Helicobacter pylori infection in havana,cuba

Helicobacter pylori infection in Havana, Cuba. Prevalence
and cagA status of the strains
Beatriz Gutiérrez1,2,3, Teresita Vidal2,3, Carlos Ernesto Valmaña2,3, Christine Camou-Juncas3, Adriana
Santos3, Françis Mégraud3, Nery González4, Ibrahim Leonard4, Rolando Martínez5, Osvaldo Díaz-Canel5,
Manuel Paniagua6, María del Pilar Escobar6 and George L. Mendez3,7.
1Academia de Ciencias de La República Dominicana, Santo Domingo, República Dominicana.
2Instituto Finlay, Centro de Investigación-Producción de Vacunas, Ciudad de La Habana, Cuba.
3Laboratoire de Bactériologie,Université Victor Segalen Bordeaux 2, Bordeaux, France.
4.Instituto de Oncología, Ciudad de La Habana, Cuba.
5 CCE Hospital Universitario Calixto García, Ciudad de La Habana, Cuba.
6Instituto de Gastroenterología, Ciudad de La Habana, Cuba.
7School of Biotechnology and Biomolecular Science, University of New Sout Wales, Sydney, Australia.
There is a great paucity of information about Helicobacter pylori infection in the countries ofthe Caribbean basin. Almost no studies have been performed to determine the prevalence, antibiotic resistance or virulence factors of the bacterium. To measure the prevalence of H.
pylori infection among patients attending endoscopy in three clinics in Havana, Cuba, toevaluate clarithromycin resistance, and to determine the cagA status of the strains obtained.
Endoscopy was performed and biopsies were obtained from 117 successive patients attendingthe Institute of Oncology, the Institute of Gastroenterology, and the Calixto Garcia Hospital inHavana, Cuba. Biopsies were maintained at –70 ºC before being cultured on three differentmedia (two selective and one non-selective) and incubated for 7 days at 37 °C under a microaerobic atmosphere. The presence of H. pylori was identified by oxidase, catalase andurease activities. DNA was extracted, and PCR was performed with primers H2761676 whichamplify a 397 bp fragment of the cagA gene. Clarithromycin susceptibility was measured by the gel diffusion method. The diagnoses of patients were: 1 gastric carcinoma; 19 duodenalulcers; 8 gastric ulcers; and 89 non-ulcer dyspepsia, including (62) gastritis, (9) hiatalhernia,(2) biliary reflux, (1) gastric polyps, and (15) no abnormality. Among the 117 biopsiestested, 83 were H. pylori positive (70.9%). The cagA status determined for 35 cases gave a positive result in 31 cases (88.5%). Only 3% of the strains were resistant to clarithromycin.
The prevalence of Helicobacter pylori infection in the symptomatic population of La Habana isthe same as reported for other developing countries. Most strains were cagA positive and are likely harbour the cag pathogenicity island. The low resistance to clarithromycin in the strains
studied probably reflects the low degree of use of the antibiotic in this population.
Keywords: H. pylori, prevalence, endoscopic diagnosis
Introduction
(1-5). The phenotype and genotype study ofHelicobacter pylori include putative bacterial The infection caused by Helicobacter pylori plays virulence factors. The most common bacterial an important role in the development of several virulence factors studies are the cag pathogenicity digestive diseases and it has a worldwide island, for which cagA is a marker and the distribution and is significantly more frequent in vacuolating cytotoxin VacA (6-8). Few studies developing countries than in industrialized country.
This microorganism has been recently accepted as determine the prevalence of Helicobacter pylori related to active chronic gastritis, peptic disease infection and to get an insight into the type of (both gastric and duodenal ones), gastric strains which are circulating (9-12).
adenocarcinoma and gastric lymphoma type MALT VacciMonitor
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Our aims were
cagA gene detection: Primers used for cagA
typing were H276/676. For PCR of each sample,
 To determine the prevalence of H. pylori infection among patients attending endoscopy the mixture contained 25 pmol of each primer, 100 ng of genomic DNA, each deoxynucleosidetriphosfate at a concentration of 100 µM, and 1U  To evaluate the cagA status of the strains of Taq DNA. The reactions were performed in anautomated DNA thermal cycler, with 30 cycles of  To evaluate clarithromycin susceptibility.
denaturation 94 ºC for 1 min, annealing at 52 ºC Material & Methods
for 1 min, and extension at 72 ºC for 2 min(21,22).
Biopsy specimens: We examined H. pylori strain
PCR product detection: All amplified PCR products
isolated from 117 patients who were subjected to were resolved in 0.8% agarose gels, stained with upper gastrointestinal endoscopy for clinical ethidium bromide and detected under a short - reasons at the Institute of Oncology, the Institute wavelength UV Light source. We consider strain of Gastroenterology and Calixto García Hospital in cagA positive when it show a product of 397 bp endoscopic diagnosis: gastric carcinoma (1); duodenal ulcer (19); gastric ulcer (8); non ulcer Results & Discussion
dyspepsia: 89 including erosive gastritis (62),hiatal hernia (9), biliary reflux (2 ), gastric polyps H. pylori status of the Cuban population has not (1), no abnormality (15). All patients signed been explored. Endoscopy was performed and biopsies were obtained from 117 successive procedure three biopsy specimen (two the antrum patients attending the Institute of Oncology, the and one from the corpus) and were maintained at Institute of Gastroenterology, and the Calixto García Hospital in Havana, Cuba. Biopsies were Culture of H. pylori: Biopsies specimens were
maintained at –70 °C before being cultured on placed in 0.5 mL of Brucella broth (Biomérieux) three different media ( two selective and one non- selective) and incubated for 7 days at 37 °C homogeniser Ultraturax; LaboModerne, Paris, under a microaerobic atmosphere. The presence of France for 30s. Aliquots were placed for isolation H. pylori was identified by oxidase, catalase and on each of three different media (selective in urease activities. DNA was extracted, and PCR house medium, pylori Agar Biomérieux, Marcy L was performed with primers H276/676 which ’Etoile, France), and a non-selective Columbia amplify a 397 bp fragment of the cagA gene.
medium (Oxoid Unipath, Basingstoke, Hants,UK) Clarithromycin susceptibility was measured by the enriched with 10% human blood. All media were gel diffusion method. The diagnoses of patients incubated for 7 days at 37 ºC in a microaerobic were: 1 gastric carcinoma; 19 duodenal ulcers; 8 atmosphere. They were identified by colony gastric ulcers; and 89 non-ulcer dyspepsia, morfology, oxidase, catalase and urease activities.
including (62) gastritis, (9) hiatal hernia, (2) biliary Clarithromycin susceptibility was measured by the reflux, (1) gastric polyps, and (15) no abnormality.
gel diffusion method. Table 1 (13-19).
Among the 117 biopsies tested, 83 were H. pyloripositive (70.9%). The cagA status determined for DNA extraction: A pool of colonies obtained from
35 cases gave a positive result in 31 cases 35 patients were transferred to a microcentrifuge (88.5%). Only 3% of the strains were resistant to tube that contained 500 µl of sterile distilled water and centrifugated for five min at 10 000 g. The The prevalence of H. pylori infection in the pellet was resuspended in 300 µl of extraction symptomatic population of La Habana is the same buffer (20 µM Tris/HCL,pH 8, 0.5% Tween 20) as reported of other developing countries (9-12).
and proteinase K was added at final concentration Most strains were cagA positive and are likely of 0.5 µg/mL. The mixture was incubated at 55 ºC harbour the cag pathogenicity island (Tabla 2).The for one hour. Finally the enzyme was inactivated low resistant to clarithromycin in the strains by heating the sample for 10 minutes at 98 ºC studied probably reflects the low degree of use of the antibiotic in this population (25-27).
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Table 1. Growth of Helicobacter pylori isolated
2. Boixeda de Miguel D, Martín de Argila C.
Epidemiología de la infección por Helicobacter pylori. En: Boixeda de Miguel D, Gisbert JP, Endoscopic diagnosis
No. Positives/No.
Martín de Argila C. Infección por Helicobacter pylori. Dónde está el límite? Barcelona: 3. F. Mégraud, N. Broutet. Epidémiologie, acquisition et transmission de Helicobacter pylori. La Revue du Praticien. 2000;50:1414- 4. Pounder RE, Ng D. The prevalence of H. pylori infection different countries. Aliment Pharmacol Therapy. 1995; (Supp.2):33-39.
5. .Eurogast Study Group. Epidemiology of, and risk factors for, Helicobacter pylori infection Table 2. Genotype cagA from Cuban isolated of
populations. Gut. 1993; 34:1672-1676.
6. .A. Occhialini, A. Marais, M. Urdaci, F. Garcia, Endoscopic diagnosis
No. isolated tested/
R. Sierra, N. Muñoz, A. Covacci, F. Mégraud.
cagA+
Composition and gene expression of the cag pathogenicity island in Helicobacter pylori strains isolated from gastric carcinoma and gastritis patients in Costa Rica. Infection and 7. L. J. Van Doorn, C. Figueiredo, F. Mégraud, S.
Pena, P. Midolo, D.M. Queiroz, F. Carneiro, B.
Vanderborght, M. F. Pegado, R. Sanna, W. De Boer, P. Schneeberger, P. Correa, E.K.W. Ng, J.
Atherton, M.J. Blaser, W.G.V. Quint.
Conclusion
Geographic distribution of vacA allelic types ofHelicobacter pylori. Gastroenterology. 1999,  The prevalence of Helicobacter pylori infection in the symptomatic population of La Habana 8. M. Heikkinen, K. Mayo, F. Mégraud, M.
is the same as reported for other developing Vornanen, S. Marin, P. Pikkarainen, R.
Julkunen. Association of cagA-positive andcagA-negative Helicobacter pylori strains with patients' symptoms and gastritis in primary care patients with functional upper abdominal  The cagA status determined for 35 cases gave a positive result in 31 cases (88.5%).
Gastroenterology. 1998, 33:31-38.22. 9. B. Gutiérrez,T. Vidal, C.E.Valmaña, N.
 Most strains were cagA positive and are likely Santiesteban, N. González, I. Leonard, J.
harbour the cagA pathogenicity island. Ruiz, O. Díaz Canel, R. Martínez, M.P.
 The low resistance to clarithromycin in the Escobar, B. Grá, E. Galbán, M. González, G.
strains studied probably reflects the low Sierra. Primer informe sobre el aislamiento de Helicobacter pylori asociado a enfermedades digestivas en Ciudad de La Habana.
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Infección por Helicobacter pylori en la Ciudad de La Habana, Cuba. Prevalencia de las
cepas cagA positivas
Resumen
Existe una gran falta de información acerca de la infección por Helicobacter pylori en los países de la
región del Caribe. Nuestros objetivos en este estudio fueron determinar la prevalencia, la resistencia alos antibióticos y los factores de virulencia de la bacteria. La medida de la prevalencia de la infecciónpor H. pylori se determinó en un grupo de pacientes a los que se les practicó una endoscopia en tres centros hospitalarios de La Ciudad de La Habana, lo que nos permitió evaluar la resistencia a laclaritromicina y la presencia de cagA + en las cepas obtenidas. De las endoscopias realizadas seobtuvieron 117 biopsias gástricas, procedentes de tres centros hospitalarios de La Ciudad de LaHabana, Cuba: Instituto de Oncología, Instituto de Gastroenterología y el Hospital Calixto García. Las biopsias fueron mantenidas a –70 ºC para posterior cultivo en tres medios diferentes (dos selectivos yuno no selectivo) y su posterior incubación por 7 días a 37 ºC en una atmósfera de microaerofilia. Lapresencia de H. pylori fue identificada por la presencia de diferentes enzimas (oxidasa, catalasa, ureasa). Se realizó la extracción del DNA y la PCR, donde se utilizó el primer H2761676 y se amplificócon 397 fragmentos del gen cagA. La susceptibilidad a la claritromicina fue medida por el método dedifusión en gel. Diagnóstico endoscópico: (1) cáncer gástrico; (19) úlcera duodenal; (8) úlcera gástrica;(89) dispepsias no ulcerosas, incluyendo (62) gastritis; (9) hernia hiatal; (2) reflujo biliar; (1) pólipo gástrico; (15) panendoscopias normales. Del total de 117 biopsias realizadas, 83 fueron positivas a lainfección por H.pylori (70,9% ). De las 35 cepas a las que se les realizó presencia de cagA+ resultaronpositivas 31 (88,5%). Solo el 3% de las cepas fueron resistentes a la claritromicina. La prevalencia de la infección por H. pylori en la población sintomática de La Ciudad de La Habana es la misma que la
reportada en países desarrollados. La mayoría de las cepas fueron cagA positivas y son probablemente
el puerto de la Isla de patogenicidad (cagPAI). La baja resistencia a la claritromicina en las cepas
estudiadas, probablemente refleja la escasa utilización de este antibiótico en la población estudiada.
Palabras clave: H. pylori, prevalencia, diagnóstico endoscópico
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