Features and trends in Helicobacter pylori antibiotic resistance in Lisbon area, Portugal (1990–1999)

J. Cabritaa,b,*, M. Oleastroa, R. Matosa, A. Manhentea, J. Cabralc, R. Barrosc, A. I. Lopesd, P. Ramalhod, B. C. Nevese and A. S. Guerreiroe

a Laboratório de Bacteriologia, Instituto Nacional de Saúde; b Faculdade de Farmácia de Lisboa; c Unidade de Gastrenterologia, Serviço de Patologia Clínica, Hospital de D. Estefânia; d Unidade de Gastrenterologia Pediátrica, Hospital de Santa Maria; e Clínica Universitária de Medicina Interna e Gastrenterologia, Hospital de Pulido Valente, Lisboa, Portugal


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The features of Helicobacter pylori antibiotic resistance in Lisbon from 1990 to 1999 were studied. Overall resistance rates to amoxycillin, tetracycline, metronidazole, clarithromycin and ciprofloxacin were 0, 0, 30.6, 19.0 and 9.6%, respectively. The incidence of resistance to clarithromycin was much higher in isolates from children (44.8%) than adults (14.6%). For metronidazole, the contrary was observed (children: 19.0%, adults: 32.3%). Ciprofloxacin-resistant isolates were all from adult patients. Concerning the adult population, the resistance rate to metronidazole showed a slight increase during the decade, while for clarithromycin and ciprofloxacin a significant increase was observed (4.6 to 22.0% and 0 to 20.9%, respectively).


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Helicobacter pylori is recognized as a major cause of human gastritis and peptic ulcer as well as an important risk factor for gastric cancer. Eradication of this organism is indicated in all patients with active or recurrent peptic ulceration.1

Dual or triple therapy, including two of the following antibiotics—amoxycillin, tetracycline, metronidazole or clarithromycin, plus a proton pump inhibitor, bismuth salt or ranitidine bismuth citrate—is the therapy most frequently used to eradicate H. pylori.1

Resistance to metronidazole is reported worldwide with a prevalence ranging from 20 to 35% in developed countries and 80 to 90% in developing regions. The prevalence of clarithromycin resistance is usually <10% in developed countries. However, in southern Europe the rate of resistance to clarithromycin and metronidazole is higher than in northern or central Europe.2

The infection eradication rate decreases from 95% in sensitive strains to 75% in metronidazole-resistant H. pylori isolates, and to 40% in clarithromycin-resistant strains.3

The aims of this study were to assess the evolution of resistance to antibiotics of choice in anti-H. pylori treatment over the last 10 years in the Lisbon area and to correlate resistance profiles with demographic and therapeutic features. Although fluoroquinolones are not frequently used to eradicate H. pylori strains, ciprofloxacin was included in this study because its use has been suggested in failure of treatment with the antibiotics previously mentioned.4 In Portugal, the prevalence of resistance to quinolones in other organisms has increased considerably in the last few years.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Between January 1990 and December 1999, a total of 473 clinical isolates of H. pylori were collected from dyspeptic patients in the Instituto Nacional de Saúde in Lisbon. Of these, 58 were collected from children (age <= 15 years) and only during 1998–99.

Demographic and clinical status data were collected for each patient. The distribution of patients by gender was 45% female and 55% male, with age ranging from 5 to 89 years (mean = 46; mode = 50).

Strains were isolated from gastric biopsy samples, taken during endoscopy. Identification was performed according to conventional tests and the MICs for isolates, of metronidazole (n = 448), clarithromycin (n = 394), amoxycillin (n = 394), tetracycline (n = 394) and ciprofloxacin (n = 371), were assessed by the Etest on Mueller–Hinton agar plus 10% horse blood.

The breakpoints used to define resistance were: metronidazole (>8 mg/L), clarithromycin (>1 mg/L), amoxycillin (>0.5 mg/L) according to Glupczvnski et al.,2 tetracycline (>4 mg/L) and ciprofloxacin (>1 mg/L) according to the NCCLS.

Statistical analyses were performed with chi-squared and Fisher's exact tests.


    Results
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 Materials and methods
 Results
 Discussion
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Of the patients, 8.9% presented with gastric ulcer, 20.5% with duodenal ulcer, 60.9% with non-ulcer dyspepsia and 9.7% with gastric cancer. Of the 140 patients questioned about previous eradication therapy to H. pylori, 50 confirmed exposure to amoxycillin and metronidazole or clarithromycin in the previous year.

All the isolates tested were sensitive to amoxycillin (MIC90 0.023 mg/L) and tetracycline (MIC90 0.125 mg/L). Clarithromycin also showed a high in vitro activity (MIC50 0.023 mg/L), but 19.0% of the isolates were resistant and about 50% had high-level resistance (MIC >= 256 mg/L). Metronidazole had reduced activity (MIC50 1.0 mg/L) and 30.6% of the isolates were resistant to this compound. An overall resistance rate of 9.6% to ciprofloxacin was found (MIC50 0.094 mg/L).

In isolates from patients previously exposed to metronidazole or clarithromycin, the prevalence of resistance was 75 and 47%, respectively, while in the remaining isolates the rate of resistance was 28.8 and 13.5% (P < 0.01).

In adult patients, 30.8% of H. pylori strains isolated from women were resistant to metronidazole, 20.1% to clarithromycin and 11.7% to ciprofloxacin. In isolates from men the prevalence of resistance was lower to clarithromycin (16.9%) and ciprofloxacin (8.4%), but slightly higher to metronidazole (31.7%). However, no statistically significant association was found between gender and resistance to any of those antibiotics.

Table IGo presents the prevalence of resistance and antimicrobial susceptibility of H. pylori by age group in strains isolated in 1998–99. In paediatric patients, the frequency of strains resistant to clarithromycin (44.8%) was much higher than in adults (22.0%) (P < 0.01). On the other hand, the prevalence of strains resistant to metronidazole was significantly higher in isolates from adult patients (34.1%) than that observed in isolates from children (19.0%) (P < 0.05). Simultaneous resistance to these antibiotics was found in 8.6% of the isolates from paediatric patients and in 11.4% from adult patients. None of the 58 isolates from children was resistant to ciprofloxacin, while in isolates from adult patients the rate of resistance was 20.9%.


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Table I. Antimicrobial susceptibility (MIC, mg/L) of H. pylori isolates to metronidazole, clarithromycin and ciprofloxacin by age group, in isolates from 1998 to 1999
 
The prevalence of resistance of H. pylori strains from adult patients to metronidazole, clarithromycin and ciprofloxacin according to the period of isolation is shown in Table IIGo. Resistance to metronidazole increased slightly during the decade. The prevalence of resistance to clarithromycin and to ciprofloxacin showed a significant increase, rising from 4.6 to 22.0% and from 0 to 20.9%, respectively, from 1990 to 1999. All H. pylori strains resistant to both clarithromycin and metronidazole were isolated during the last 3 years. The overall resistance rates (adults and children, 1998–99) were 29.5% to metronidazole, 28.9% to clarithromycin and 13.8% to ciprofloxacin.


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Table II. Evolution of prevalence of resistance to metronidazole, clarithromycin and ciprofloxacin in H. pylori strains isolated from adult patients from 1990 to 1999
 

    Discussion
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 Materials and methods
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In Lisbon, resistance to amoxycillin and tetracycline in H. pylori strains has not been detected. On the other hand, the prevalence of strains resistant to clarithromycin has increased steadily over the 1990s reaching a rate of 28.9%. This prevalence is considerably higher than that recently reported in other southern European countries, such as Spain 3.5%, Italy 8%, Greece 15% and France 15%.58 In paediatric patients the percentage of strains resistant to this macrolide reached 44.8%, a value eight times higher than that reported for Greek children.9 Resistance to metronidazole did not rise over the decade, remaining at 30%, a lower value than that reported by other authors.7,8 Recently, Glupczvnski et al.2 reported a prevalence of resistance to metronidazole of 37% in the southern European region. Overall, the growth in prevalence of clarithromycin- and ciprofloxacin-resistant strains over the decade is most probably correlated with the increased use of these antibiotics in Portugal. The lower prevalence of resistance to metronidazole and to ciprofloxacin and the higher resistance rate to clarithromycin in isolates from children also reflects the pattern of antibiotic use in Portugal.10

In conclusion, in the Lisbon area the isolation of strains of H. pylori resistant to antibiotics used in eradication therapy (namely nitroimidazoles and macrolides), as well as to fluoroquinolones, is frequent. Resistance to clarithromycin is particularly common in strains isolated from paediatric patients. These findings stress the importance of monitoring the prevalence of resistance in order to select the most effective treatment.


    Notes
 
* Correspondence address. Instituto Nacional de Saúde, Dr Ricardo Jorge Av., Padre Cruz 1649-016 Lisboa, Portugal. Tel: +351-21-75-19231; Fax: +351-21-75-90441; E-mail: jcabrita{at}ff.ul.pt Back


    References
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1 . NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease. (1994). Helicobacter pylori in peptic ulcer disease. NIH Consensus Conference. Journal of the American Medical Association 272, 65–9.[ISI][Medline]

2 . Glupczvnski, Y., Mégraud, F., Anderson, L. P. & Lopez-Brea, M. (1999). Antibiotic susceptibility of Helicobacter pylori in Europe in 1998: results of the third multicentre study. Gut 45, Suppl. 3, A3.[ISI]

3 . Bazzoli, F., Berretti, D., De Luca, L., Nicolini, G., Pozzato, P., Fossi, S. et al. (1999). What can be learnt from the new data about antibiotic resistance? Are there any practical consequences of Helicobacter pylori antibiotic resistance? European Journal of Gastroenterology and Hepatology 11, Suppl. 2, S39–42.[ISI][Medline]

4 . Dresner, D., Coyle, W., Nemec, R., Peterson R., Duntemann, T. & Lawson, J. M. (1996). Efficacy of ciprofloxacin in the eradication of Helicobacter pylori. Southern Medical Journal 89, 775–8.[ISI][Medline]

5 . López-Brea, M., Domingo, D., Sanchez, I. & Alarcon, T. (1997). Evolution of resistance to metronidazole and clarithromycin in Helicobacter pylori clinical isolates from Spain. Journal of Antimicrobial Chemotherapy 40, 279–81.[Abstract]

6 . Iovene, M. R., Romano, M., Pilloni, A. P., Giordano, B., Montella, F., Caliendo, S. et al. (1999). Prevalence of antimicrobial resistance in eighty clinical isolates of Helicobacter pylori. Chemotherapy 45, 8–14.[ISI][Medline]

7 . Rokkas, T. (2000). Prevalence of Helicobacter pylori resistance. (Personal communication) in GEPH & EHPSG, Estoril, Portugal.

8 . Birac, C., Bouchard, S., Camou, C., Lamouliatte, H., Lamireau, T. & Mégraud, F. (1999). Six year follow-up of resistance to antibiotics of Helicobacter pylori in Bordeaux, France. Gut 45, Suppl. 3, A107.

9 . Mentis, A. F., Roma, E., Pangalis, A. & Katsiyiannakis, E. (1999). Susceptibilities of Helicobacter pylori strains isolated from children with gastritis to selected antibiotics. Journal of Antimicrobial Chemotherapy 44, 721–2.[Medline]

10 . Caldeira, L., Silva, E., Santos, P., Inês, M. & Raposo, P. (1999) Dispensa de antibacterianos em Medicina Humana Comunitária (INFARMED. Ministério da Saúde), Lisbon.

Received 26 April 2000; returned 21 July 2000; revised 7 August 2000; accepted 24 August 2000