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Incidencia y factores asociados de Helicobacter pylori en lapoblación infantil del departamento de Cajamarca - Perú’
Item Type info:eu-repo/semantics/bachelorThesis
Authors Calderón Rivera, Andrea Ximena; Espinal Reyes, Maria DeFátima; Palacios Cuervo, Fernando André
Publisher Universidad Peruana de Ciencias Aplicadas (UPC)
Rights info:eu-repo/semantics/openAccess
Download date 27/06/2022 20:30:05
Item License http://creativecommons.org/licenses/by-nc-nd/4.0/
Link to Item http://hdl.handle.net/10757/621826
FACULTAD DE CIENCIAS DE LA SALUD
ESCUELA DE MEDICINA
‘’Incidencia y factores asociados de Helicobacter pylori en la población infantil
del departamento de Cajamarca - Perú’’
Proyecto de Investigación
Para optar el título profesional de:
MEDICO CIRUJANO
AUTORES:
CALDERÓN RIVERA, ANDREA XIMENA
ESPINAL REYES, MARIA DE FÁTIMA
PALACIOS CUERVO, FERNANDO ANDRÉ
ASESORES:
Ph.D. JUANA DEL VALLE
Ph D. ALONSO SOTO
CALIFICACIÓN: APROBADO
FECHA DE SUSTENTACION 12 DE FEBRERO DEL 2016
LIMA – PERÚ 2016
2
3
DEDICATORIA
Gracias a dios por permitirnos vencer cada obstáculo puesto en este largo viaje
Gracias a nuestros padres, familiares, amigos y asesores
por confiar y retarnos en esta aventura.
4
AGRADECIMIENTOS
A nuestros maestros, amigos y padres
por todo el apoyo brindado, por que sin ellos
todo este esfuerzo no hubiera sido tan gratificante.
5
Tabla de Contenido
Pag.
1. Artículo Científico Publicado 6
1.1. Resumen 8
1.2. Introducción 9
1.3. Materiales y Métodos 11
1.4. Resultados 13
1.5. Discusión 14
1.6. Conclusión 16
1.7. Bibliografía 17
1.8. Anexos 25
1.8.1. Tablas 25
1.8.2. Flujograma 30
1.8.3. Carta de Comité de Ética 31
1.8.4. Consentimiento informado 32
2. Revista de Publicación Científica 34
3. Proceso de Revisión 37
3.1. Envío original de artículo
4. Estado de Publicación 39
6
1.0 Artículo Científico
7
FULL TITLE:
Prevalence and associated factors to Helicobacter pylori infection in children from a
community of Cajamarca-Peru
AUTHORS:
Fatima Espinal-Reyes1; Andrea Calderón-Rivera
1; Fernando Palacios-Cuervo
1; Carlos
Palomares-Reyes2,3
; Fernando Vásquez-Achaya,2,3
; Jorge Bazán-Mayra4, Gabriela Ulloa-
Urizar2,3
; Alonso Soto1; Miguel Angel Aguilar-Luis
1,2,3; Juana del Valle Mendoza
1,2,3
AFFILIATIONS:
1Medicine School, Health Sciences Faculty, Universidad Peruana de Ciencias Aplicadas - UPC.
Av. San Marcos cdra. 2. Cedros de Villa. Lima, Perú.
2 Investigation Center of the Health Sciences Faculty, Universidad Peruana de Ciencias
Aplicadas (UPC), Av. San Marcos cdra 2 Cedros de Villa, Lima, Peru.
3 Molecular Biology Laboratory, Instituto de Investigación Nutricional. Av. La Molina 1885.
Lima 12, Perú.
4Dirección Regional de Salud de Cajamarca (DIRESA-Cajamarca), Cajamarca, Perú.
Corresponding author
Juana del Valle
Universidad Peruana de Ciencias Aplicadas
Av San Marcos Cdra 2, Chorrillos, Lima-Peru
E-mail: [email protected]
Phone. +51 1988040532
Fax. +51 1 3496023
Miguel Angel Aguilar-Luis
Universidad Peruana de Ciencias Aplicadas
Av San Marcos Cdra 2, Chorrillos, Lima-Peru
E-mail: [email protected]
Phone. +51 1985032252
Fax. +51 1 3496023
8
ABSTRACT
Background: The prevalence of H. pylori is greater than 50% in low development countries. In
children, infection with H. pylori produces effects such as low height, impairment in growth
especially in pubertal age. Polymerase Chain Reaction has a high sensitivity and specificity of
89.6% and 100% respectively, compared to other invasive methods. The objective of this study
is to determine the frequency of patients with presence of H. pylori in asymptomatic children in a
rural community in the north of Peru, using PCR technique in stool samples.
Methods: A cross-sectional study with a convenience sampling was performed, resulting in 147
children aged between 6-14 years old of the district of San Pablo in Cajamarca, Peru. Using stool
samples H. pylori DNA was obtained, then PCR amplification was done, and sequencing those
with positive results, in an agreement with Macrogen, Seoul - Korea.
Results: It was found a frequency of H. pylori positive cases of 21.1% with a CI 95% of 14.8 to
28.6. It was found a frequency (p=0.01) of positive for H. pylori among males (29% IC95: 19.1-
40.5) compared to women (12.7%; IC95: 6.0-22.7). There was no statistically significant
differences between those positive and negative patients with H. pylori compared to age (p =
0.57), presence of overweight (p = 0.09), or excreta disposal (p = 0.71). It was not found
significance difference among other variables.
Conclusion: Our study found a high frequency of H. pylori determined by a molecular test with
high sensitivity and specificity, demonstrating the feasibility of its implementation in rural
communities. It can be considered as a tool for surveillance in areas of high prevalence of H.
pylori infection.
Keywords: Helicobacter pylori, PCR, gastric disease, duodenal peptide ulcer disease
9
INTRODUCTION
Helicobacter pylori is the main cause of gastric and duodenal peptide ulcer disease; as well as
the most important factor associated to chronic gastritis (1). Worldwide, the estimated prevalence
of H. pylori is greater than 50% in the population (2-5), with a higher number of cases observed
in under developed countries (5,7). Moreover, individuals infected with H. pylori have an
increased risk of gastric adenocarcinoma and mucosa associated lymphoid tissue (MALT)
lymphoma (4, 5, 8-10). In children, H. pylori infections are also linked to low height, failure to
thrive and growth retardation especially during puberty (6). Therefore, an early detection and
treatment of the bacteria is essential to avoid chronic disease and further complications; since the
World Health Organization (WHO) considers the H. pylori infections as group 1 carcinogens (7,
10, 11).
In Latin America, the H. pylori is recognized as a major public health problem, with endemic
areas that can reach a prevalence of 80% of the adult population (12). This increase number of
cases has been linked to numerous factors including: low income, malnutrition, poor hygiene,
among others (6). All these risk factors facilitate an early exposure to the bacteria and
asymptomatic infection.
Both invasive and non-invasive methods have been described for the H. pylori detection (13).
Invasive tests require an endoscopic sample and include: histologic analysis, urease test and
cultures. On the other hand, the urea breath test, serologic tests and the antigen detection in feces
are non-invasive methods with limited applications (14). In that sense, a positive serologic result
cannot differentiate between a current and a past infection (15); and the urea breath test requires
very sophisticated equipment for spectrophotometry analysis, which is not always available in
the low-income settings (16). A stool sample can be very convenient for H. pylori detection,
especially in children, due to their easy and fast sample collection; and a few methods are used
for this bacterial detection: cultures, polymerase chain reaction (PCR) and enzymatic
immunoassays (17). However, a very wide range of detection rates have been reported with these
methods, due to the fast degradation of the bacteria in the gastrointestinal tract which leads to a
very low concentration of the H. pylori in the feces (18,19).
10
In the last few decades, non-invasive test have been gaining more importance, for the early
detection of H. pylori, especially in the after-treatment infants population (18). In addition, PCR
has shown a high sensitivity and specificity of 89.6% and 100% respectively, in comparison with
other invasive methods (20). The H. pylori eradication can drastically reduce both gastric and
duodenal ulcers rates in children, as well as prevent further complications. Therefore, a specific
and fast diagnostic method is crucial (21).
The main objective of this study is to determine the frequency of children infected with H. pylori
in a rural community in the north of Peru, by PCR detection in stool samples, as well as to
identify the most important associated factors to this infection in children population.
11
MATERIALS AND METHODS
Patients and ethics
A cross-sectional study was conducted in 147 children between 6 to 14 years old from the district
of San Pablo in Cajamarca-Peru. Informed consent was obtained from the children before
samples collection. The protocol of the study was approved by the Ethics committee of the
“Hospital Regional de Cajamarca-Peru”.
Samples preparation
The sample was obtained from each participant and all of them were transported to the molecular
biology laboratory of the Universidad Peruana de Ciencias Aplicadas (UPC), careful under cold
chain. Upon arrival, three aliquots of 200 µl each were prepared from each patient sample, and
stored at -70 °C until processing.
DNA extraction
Extracted DNA was from a volume of 200 µl of each aliquot using a commercial kit (High Pure
Template Preparation Kit, Roche Applied Science, Germany) (24) according to the manufacturer
instructions. DNA extraction was subjected to a dilution of 100 µl nuclease free buffer, assayed
immediately or stored at -70°C until use.
PCR amplification for detection H. pylori
PCR was target to the 23S rRNA region for H. pylori detection in feces samples. The primers,
forward primers (FP-1) and reverse primers (RP-1) previously designed and verified, were
synthesized for the amplification of a 320 bp fragment from the 23S rRNA (25,26) (Table 1). For
the amplification of the genomic DNA, a 20 µl mix was prepared, containing 10 µl of Ready
Mix Enzyme Solution (Taq polymerase, 2.5 mM MgCl2; 15 mM Tris/HCl PH 8.3, 50 mM KCl,
200 mM each deoxynucleotide) (KAPA Taq ReadyMix PCR Kit, Kappa Biosystems,
Massachusetts, United States), 20 pmol of each primer (Macrogen, Seoul, Korea), free water and
8µl of DNA; using a thermocycler Verity (Applied Biosystems, Foster City, CA, USA). The
amplified products were analyzed, recovered and sequence as mentioned above to confirm the
PCR results. All samples were compared to a positive control, and also they had a base pair
12
marker control. The adaptation of the thermocycler conditions for this process is presented in the
graph.
The presence and size of the amplified product is analyzed by electrophoresis on 2.5% agarose
gel (FMC, Rockland, ME) which was previously dyed with 3 µg / ml ethidium bromide and
separated in 1x Tris buffer - acetic EDTA. The bands were using a KODAK 1500 LOGIC
(Eastman Kodak Company, Rochester, NY, USA) Documenting system. To confirm the
specificity of the amplicons, all positive samples were sequenced in the service of Macrogen -
Korea, to verify the results.
Statistical analysis
The data obtained through questionnaires provided by DIRESA were entered into a database of
Microsoft Excel 2012, and coded according to the categories belonging to each variable. The
variable Body Mass Index (BMI) was categorized by percentiles established by WHO (27, 28).
Categorical variables, expressed as absolute frequencies and percentages. Numeral variables
expressed as median and interquartile range. A secondary analysis was performed to explore the
association of possible predictors. P-values <0.05 were considered to denote statistical
significance.
To assess association between categorical variables, we used Chi squared test or Fisher exact
test, and for nonparametric variables Wilcoxon test was used. The analysis was performed using
the statistical package STATA v11.
13
RESULTS
The prevalence of H. pylori was 21.1% (31/147) (Table 2). Demographic characteristic in the
147 children are summarized in table 2. The male to female proportion was 76:71 with an
average age of 9 years old, with an interquartile range (IQR) of 4 years, corresponding 76
(51.7%) male participants (Table 3). The children age was not associated with the H. pylori
prevalence. However, a significant association was observed between male gender and positives
cases of H. pylori with 70.9% (22/31, 2=5.84, p=0.01).
The BMI did not show any association with the prevalence of the H. pylori. A higher positives
cases of H. pylori was observed in the group of children with normal weight (80.6%, 25/31),
while 11.6% was found in overweight (percentile p > 85).
Furthermore, an association between the use of latrines and a higher rate of positives cases was
observed (2=4.08, p=0.043), with a prevalence of 80.6% (25/31). On the contrary, children who
had access to a drain system presented fewer infections in 19.3% (6/31).
On table 4, the frequency of positive samples for H. pylori is compared to the dietary
characteristics; and none of them were associated to a higher rate of cases. However, consuming
raw lettuce and having domestic animals showed a prevalence of 90.32% (28/31) and 77.42%
(24/31), respectively (Table 4).
Seven clinic symptoms were constantly observed in children with H. pylori detected including
headache, fever, nauseas, vomits, diarrhea, loss of weight and abdominal pain in the last 3
months (Table 5).
14
DISCUSSION
In recent years, emphasis has been placed on the use of non-invasive diagnostic methods for the
detection of H. pylori in pediatric population (29, 30); however, their use is not yet standardized
in routine clinical practice. Among the most promising techniques is set the PCR, however, so
far there have not been studies at the national level to assess the feasibility and utility of it use.
The PCR technique used in this study to find the frequency of H. pylori have proven an
advantage in the detection of bacteria, especially pathogens difficult to culture in vitro (31). This
method can detect molecular pathogen regardless of its properties bacterial viability with high
sensitivity and specificity of 95% (31). These benefits of PCR have been described in several
studies, for example Montealegre et al; they reported a prevalence of H. pylori in 78.3% with
histological diagnosis and 68.3% for detection by PCR (32). Another study by Gonzales et al;
they compared the detection of H. pylori antigen in feces with ELISA against other invasive
methods, demonstrating the presence of the antigen with a 90% sensitivity and 100% specificity;
and a positive predictive value of 100% (33). Another study by Smith et al; they compared the
detection of H. pylori in dyspeptic patients by urease breath test and stool PCR, which showed
the presence of H. pylori by PCR in 20.6% (34).
Our study demonstrates the feasibility of using the PCR as a method to detect the genome of H.
pylori in stool samples, in children in a rural area. We found a prevalence of 21.1%. We did not
find in the literature previous studies that used PCR to detect H. pylori, however, studies have
assessed the frequency of H. pylori antigen in stool samples in 18.9% (35).
They have also performed serological studies in Brazil at a rate of 35.6% (36); in Taiwan with
15.1% (37). In Peru, there are not studies evaluating the frequency of H. pylori by noninvasive
tests in pediatric population. Peruvian literature has only evaluated the presence of H. pylori in
histopathologic specimens. A study in the Cayetano Heredia Hospital between the periods 2003
to 2006, found a prevalence of 46% in gastric biopsies (38).
15
Our study showed a higher frequency of H. pylori positivity in males (29% vs. 12.7%). The
literature shows discordant evidence about a study by Kate et al; demonstrating an association
with male gender and the frequency of H. pylori (57%) (39). By another hand; other studies such
as De Martel and Parsonnet and Çınar et al; they found no significant difference between gender
and H. pylori (40, 41).
There are other factors associated with the presence of H. pylori, including age (41, 42), BMI
(43), level of sanitation (44), and dietary consumption. Regarding the relationship between H.
pylori infections and nutritional status, we found that the nutritional status of a population with
greater than 85 percentiles were infected in 54.5% compared to 23.8% of the population with
adequate percentile for age (p10-p85 as WHO tables). Some studies suggest that H. pylori
colonization may be associated with growth retardation, by compromising the acid barrier of the
stomach, reducing the defenses against pathogens, and predisposing the host to diarrheal
diseases, resulting in mal-absorption of some nutrients (45-47). However, in our study there was
no significant difference related to growth percentile for height, but further investigations with a
larger population are required. Our study found no association with any of these factors.
Although, it must be emphasized the exploratory nature of our analysis which probably does not
have enough power to detect these associations. It is important to generate larger studies to
formally evaluate the possible factors associated with H. pylori infections, considering that our
country has a high prevalence of this infection and its complications including gastric cancer in
adults (21.2 cases per 100 000 inhabitants) (47).
The study presented in their main limitations, the population belonging to the district of San
Pablo which represents 1.7% of Cajamarca population (48), the smallest of the department; as
well as belonging to a poverty area, so those results cannot be extrapolated to other regions. We
believe it is an event unlikely because of the staff was previously trained.
16
CONCLUSION
Our study found a high frequency of H. pylori in a specific population with a molecular test with
high sensitivity and specificity, demonstrating the feasibility of its implementation in rural
communities. This test can be considered as a tool for surveillance in areas of high frequency of
infection for H. pylori.
ACKNOWLEDGMENTS
Grant-UPC-501-2016. JDVM support was provided by 5to
Incentives for Research of the
Peruvian University of Applied Sciences (UPC). Lima-Peru.
AUTHORS CONTRIBUTIONS
JdVM, CPR. FVA, and FP performed the PCR for H. pylori; JdVM and MAAL designed the
study protocol and they are responsible for obtaining funding and laboratory work supervision.
FPC, ACR, FER, GUU was responsible for the clinical assessment, samples collection and
database completion. FP, ACR, FE, JdVM, MAAL drafted the manuscript. All authors critically
revised the manuscript for intellectual content. All authors read and approved the final
manuscript.
CONFLICT OF INTERESTS
On behalf of all authors, the corresponding author states that there are no conflicts of interest or
funding related to this study.
17
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ANEXOS:
TABLES
Table 1. Oligonucleotide sequence
Primer Sequence (5´3´) Target
FP-1 TCGAAGGTTAAGAGGATGCGTCAGTC 23S rRNA
RP-1 GACTCCATAAGAGCCAAAGCCCTTAC
26
Table 2. Prevalence of H. pylori by PCR in children of school age in a district of San Pablo
in Cajamarca-Peru 2015.
Identified of H. pylori
by PCR
Cases Frequency
(n)
Prevalence
(%)
Positives 31 21.1
Negatives 116 78.9
Total 147 100.0
27
Table 3. Demographics characteristics in children from a district of San Pablo in Cajamarca-
Peru 2015.
CHARACTERISTICS Total of cases
n=147 (%)
Positives for
H. pylori by
PCR
n=31 (%)
Negatives for
H. pylori by
PCR
n=116 (%)
P value*
Age a 9 (4) 8 (3) 9 (4)
Gender* 0.01
Masculine 76 (51.7) 22 (70.9) 54 (46.5)
Feminine 71 (48.3) 9 (29.0) 62 (53.4)
BMI (kg/m2)* 0.12
Normal (p10-p85) 130 (88.4) 25 (19.2) 105 (80.8)
Overweight (p85- p95) 17 (11.5) 6 (35.3) 11 (64.7)
Drain system at
home* 0.71
Bathroom 51 (34.6) 6 (19.3) 45 (31.0)
Latrine 96 (65.3) 25 (80.6) 71 (68.9)
a: Median and Interquartile range
* Chi-Square of Pearson (2), p<0.05
28
Table 4. Dietary characteristics in children infected with Helicobacter pylori in a district of
San Pablo-Cajamarca, 2015.
ASSOCIATED FACTORS
Positives for H.
pylori
n=31 (%)
Negatives for H.
pylori
n=116 (%)
p-value*
Unboiled water consumption** 4 (12.9) 18 (15.5) 1.00
Boiled water consumption** 4 (12.9) 28 (24.1) 0.17
Washing hands before eating* 31 (100.0) 115 (99.1) 1.00
Washing hands after eating* 31 (100.0) 114 (98.2) 1.00
Domestic animal raising* 24(77.4) 76(65.5) 0.20
Chewing grass** 4 (12.9) 13 (11.2) 1.00
Raw lettuce consumption* 28 (90.3) 112 (96.5) 0.16
Watercress consumption* 12 (38.7) 34 (29.3) 0.31
Meat consumption* 31 (100.0) 115 (99.1) 1.00
Legume consumption* 14 (45.1) 61 (52.5) 0.46
Tubers consumption* 7 (22.5) 40 (34.4) 0.20
Fruits consumption* 12 (38.7) 46 (39.6) 0.92
Vegetables consumption* 16 (51.6) 60 (51.7) 0.99
Egg consumption* 17 (54.8) 79 (68.1) 0.16
Milk consumption* 20 (64.5) 76 (65.5) 1.00
Emollient consumption* 12 (38.7) 35 (30.1) 0.36
* Chi-Square of Pearson (2)
** Fisher’s exact test
29
Table 5. Clinical symptoms in children infected with Helicobacter pylori in a district of San
Pablo-Cajamarca, 2015.
CLINICAL SYMPTOMS
Positives for H.
pylori
n=31 (%)
Negatives for H.
pylori
n=116 (%)
p-value*
Diarrhea in the last 3 months* 6 (19.4) 29 (25.0) 0.51
Weight loss in the last 3 months* 15 (48.4) 58 (50.0) 0.87
Abdominal pain in the last 3
months*
20 (64.5) 87 (75.0) 0.24
Nauseas** 2 (6.5) 9 (7.8) 1.00
Vomiting** 2 (6.5) 11 (9.5) 0.73
Fever** 3 (9.7) 9 (7.8) 0.71
Headache** 4 (12.9) 7 (6.0) 0.24
* Chi-Square of Pearson (2)
** Fisher’s exact test
30
Flujograma de la población
31
Carta de autorización del Comité de Ética
32
Documento de Consentimiento Informado y Asentimiento:
33
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__________________________ __________________________
Calderón Rivera, Andrea Del Valle Mendoza, Juana
__________________________
Espinal Reyes, María de Fátima
__________________________
Palacios Cuervo, Fernando