Detection of Helicobacter pylori and the genotypes of resistance to clarithromycin and the heterogeneous genotype to this antibiotic in biopsies obtained from symptomatic children

Highlights

• 81.6% of biopsies from symptomatic children were H. pylori positive by PCR.

• Clarithromycin genotype: 42.3% wild-type, 47.7% resistant and 9.9% heterogeneous.

• Mutations detected: A2143G (87.5%), A2142G (7.5%) and double A2142C-A2143G (5%)

• Heterogeneous genotypes showed wild-type genotype plus mutation, A2143G or A2142G

Abstract

The aim of this study was to use a commercially available kit (GenoType® HelicoDR; Hain Life Science, Germany) to detect Helicobacter pylori infection and clarithromycin resistance genotype in biopsies obtained from symptomatic children. Results: 111 out of 136 (81.6%) biopsies were H. pylori positive by genotype: 47 (42.3%) showed wild-type genotype, 53 resistant genotype (47.7%) and 11 heterogeneous genotype (9.9%). Culture was negative in 27 out of the 111 genotyped biopsies. Mutation A2143G (87.5%), followed by A2142G (7.5%) and double mutant A2142C-A2143G (5%) were found. The 11 heterogeneous genotype biopsies showed wild-type plus A2143G in 9 and plus A2142G in 2. Conclusions: This kit is a rapid, culture-independent method for routine application in biopsies from the pediatric population that allows detection of clarithromycin resistance and heterogeneous genotypes. It is important to know the clinical impact of infection with this type of strains as well as the role in treatment success.

Introduction

Helicobacter pylori is a spiral-shaped microaerophilic gram-negative bacterium that colonizes the gastric mucosa of humans and it is estimated that it infects 50% of the population worldwide (Marshall, 2002). Chronic infection with H. pylori can cause several gastrointestinal diseases, such as severe gastritis, peptic ulcer disease and gastric mucosa-associated lymphoid tissue lymphoma (MALT) (Campuzano-Maya, 2014). Moreover, it is identified as a Group I carcinogen by the World Health Organization International Agency for Research on Cancer (Herrero et al., 2014).

Numerous treatment regimens have been used to eradicate this microorganism. Triple therapy including a proton pump inhibitor and amoxicillin and clarithromycin, is the most common treatment used (Wu et al., 2014). The main cause of treatment failure of the H. pylori infection is the macrolide resistance (mainly clarithromycin) (Rimbara et al., 2007). The prevalence of clarithromycin resistance varies with the population. The high clarithromycin resistance rates in strains isolated from children is strikingly alarming (Vega et al., 2007). A high level of macrolides resistance in Streptococcus pyogenes, has been reported in Spain since 1986 (Granizo et al., 2000) and this high resistance seems to be related to the antibiotic consumption that had risen during the previous decade (Carrasco-Garrido et al., 2009). In a European study describing the antimicrobial susceptibility in H. pylori, an association was found between the use of long-acting macrolides and clarithromycin resistance; resistance was higher in Western/Central and Southern Europe (higher than 20%) than in Northern European countries (lower than 10%) (Megraud et al., 2013).

Mutations in the peptidyltransferase-encoding region of 23S rRNA confer resistance to clarithromycin by decreasing the binding of clarithromycin to ribosomes in H. pylori (Wang et al., 1999). A2142G, A2143G or A2142C are the most frequently described (De Francesco et al., 2011), with other such as A2515G, T2717C, A2116C, G2141A, A2144T, T2182C, G2224A, C2245, C2136T, C2310A and C2428T which are described sporadically (Herrero et al., 2014, Granizo et al., 2000). Mutations in other genes, such as translation initiation factor IF-2 (infB) and ribosomal protein L22 (rpl22) genes were also described (Bihn et al., 2014).

During the H. pylori infection, multiple strains could colonize the same host (Blaser and Kirschner, 2007) and heterogeneous genotypes refers to infection with one or multiple strains presenting different genotypes of resistance to clarithromycin (Kao et al., 2014).

Clarithromycin resistance is mainly detected by phenotypic methods performed after culture such as E-test or agar dilution (Mégraud and Lehours, 2007). These methods are time-consuming and can take up to 2 weeks from the endoscopy (Agudo et al., 2010a). Detection of point mutations conferring resistance to clarithromycin by molecular methods may constitute a quicker approach. One commercially available test, combines polymerase chain reaction (PCR) and hybridization. It allows the molecular detection of H. pylori (by targeting a specific region of the 23S rRNA gene) and characterization of common mechanisms of resistance to clarithromycin (mutations in 23S rRNA gene in positions 2142 and 2143) and for fluoroquinolones (most significant mutations of the quinolone-resistance region of the gyrA gene, especially at codons 87 and 91) within 6 h (Herrero et al., 2014, Mégraud and Lehours, 2007, Megraud et al., 2013, Mazigh et al., 2012). Furthermore, this test allows detecting strains with heterogeneous genotype (Kanai et al., 2004, Mégraud and Lehours, 2007, Miendje et al., 2011). The frequency of these strains in children is poorly studied as well as the clinical implication in patients infected with this type of strains.

The aim of this study was to use a commercial available kit (GenoType® HelicoDR, Hain Life Science, Germany) to detect H. pylori infection and the genotypes of resistance to clarithromycin and the heterogeneous genotype to this antibiotic in biopsies obtained from symptomatic children.