Letter to the Editor

Analysis of resistance to clarithromycin and iceA status in Helicobacter pylori isolates from San Luis, Argentina

To characterize Helicobacter pylori isolates from western Argentina using virulence markers and antimicrobial susceptibility patterns in order to assess the association between virulent genotypes, antimicrobial resistance, and disease. DNA fingerprinting was also evaluated for the segregation of virulent or resistant strain clusters.

Genotyping of 299 H. pylori isolates was performed by PCR using specific primers for the cagA, vacA and iceA genes. Random amplification of polymorphic DNA (RAPD)-PCR and rep-PCR genetic clustering were assessed using five random primers and BOXA1R and ERIC primers, respectively. Resistance to clarithromycin (Cla) and metronidazole (Mtz) was assessed by the agar dilution method.

It was observed that 40.8% of the genotypes were cagA-positive; 66.9% were vacA s1m1 genotype and the iceA1 allele was found in 40.8%. A significant correlation (p = 0.0000) was observed between cagA positivity and vacA s1m1/iceA1 genotypes. Triple virulent genotypes were statistically associated with peptic ulcer (PU) (p = 0.0001) and Cla resistance (p = 0.0000). RAPD fingerprints obtained with AO2 primers identified clusters that were strongly associated with PU, virulence markers, and resistance to Cla and Mtz.

The H. pylori isolates that harbored two or three virulence markers were more resistant to Cla and Mtz. Combined analysis of virulent genotypes and resistance patterns may permit identification of high-risk patients to prevent PU later in life or to avoid antimicrobial treatment failure.

Little information is available concerning the relationships between clarithromycin resistance and virulence marker genes (iceA, cagA and vacA) in Helicobacter pylori isolated in Taiwan. The aim of this study was to evaluate the possible association between clarithromycin resistance and genotypes of the virulence markers on clarithromycin-resistant H. pylori isolates obtained in eastern Taiwan

The genotypes of the virulence marker genes (iceA, cagA and vacA) were analyzed by PCR, and the 23S rDNA region from 18 clarithromycin-resistant clinical isolates of H. pylori was amplified by PCR and sequenced.

Point mutations were found to occur in all isolates. Two isolates had A2143G, six had T2182C, one had C2227T, six had A2143G plus T2182C, and three had heterozygous alleles. The latter included a wild-type allele (A2143) plus (i) an A2143G, (ii) an A2143G plus an A2223G, and (iii) an A2143G plus a T2182C. The prevalence of the marker genes cagA, iceA1, iceA2, and both iceA1 and iceA2, in the isolates was 95.5%, 66.9%, 7.5%, and 25.6%, respectively. The vacAs1 allele was detected in all isolates, whereas the m1 and m2 alleles were found in 44.4% and 55.6% of the isolates, respectively

There were no significant associations between clarithromycin resistance and the presence of the cagA gene, vacA allele mosaicism, and the iceA genotypes. The most notable finding of our study was that the C2227T single mutation in 23S rDNA could also be related to the high clarithromycin minimal inhibitory concentrations in clinical isolates from eastern Taiwan.

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.