Helicobacter pylori infection has been implicated in the development of peptic ulcer disease in children. A U.S. expert guideline protocol for the eradication of H. pylori organisms in symptomatic children recommended a therapeutic course of 7-14 days of dual antibiotics with a proton pump inhibitor (PPI) (1). Unfortunately, following treatment recommendations, a poor eradication rate of H. pylori organisms was reported in children (2), which was partially explained by the increased rate of bacterial resistance to clarithromycin (3). Clarithromycin resistance is mainly related to 3 specific point mutations within the bacterial 23S ribosomal ribonucleic acid (rRNA) (A2143G, A2144G and A2143C) (4). Previous reports demonstrated that those mutations could be accurately identified by fluorescent in situ hybridization (FISH) technique on fresh shock frozen tissue or on formalin fixed paraffin-embedded biopsies (5). Using the same technology, in the present study, we assessed the resistance rate against clarithromycin in H. pylori-infected children from West Virginia.
Paraffin-embedded gastric biopsy blocks from 16 H. pylori-positive children, obtained from a previous prospective clinical study (6), constitute our test samples. For each patient, 1 biopsy from the antrum and 1 from the corpus were used to identify the clarithromycin-susceptible "wild type“ of H. pylori bacterium.
All positive children were treated for 2 weeks with a standard antibiotic protocol for H. pylori infection, including amoxicillin (50 mg/kg), clarithromycin (15 mg/kg) and a proton pump inhibitor. Repeat endoscopy with biopsies was offered to children who remained symptomatic 2 months after completing therapy, but for ethical reasons, not to patients in whom symptoms were cleared.
FISH technique was performed in the paraffin-embedded gastric biopsies, as previously described (5). The oligonucleotide probe H py-1 was used to specifically identify H. pylori, and the oligonucleotide probes ClaR1, ClaR2 and ClaR3 were used to determine the bacterial 23S rRNA point mutations. Paired samples of gastric biopsies (antral and body) were available from all 16 children who were positive for H. pylori by histology. There were 8 boys and 8 girls with a mean age of 11.7 ± 4.0 years. In 3 pairs, H. pylori organisms were not identified by the H. pylori-specific probe (H py-1), suggesting a false-positive reading by the pathologist. Overall, in 7 patients, clarithromycin-susceptible strains were identified in both locations (antrum, body); in 2 patients clarithromycin-resistant strains were found; and in the remaining 3 children mixed colonies were identified (clarithromycin-sensitive and -resistant).
Clinical follow-up showed the following: of the 8children who harbored only clarithromycin-sensitive bacteria, 2 were lost to follow-up whereas the remaining 6 were asymptomatic. In one of those patients, a repeat endoscopy was performed due to duodenal ulcer found in the initial procedure. This patient was still positive for H. pylori organisms, indicating a failed therapy. Of the 5 children who harbored a clarithromycin-resistant strain, all but 1 showed no clinical improvement and were positive for H. pylori organism on repeat endoscopy. Of the 3 patients who mistakenly were identified as H. pylori positive by the pathologist, only 1 remained symptomatic. This patient refused repeat endoscopy but was tested negative by C-urea breath test.
Our results raise several issues which are relevant to the treatment of H. pylori infection in children, namely, H. pylori resistance to clarithromycin therapy and its effect on eradication success.
Previous studies have implicated H. pylori resistant to clarithromycin in the failure of bacterial eradication (7-9). The primary resistance rate in our small cohort of H. pylori-infected children is very high: between 31% (5/16) (if we consider the 3 children with negative FISH results as H. pylori-infected with a clarithromycin-susceptible strain) and 38% (5/13) (if we assume a false-positive histology in these 3 patients). The reported data of macrolide resistance in H. pylori-infected children from the United States are very limited. Previous pediatric studies, describing a similar referral-biased population (symptomatic children referred to an academic center), reported a primary clarithromycin resistance rate of 18% and 45% (9, 10). In those studies, clarithromycin resistance was determined after bacterial culture was obtained. In contrast, we present resistance rate to clarithromycin utilizing FISH technique on paraffin-embedded blocks. To our knowledge, this is the first description of clinical usage of FISH technique in H. pylori-infected children from the United States. Concurring with previous reports (11), we also report 3 patients who harbored a mixed bacterial population. Unfortunately, genotyping of the bacteria was not performed in this study; thus, it cannot be determent whether the mixed infection was related to 2 genetically different strains or 1 single strain in which a portion developed the resistance to clarithromycin in vivo by spontaneous or macrolide-induced mutation (12).
Bacterial resistance and a low compliance for drug intake are the main factors for the treatment failure (13). In the present study, we documented that triple treatment had failed in at least 4 of 5 children (80%) with an H. pylori-resistant strain. Clinical service for culture and bacterial susceptibility testing for H. pylori organisms is not available for the primary care physicians practicing in the United States. The common practice among physicians in the United States is to initiate therapy based on a positive histology without the guidance of culture and antibiogram. In our sample of 16 children, this policy resulted in an unnecessary therapy in 3 patients (possible false-positive) and a treatment failure proven by second endoscopy in at least 4 other children. In the face of the high primary resistance rate in our population, a therapy guided by the antibiogram would be beneficial; however, until such service is widely available, adopting FISH technique for clarithromycin resistance may be a feasible alternative to detect at least 1 antibiotic resistant. Using paraffin-embedded sections with this technique may provide an important advantage for such practice.
In conclusion, in the present study we demonstrated that clarithromycin-resistant bacteria are common in H. pylori-infected children from West Virginia and may affect the clinical outcome of these children. Applying FISH technique on paraffin-embedded biopsies to detect clarithromycin-resistant mutants would be an important step toward cost containment and improving patient's standard of care.
Marshall University Joan C. Edwards
School of Medicine
Max von Pettenkofer-Institute
for Hygiene and Medical Microbiology
Dr. v. Haunersches Kinderspital,
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