s pathologies. MATERIALS AND METHODS: DNAs from 95 H. pylori cagA-positive strains were used to amplify the 3´ variable region of cagA gene by PCR using two different strategies. Additionally, new primers were designed to identify either Western or Eastern CagAEPIYA motiftype by PCR. To confirm the PCR results, PCR products from 14 representative isolates were purified and sequenced. RESULTS: The distribution of the EPIYA motif found was: 2 AB (2.1 %), 1 AC (1.1 %), 1 BC (1.1 %), 70 ABC (73.6 %), 19 ABCC (20 %), and 2 ABCCC (2.1 %). Sequencing analysis confirmed the PCR classification in the 14 studied strains and showed three strains with unusual nucleotide sequences, not reported before. Distribution of the EPIYA-ABC pattern was equivalent in all pathologies (78.9 % in gastric ulcer, 72.5 % in duodenal ulcer and 72.2 % in non-ulcer dyspepsia). CONCLUSION: The PCR results using the new primers confirmed that all studied strains carried the Western CagA type. No specific EPIYA motif was associated with peptic ulcer. This is the first report that shows EPIYA motif distribution in H. pylori isolates from the Caribbean region.

EPIYA motif patterns among Cuban Helicobacter pylori CagA positive strains / Patrón de los motivos EPIYA de cepas cubanas de Helicobacter pylori positivas para CagA

Introduction. It is known that polymorphisms in C-terminal region of CagA influence gastric disease development on Helicobacter pylori infection. Additionally, the geographic distribution of these polymorphisms has been associated with the appearance of more severe gastroduodenal pathologies. Objective. To determine the CagA phosphorylation motifs pattern (EPIYA pattern) in Cuban H. pylori isolates, and to study its association with patient´s pathologies. Materials and methods. DNAs from 95 H. pylori cagA-positive strains were used to amplify the 3´ variable region of cagA gene by PCR using two different strategies. Additionally, new primers were designed to identify either Western or Eastern CagAEPIYA motiftype by PCR. To confirm the PCR results, PCR products from 14 representative isolates were purified and sequenced Results. The distribution of the EPIYA motif found was: 2 AB (2.1 %), 1 AC (1.1 %), 1 BC (1.1 %), 70 ABC (73.6 %), 19 ABCC (20 %), and 2 ABCCC (2.1 %). Sequencing analysis confirmed the PCR classification in the 14 studied strains and showed three strains with unusual nucleotide sequences, not reported before. Distribution of the EPIYA-ABC pattern was equivalent in all pathologies (78.9 % in gastric ulcer, 72.5 % in duodenal ulcer and 72.2 % in non-ulcer dyspepsia). Conclusion. The PCR results using the new primers confirmed that all studied strains carried the Western CagA type. No specific EPIYA motif was associated with peptic ulcer. This is the first report that shows EPIYA motif distribution in H. pylori isolates from the Caribbean region.

Introducción. Se sabe que el polimorfismo en la región C-terminal de la citotoxina asociada al gen A (CagA) influye en el desarrollo de la enfermedad gástrica durante la infección por Helicobacter pylori. Objetivo. Determinar el número y el tipo de patrones de fosforilación de CagA (patrón EPIYA) en aislamientos cubanos de H. pylori, y estudiar su asociación con las enfermedades gástricas. Materiales y métodos. Se empleó el ADN de 95 cepas de H. pylori positivas paraCagA, para amplificar la región 3´ variable del gen cagA por PCR, mediante el empleo de diferentes estrategias. Además, se diseñaron nuevos cebadores para clasificar por PCR los aislamientos según el tipo de CagA, occidental o del este asiático. Los productos de PCR obtenidos de 14 aislamientos representativos se purificaron y secuenciaron para confirmar los resultados de la PCR. Resultados. La distribución de los patrones EPIYA encontrada, fue: 2 AB (2,1 %), 1 AC (1,1 %), 1 BC (1,1 %), 70 ABC (73,6 %), 19 ABCC (20 %), y 2 ABCCC (2,1 %). El análisis de la secuenciación confirmó las clasificaciones hechas por PCR en las 14 cepas estudiadas y demostró tres cepas con secuencias únicas de nucleótídos, no reportadas anteriormente. La distribución del patrón EPIYA-ABC fue equivalente en todas las enfermedades encontradas: 78,9 % en úlcera gástrica, 72,5 % en úlcera duodenal y 72,2 % en dispepsia no ulcerada. Conclusión. La mayoría de los aislamientos cubanos presentaron las combinaciones de motivos EPIYA menos virulentas (ABC). Los resultados del empleo de los nuevos cebadores y el análisis de la secuenciación, confirmaron que todas las cepas estudiadas portaban el gen cagA de tipo occidental. Ninguno de los patrones específicos de EPIYA se asoció con úlcera péptica. Este es el primer reporte que muestra la distribución de los motivos EPIYA en los aislamientos de H. pylori de la región del Caribe.

Prevalence of vacA, cagA and babA2 genes in Cuban Helicobacter pylori isolates.

AIM: To investigate the prevalence of vacuolating cytotoxin (vacA), cytotoxin associated gene A (cagA) and blood adhesion binding antigen (babA2) genotypes of Helicobacter pylori (H pylori) isolates from Cuban dyspeptic patients. METHODS: DNA was extracted from H pylori-positive cultures taken from 130 dyspeptic patients. Genotyping was performed by PCR, using specific primers for vacA (s1, s2, m1, m2), cagA and babA2 genes. Endoscopic observations and histological examinations were used to determine patient pathologies. RESULTS: vacA alleles s1, s2, m1 and m2 were detected in 96 (73.8%), 34 (26.2%), 75 (57.7%) and 52 isolates (40%), respectively, while the cagA gene was detected in 95 isolates (73.2%). One hundred and seven isolates (82.3%) were babA2-positive. A significant correlation was observed between vacAs1m1 and cagA and between vacAs1m1 and babA2 genotypes (P < 0.001 and P < 0.05, respectively) and between babA2 genotype and cagA status (P < 0.05); but, no correlation was observed between vacAs1 and babA2 genotypes. Eighty five (65.4%) and 73 (56.2%) strains were type 1 (vacAs1-cagA-positive) and "triple-positive" (vacAs1-cagA-babA2-positive), respectively, and their presence was significantly associated with duodenal ulcer (P < 0.01 and P < 0.001, respectively). CONCLUSION: The distribution of the main virulence factors in the Cuban strains in this study resembled that of the Western-type strains, and the more virulent H pylori isolates were significantly associated with duodenal ulcer, ulcer disease being the worst pathology observed in the group studied.