RESUMO
Helicobacter pylori is a gastric pathogen that is widely recognized as a causative agent of gastric disease. Its eradication is variable, mainly due to increased resistance to clarithromycin. Our objective was: to evaluate (i) if the biopsy specimen used for the rapid urease test is a useful sample to detect resistance to clarithromycin by PCR-RFLP and (ii) the distribution of A2142G and A2143G point mutations in the 23S rRNA gene, in relation to virulence factors in our region. Gastric specimens were collected from adult dyspeptic patients (n = 141) and H. pylori was investigated by the rapid urease test, histopathological analysis and PCR for the hsp60 gene. Clarithromycin resistance was detected by PCR-RFLP in 62 H. pylori (+) paired biopsy specimens submitted to molecular analysis and the rapid urease test. H. pylori virulence factors were analyzed by multiplex PCR using specific primers for the cagA, vacA and babA2 genes. Thirteen out of 62 strains (20.9%) were resistant to clarithromycin: 6/13 (46.2%) harbored the A2143G mutation whereas 7/13 (53.8%) carried the A2142G point mutation. vacA m1s1 was the most frequent genotype among the resistant strains. In conclusion, the biopsy specimens used for the rapid urease test were suitable samples for clarithromycin resistance detection in patients infected with H. pylori, which became especially useful in cases where the number or size of the biopsies is limited. In addition, this is the first report of a molecular analysis for clarithromycin resistance performed directly from gastric biopsies in our region.
Helicobacter pylori es un patógeno ampliamente reconocido como causante de enfermedad gástrica. Su erradicación es variable, principalmente debido al incremento de la resistencia a claritromicina. Nuestros objetivos fueron evaluar la utilidad de la biopsia usada para realizar el test rápido de ureasa en la detección de resistencia a claritromicina por PCR-RFLP y conocer la distribución de las mutaciones puntuales A2142G y A2143G en el gen ARNr 23S, en relación con los factores de virulencia en nuestra región. Se recolectaron muestras gástricas (n=141) provenientes de pacientes adultos dispépticos y se investigó la presencia de H. pylori mediante el test rápido de ureasa, análisis histopatológico y PCR para el gen hsp60. La resistencia a claritromicina se analizó por PCR-RFLP en 62 muestras pareadas de biopsias gástricas H. pylori+ destinadas al análisis molecular y al test rápido de ureasa. Los factores de virulencia de H. pylori fueron analizados mediante PCR multiplex usando oligonucleótidos específicos para los genes cagA, vacA y babA2. Trece de 62 cepas (20,9%) fueron resistentes a claritromicina, 6/13 (46,2%) llevaron la mutación A2143G, mientras que 7/13 (53,8%) presentaron la mutación A2142G. El genotipo vacA s1m1 fue el más frecuente entre las cepas resistentes a claritromicina. En conclusión, las biopsias destinadas al test rápido de ureasa fueron muestras apropiadas para la detección de la resistencia a claritromicina en pacientes infectados con H. pylori. Esto es especialmente útil en aquellos casos en los que el número o el tamaño de las muestras son limitados. Además, este es el primer reporte de estudio de resistencia a claritromicina (mediante técnicas moleculares), directamente de biopsias gástricas en nuestra región.
Assuntos
Humanos , Helicobacter pylori/efeitos dos fármacos , Infecções por Helicobacter/diagnóstico , Claritromicina/farmacologia , Fatores de Tempo , Urease/metabolismo , Polimorfismo de Fragmento de Restrição , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Helicobacter pylori/enzimologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Infecções por Helicobacter/microbiologia , Mutação Puntual , Farmacorresistência Bacteriana , Testes Diagnósticos de Rotina/métodosRESUMO
Our goals were: a) to detect Helicobacter pylori in gastric biopsies of symptomatic adults by PCR, b) to detect the presence of the cagA gene as well as of the allelic variants of the vacA gene, and c) to correlate genotypes with the endoscopic diagnoses. H. pylori was detected in 81
(39/48) of patients by nested PCR for hsp60. The presence of cagA was detected in 15/22 of samples and vacA s1 - m1 was the most frequent allelic combination (15/22). Gastritis, the most frequent diagnosis, was associated with genotype cagA+ in 10/13 of patients. In this group, 9/13 showed the allelic variant vacA s1- m1. The variant vacA s2 - m2 was detected in 3/3 of gastritis cases by H. pylori with the cagA- genotype. These results are the first reported in our region and provide data of epidemiological interest.