Helicobacter ISSN 1523-5378 doi: 10.1111/hel.12098
Present and Past Helicobacter pylori Infection in Mexican School Children Eugenia Mendoza,* Margarita Camorlinga-Ponce,* Guillermo Perez‐Perez,† Robertino Mera,‡ Jenny Vilchis,§ Segundo Moran,* Octavio Rivera,* Rafael Coria,¶ Javier Torres,* Pelayo Correa‡ and Ximena Duque* *Mexican Institute of Social Security, Mexico City, Mexico, †New York University School of Medicine, New York, NY, USA, ‡Vanderbilt University Medical Center, Nashville, TN, USA, §Hospital Infantil “Federico Gomez”, Mexico City, Mexico, ¶National Institute of Pediatrics, Mexico City, Mexico
Keywords Active infection by H. pylori, CagA H. pylori strains, evidence of past infection by H. pylori, Helicobacter pylori, serological tests to H. pylori, urea breath test. Reprint requests to: Ximena Duque, Unidad de Investigacion en Enfermedades Infecciosas y Parasitarias, Hospital de Pediatria, CMN Siglo XXI, IMSS, Av. Cuauhtemoc No. 330, Col Doctores, Del Cuauhtemoc, Cp 06725 Mexico City, Distrito Federal, Mexico. E-mail: [email protected]
Abstract Background: In developing countries, more than 50% of children have serological evidence of Helicobacter pylori infection. However, serological tests for H. pylori did not differentiate between active and past infection. The objectives of this study were to estimate the frequency of active and past H. pylori infection utilizing functional urea breath test (UBT) and serological tests and evaluate factors associated with the infection. Methods: A total of 675 school children, 6–13 years of age, participated. UBT was performed to detect active H. pylori infection. Blood samples were obtained to determine iron status and Immunoglobulin G (IgG) responses to the H. pylori whole-cell and to Cag A antigens by antigen-specific enzymelinked immunosorbent assays. Weight, height, and sociodemographic characteristics were recorded. Results: A total of 37.9% (95% Confidence Intervals (CI): 34.2–41.6) of school children had active or past H. pylori infection; of them, 73.8% (CI95% 68.4 79.2) were carrying CagA-positive strain, 26.5% (CI95% 23.2–29.8) had active infection, and 11.4% (95%CI: 9.0–13.8) had evidence of past H. pylori infection. School children with iron deficiency and low height for age had higher risk of H. pylori infection: [OR to active or past infection was 2.30 (CI 95% 1.01–5.23) and to active infection it was 2.64 (CI 95% 1.09–6.44)] compared to school children with normal iron status and height for age or with normal iron status but low height for age or with iron deficiency and normal height for age. Conclusions: The estimated prevalence of infection depends of the test utilized. Frequency of H. pylori infection and carrying CagA-positive strains was high in this population. Malnutrition was associated with active H. pylori infection.
Helicobacter pylori (H. pylori) is a gram-negative, curvedshaped bacterium, classified in Group I carcinogen, clinically associated with gastritis, peptic ulcer disease and gastric cancer [1, 2]. In developing countries, more than 80% of adults and 50% of children are colonized by H. pylori compared to 30% of adults and 10% of children in developed countries [3]. In Mexico, in 1988 a seroepidemilogical survey estimated H. pylori prevalence of 66% [4, 5]. Twenty percent of infants of 1 year and younger were colonized by H. pylori, and colonization had reached 50% in children before they reached 10 years of age [6].
© 2013 John Wiley & Sons Ltd, Helicobacter 19: 55–64
In a study carried out in 2001 in boarding schools of the National Indigenous Institute of Hidalgo State in Mexico, prevalence of active H. pylori infection was 52% [7]. In a population study, in Mexico City, 38% of school children had active H. pylori. Children with H. pylori infection averaged 1.32 cm (CI 95% 2.22 to 0.42) less in height than children without infection [8]. In the same population, the colonization by H. pylori was a dynamic phenomenon, with an incidence rate of 64 new cases/ year/1000 school children and a spontaneous infection clearance rate of 47 cases/year/1000 school children [9].
55
Active and Past H. pylori Infection in Children
There are different H. pylori strains with genetic variability. Bacterial characteristics, host characteristics, and environmental factors determine the degree of damage that the infection can cause in the gastric mucosa [10]. H. pylori displays factors that determine its virulence; one of them is the cytotoxin-associated gene A (cagA) [11]. In most populations, approximately 50% of H. pylori strains have this virulence factor. The cagA island encodes a bacterial type IV secretion system that translocates CagA into host cells. Intracellular CagA affects multiple pathways that alter host cell morphology, signaling, and inflammatory responses [11]. H. pylori infection with this virulence factor has been associated with the development of severe diseases such as gastric and duodenal ulcer, gastric atrophy, and gastric cancer [12–14]. The infection by H. pylori in children has also been associated with extra-gastric manifestations such as lower growth rate and iron deficiency (ID) or iron deficiency anemia (IDA) [15–21]. Some authors suggest that a chronic infection is a prerequisite for the development of diseases such as symptomatic gastritis, gastric and duodenal ulcers, gastric cancer [22], ID or IDA [23, 24]. Studies on the effect of active infection on the speed of child growth have shown that there is a greater negative effect in the months after the onset of the infection. This effect is maintained and affects infected children’s growth cumulatively throughout time [18, 19, 21]. The majority of H. pylori-infected people remain asymptomatic; thus, the infection is not detected in the acute phase. Several types of tests are commonly used for assessing H. pylori status, including the more invasive methods of culturing samples obtained during endoscopy and the less invasive methods of serologic antibody tests, the UBT, and the stool antigen test. All methods have advantages and disadvantages, none can be considered as the gold standard. The invasive method requires a gastric biopsy. The culture of H. pylori is the most specific method but has low sensitivity. The histology biopsy evaluation has sensitivity and specificity higher than 80%, but because of the nonhomogeneous bacterium colonization, it depends on the number and location of biopsies [25]. Invasive methods are not justifiable in studies with asymptomatic subjects. The use of noninvasive tests such as UBT or stool antigen is recommended at the clinical level for subjects in whom the direct evaluation of the gastric mucosa is not always indicated (e.g., in monitoring the clearance of the infection after eradication therapy) [26]. Serological testing by enzyme immunoassay is useful in epidemiological studies. These tests are based on the
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Mendoza et al.
detection of serum antibodies to H. pylori-specific antigens. These antibodies are present some weeks after acquiring the infection and decline slowly after bacterium eradication [10, 27, 28]. The major disadvantage of these serological tests (IgG antibodies to whole-cellH. pylori or CagA antigens) is that they cannot distinguish active from past infection [23, 25, 29, 30]. CagA is a highly immunogenic protein; in fact, more than 95% of subjects infected by CagA-positive H. pylori strain develop a serologically detectable response against the CagA antigen [31]. The quantitation of antibodies to CagA antigen can be carried out by ELISA or Western Blot. This detection has been utilized to discard cases of false-negative H. pylori infection when detection of whole-cell H. pylori antibodies is used [28, 31]. The objectives of this study were to estimate the frequency of active and past H. pylori infection utilizing functional urea breath test (UBT) and serological tests and evaluate factors associated with the infection. This information may be useful in determining the natural history of H. pylori infection and in planning preventive strategies against the infection and its consequences.
Methods A total of 675 school children aged 6–13 years old participated in this cross-sectional study. They were tested for H. pylori infection by three different testing methods: 13C-UBT, antibodies to whole-cell H. pylori, and CagA antigens using antigen-specific enzyme-linked immunosorbent assays (ELISAs). This study is part of the main cohort study carried out in a homogeneous population of school children from low-income families. All of them attended public boarding schools at Mexico City. In that study, prevalence of H. pylori infection, incidence rate, spontaneous clearance rate, and the effect of H. pylori on growth were evaluated. The study was approved by the Ethics and Investigation Committee of the Mexican Social Security Institute and was also authorized by the Secretary of Public Education of Mexico. The parents signed an informed consent form authorizing their children’s participation; additionally, children were asked to give their consent to participate in the study. Details about this study have been published [9]. In brief, school children were tested for H. pylori infection and their iron status was evaluated. Skilled personnel drew venous blood sample to determine by enzymatic immunoassays (ELISAs), H. pylori whole-cell and CagA antigens antibodies. The UBT test was utilized to detect active H. pylori infection. At the time the samples were taken, the school children were fasting 8 hours and had not received antibiotic
© 2013 John Wiley & Sons Ltd, Helicobacter 19: 55–64
Active and Past H. pylori Infection in Children
Mendoza et al.
treatments, bismuth salts, proton pump inhibitors, or sucralfate in the previous month. Height and weight were measured. Sociodemographic information such as age, sex, and number of occupants in the dwelling was gathered by a questionnaire. 13
C-urea breath test
The UBT consisted of collecting two samples of expired air. The basal sample was obtained 10 minutes after the child had ingested a beverage containing 2 g of citric acid (Citra-LP; San Miguel Proyectos Agropecuarios S.P.R., Hidalgo, Mexico) to delay gastric emptying. Immediately afterward, children were given 50 mg of 13 C-labeled urea dissolved in 150 mL of water, and the final sample was collected 30 minutes later. Expired air samples were collected in 10-mL tubes (Exatainers, Labco Ltda, High Wycombe, UK). A difference of ≥5 parts/1000 between ratio values 13CO2/12CO2 at baseline and 30 minutes post-intake of 13C-urea was considered a positive test for active H. pylori. The samples were stored at room temperature and analyzed by a mass spectrometer (BreathMat-plus, Finnigan MAT, Bremen, Germany). The sensitivity and specificity of this test in children 6 years or older is >90% [25, 32–34].
Serological tests: antibodies to whole-cell H. pylori and CagA antigens A 4.7 mL venous blood sample was obtained. The sample was centrifuged and serum was frozen at –70 °C until its biochemical analysis. Assays for H. pylori-specific immunoglobulin (IgG) were performed by ELISA. An optical density ratio (ODR) value ≥1.0 was considered seropositive. An ELISA was performed to detect antibodies to CagA antigens using purified recombinant CagA antigen. ODR values were calculated in relation to reference sera, values ≥1.5 were considered seropositive. These tests had been previously validated in Mexican pediatric populations. The sensitivity and specificity of the tests are 85–87% for whole-cell H. pylori and 83–97% for CagA [5].
Hemoglobin and serum ferritin concentration were determined. Blood cytology samples were analyzed by the automated cyanmethehemoglobin method (Beckman Coulter, Fullerton, CA, USA). Ferritin was determined by immunoradio assay (Inmunotech SA, Marseille, France). Iron nutritional status was based on hemoglobin concentration adjusted for altitude and on serum ferritin. School children classified with normal iron status had hemoglobin ≥11.5 g/dL if they were younger than 12 years, hemoglobin ≥12.0 g/dL if they were 12 years or older, and ferritin ≥15 ng/mL. Children with iron deficiency had ferritin
Present and Past Helicobacter pylori Infection in Mexican School Children Eugenia Mendoza,* Margarita Camorlinga-Ponce,* Guillermo Perez‐Perez,† Robertino Mera,‡ Jenny Vilchis,§ Segundo Moran,* Octavio Rivera,* Rafael Coria,¶ Javier Torres,* Pelayo Correa‡ and Ximena Duque* *Mexican Institute of Social Security, Mexico City, Mexico, †New York University School of Medicine, New York, NY, USA, ‡Vanderbilt University Medical Center, Nashville, TN, USA, §Hospital Infantil “Federico Gomez”, Mexico City, Mexico, ¶National Institute of Pediatrics, Mexico City, Mexico
Keywords Active infection by H. pylori, CagA H. pylori strains, evidence of past infection by H. pylori, Helicobacter pylori, serological tests to H. pylori, urea breath test. Reprint requests to: Ximena Duque, Unidad de Investigacion en Enfermedades Infecciosas y Parasitarias, Hospital de Pediatria, CMN Siglo XXI, IMSS, Av. Cuauhtemoc No. 330, Col Doctores, Del Cuauhtemoc, Cp 06725 Mexico City, Distrito Federal, Mexico. E-mail: [email protected]
Abstract Background: In developing countries, more than 50% of children have serological evidence of Helicobacter pylori infection. However, serological tests for H. pylori did not differentiate between active and past infection. The objectives of this study were to estimate the frequency of active and past H. pylori infection utilizing functional urea breath test (UBT) and serological tests and evaluate factors associated with the infection. Methods: A total of 675 school children, 6–13 years of age, participated. UBT was performed to detect active H. pylori infection. Blood samples were obtained to determine iron status and Immunoglobulin G (IgG) responses to the H. pylori whole-cell and to Cag A antigens by antigen-specific enzymelinked immunosorbent assays. Weight, height, and sociodemographic characteristics were recorded. Results: A total of 37.9% (95% Confidence Intervals (CI): 34.2–41.6) of school children had active or past H. pylori infection; of them, 73.8% (CI95% 68.4 79.2) were carrying CagA-positive strain, 26.5% (CI95% 23.2–29.8) had active infection, and 11.4% (95%CI: 9.0–13.8) had evidence of past H. pylori infection. School children with iron deficiency and low height for age had higher risk of H. pylori infection: [OR to active or past infection was 2.30 (CI 95% 1.01–5.23) and to active infection it was 2.64 (CI 95% 1.09–6.44)] compared to school children with normal iron status and height for age or with normal iron status but low height for age or with iron deficiency and normal height for age. Conclusions: The estimated prevalence of infection depends of the test utilized. Frequency of H. pylori infection and carrying CagA-positive strains was high in this population. Malnutrition was associated with active H. pylori infection.
Helicobacter pylori (H. pylori) is a gram-negative, curvedshaped bacterium, classified in Group I carcinogen, clinically associated with gastritis, peptic ulcer disease and gastric cancer [1, 2]. In developing countries, more than 80% of adults and 50% of children are colonized by H. pylori compared to 30% of adults and 10% of children in developed countries [3]. In Mexico, in 1988 a seroepidemilogical survey estimated H. pylori prevalence of 66% [4, 5]. Twenty percent of infants of 1 year and younger were colonized by H. pylori, and colonization had reached 50% in children before they reached 10 years of age [6].
© 2013 John Wiley & Sons Ltd, Helicobacter 19: 55–64
In a study carried out in 2001 in boarding schools of the National Indigenous Institute of Hidalgo State in Mexico, prevalence of active H. pylori infection was 52% [7]. In a population study, in Mexico City, 38% of school children had active H. pylori. Children with H. pylori infection averaged 1.32 cm (CI 95% 2.22 to 0.42) less in height than children without infection [8]. In the same population, the colonization by H. pylori was a dynamic phenomenon, with an incidence rate of 64 new cases/ year/1000 school children and a spontaneous infection clearance rate of 47 cases/year/1000 school children [9].
55
Active and Past H. pylori Infection in Children
There are different H. pylori strains with genetic variability. Bacterial characteristics, host characteristics, and environmental factors determine the degree of damage that the infection can cause in the gastric mucosa [10]. H. pylori displays factors that determine its virulence; one of them is the cytotoxin-associated gene A (cagA) [11]. In most populations, approximately 50% of H. pylori strains have this virulence factor. The cagA island encodes a bacterial type IV secretion system that translocates CagA into host cells. Intracellular CagA affects multiple pathways that alter host cell morphology, signaling, and inflammatory responses [11]. H. pylori infection with this virulence factor has been associated with the development of severe diseases such as gastric and duodenal ulcer, gastric atrophy, and gastric cancer [12–14]. The infection by H. pylori in children has also been associated with extra-gastric manifestations such as lower growth rate and iron deficiency (ID) or iron deficiency anemia (IDA) [15–21]. Some authors suggest that a chronic infection is a prerequisite for the development of diseases such as symptomatic gastritis, gastric and duodenal ulcers, gastric cancer [22], ID or IDA [23, 24]. Studies on the effect of active infection on the speed of child growth have shown that there is a greater negative effect in the months after the onset of the infection. This effect is maintained and affects infected children’s growth cumulatively throughout time [18, 19, 21]. The majority of H. pylori-infected people remain asymptomatic; thus, the infection is not detected in the acute phase. Several types of tests are commonly used for assessing H. pylori status, including the more invasive methods of culturing samples obtained during endoscopy and the less invasive methods of serologic antibody tests, the UBT, and the stool antigen test. All methods have advantages and disadvantages, none can be considered as the gold standard. The invasive method requires a gastric biopsy. The culture of H. pylori is the most specific method but has low sensitivity. The histology biopsy evaluation has sensitivity and specificity higher than 80%, but because of the nonhomogeneous bacterium colonization, it depends on the number and location of biopsies [25]. Invasive methods are not justifiable in studies with asymptomatic subjects. The use of noninvasive tests such as UBT or stool antigen is recommended at the clinical level for subjects in whom the direct evaluation of the gastric mucosa is not always indicated (e.g., in monitoring the clearance of the infection after eradication therapy) [26]. Serological testing by enzyme immunoassay is useful in epidemiological studies. These tests are based on the
56
Mendoza et al.
detection of serum antibodies to H. pylori-specific antigens. These antibodies are present some weeks after acquiring the infection and decline slowly after bacterium eradication [10, 27, 28]. The major disadvantage of these serological tests (IgG antibodies to whole-cellH. pylori or CagA antigens) is that they cannot distinguish active from past infection [23, 25, 29, 30]. CagA is a highly immunogenic protein; in fact, more than 95% of subjects infected by CagA-positive H. pylori strain develop a serologically detectable response against the CagA antigen [31]. The quantitation of antibodies to CagA antigen can be carried out by ELISA or Western Blot. This detection has been utilized to discard cases of false-negative H. pylori infection when detection of whole-cell H. pylori antibodies is used [28, 31]. The objectives of this study were to estimate the frequency of active and past H. pylori infection utilizing functional urea breath test (UBT) and serological tests and evaluate factors associated with the infection. This information may be useful in determining the natural history of H. pylori infection and in planning preventive strategies against the infection and its consequences.
Methods A total of 675 school children aged 6–13 years old participated in this cross-sectional study. They were tested for H. pylori infection by three different testing methods: 13C-UBT, antibodies to whole-cell H. pylori, and CagA antigens using antigen-specific enzyme-linked immunosorbent assays (ELISAs). This study is part of the main cohort study carried out in a homogeneous population of school children from low-income families. All of them attended public boarding schools at Mexico City. In that study, prevalence of H. pylori infection, incidence rate, spontaneous clearance rate, and the effect of H. pylori on growth were evaluated. The study was approved by the Ethics and Investigation Committee of the Mexican Social Security Institute and was also authorized by the Secretary of Public Education of Mexico. The parents signed an informed consent form authorizing their children’s participation; additionally, children were asked to give their consent to participate in the study. Details about this study have been published [9]. In brief, school children were tested for H. pylori infection and their iron status was evaluated. Skilled personnel drew venous blood sample to determine by enzymatic immunoassays (ELISAs), H. pylori whole-cell and CagA antigens antibodies. The UBT test was utilized to detect active H. pylori infection. At the time the samples were taken, the school children were fasting 8 hours and had not received antibiotic
© 2013 John Wiley & Sons Ltd, Helicobacter 19: 55–64
Active and Past H. pylori Infection in Children
Mendoza et al.
treatments, bismuth salts, proton pump inhibitors, or sucralfate in the previous month. Height and weight were measured. Sociodemographic information such as age, sex, and number of occupants in the dwelling was gathered by a questionnaire. 13
C-urea breath test
The UBT consisted of collecting two samples of expired air. The basal sample was obtained 10 minutes after the child had ingested a beverage containing 2 g of citric acid (Citra-LP; San Miguel Proyectos Agropecuarios S.P.R., Hidalgo, Mexico) to delay gastric emptying. Immediately afterward, children were given 50 mg of 13 C-labeled urea dissolved in 150 mL of water, and the final sample was collected 30 minutes later. Expired air samples were collected in 10-mL tubes (Exatainers, Labco Ltda, High Wycombe, UK). A difference of ≥5 parts/1000 between ratio values 13CO2/12CO2 at baseline and 30 minutes post-intake of 13C-urea was considered a positive test for active H. pylori. The samples were stored at room temperature and analyzed by a mass spectrometer (BreathMat-plus, Finnigan MAT, Bremen, Germany). The sensitivity and specificity of this test in children 6 years or older is >90% [25, 32–34].
Serological tests: antibodies to whole-cell H. pylori and CagA antigens A 4.7 mL venous blood sample was obtained. The sample was centrifuged and serum was frozen at –70 °C until its biochemical analysis. Assays for H. pylori-specific immunoglobulin (IgG) were performed by ELISA. An optical density ratio (ODR) value ≥1.0 was considered seropositive. An ELISA was performed to detect antibodies to CagA antigens using purified recombinant CagA antigen. ODR values were calculated in relation to reference sera, values ≥1.5 were considered seropositive. These tests had been previously validated in Mexican pediatric populations. The sensitivity and specificity of the tests are 85–87% for whole-cell H. pylori and 83–97% for CagA [5].
Hemoglobin and serum ferritin concentration were determined. Blood cytology samples were analyzed by the automated cyanmethehemoglobin method (Beckman Coulter, Fullerton, CA, USA). Ferritin was determined by immunoradio assay (Inmunotech SA, Marseille, France). Iron nutritional status was based on hemoglobin concentration adjusted for altitude and on serum ferritin. School children classified with normal iron status had hemoglobin ≥11.5 g/dL if they were younger than 12 years, hemoglobin ≥12.0 g/dL if they were 12 years or older, and ferritin ≥15 ng/mL. Children with iron deficiency had ferritin
