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 studieshave been performed to determine the prevalence,
antibiotic resistance orvirulence factors of the bacterium. To measure the prevalence of H. pylori infection among patientsattending 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 from117 successive patients attendingthe Institute of Oncology, the Instituteof 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 °Cunder a
microaerobic atmosphere. The presence of H. pylori was identifiedby oxidase, catalase andurease activities. DNA was extracted, and PCRwas performed with primers H2761676 whichamplify a 397 bp fragmentof the cagA gene. Clarithromycin susceptibility was measured by
the geldiffusion method. The diagnoses of patients were: 1 gastric carcinoma; 19duodenalulcers; 8 gastric ulcers; and 89 non-ulcer dyspepsia, including (62) gastritis, (9) hiatalhernia,(2) biliary reflux, (1) gastric polyps, and (15) noabnormality. Among the 117 biopsiestested, 83 were H. pylori positive (70.9%). The cagA status determined for 35 cases gave a
positive result in31 cases (88.5%). Only 3% of the strains were resistant to clarithromycin. The prevalence of Helicobacter pylori infection in thesymptomatic population of La Habana isthe same as reported for otherdeveloping countries. Most strains were cagA positive and are
likely harbourthe cag pathogenicity island. The low resistance to clarithromycin inthe 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 15 Año 14 No. 2 julio-diciembre de 2005 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). VacciMonitor 16 Año 14 No. 2 julio-diciembre de 2005 Table 1. Growth of Helicobacter pylori isolated
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Endoscopic diagnosis No. Positives/No.
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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
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Pena, P. Midolo, D.M. Queiroz, F. Carneiro, B.
Vanderborght, M. F. Pegado, R. Sanna, W. De
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8. M. Heikkinen, K. Mayo, F. Mégraud, M.
is the same as reported for other developing
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care patients with functional upper abdominal
The cagA status determined for 35 cases gave
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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.
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Escobar, B. Grá, E. Galbán, M. González, G.
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Helicobacter pylori asociado a enfermedades
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VacciMonitor 18 Año 14 No. 2 julio-diciembre de 2005 Infección porHelicobacter pylorien 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 VacciMonitor 19 Año 14 No. 2 julio-diciembre de 2005
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