Helicobacter ISSN 1523-5378 doi: 10.1111/hel.12213
The Prevalence of Mixed Helicobacter pylori Infections in Symptomatic and Asymptomatic Subjects in Dhaka, Bangladesh Khandoker Mohammad K. Kibria,* Md Enayet Hossain,† Jinath Sultana,‡ Shafiqul A. Sarker,† Pradip Kumar Bardhan,† Motiur Rahman§ and Shamsun Nahar† *Mawlana Bhashani Science and Technology University, Shantosh, Tangail, Bangladesh, †International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh, ‡North-South University, Dhaka, Bangladesh, §Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam
Keywords Helicobacter pylori, Bangladesh, mixed infection, cagA, vacA. Reprint requests to: Shamsun Nahar, Enteric Microbiology Laboratory, Laboratory Sciences Division, ICDDR, B GPO Box-128, Dhaka 1000, Bangladesh. E-mail: [email protected]
Abstract Background: Helicobacter pylori is a highly genetically diverse bacterial species, which can persist in the gastric environment for decades. Recent studies have shown that single infections predominate in developed countries, whereas mixed infections are more prevalent in developing countries. Mixed infections of this bacterium may be important for adaptation to the hostile gastric environment and may facilitate dyspeptic symptoms. Materials and Methods: To calculate the prevalence of mixed infections in symptomatic and asymptomatic subjects, 2010 H. pylori isolates collected from 83 symptomatic and 91 asymptomatic subjects from Dhaka, Bangladesh, were analyzed by (i) random amplified polymorphic DNA fingerprinting (RAPD) and (ii) multiplex PCR amplification for cagA and vacA virulence gene alleles. Results: The overall prevalence of mixed H. pylori infection was 60.15% (77/128), indicating substantial co-colonization in this population. We additionally found that symptomatic subjects (53%) had a significantly higher rate of mixed infection than asymptomatic individuals (36.3%) (p = .016) and that the prevalence of the cagA and vacA and vacA m1/s1 and vacA m2/s1 alleles were higher in subjects with mixed infection. Conclusion: Our findings suggest that an increased diversity of the H. pylori strains in the gastric environment may contribute to the development of disease symptoms.
Helicobacter pylori is one of the most genetically diverse known bacterial species. Colonization of the stomach with H. pylori is extremely common in humans, and although generally asymptomatic, infection can cause abdominal pain, dyspepsia, peptic ulcers, and gastric cancer. Infection rates of H. pylori within human populations vary from between 20 and 30% in developed countries to between 80 and 90% in developing countries [1]. The population structure of H. pylori is atypical with respect to many bacterial species as a consequence of vertical transmission of the bacteria within families, predominantly from parents to child, or between children via oral–oral, gastric–oral, or fecal–oral transmission [2–5]. Our recent work, in Dhaka, Bangladesh, added to these date and indicated that mother to child transmission of H. pylori occurs mainly in the early childhood [6].
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The natural habitat of H. pylori is deep within the gastric mucus, an ecological niche where there are no other bacterial species. The use of molecular fingerprinting techniques, such as RAPD-PCR, to investigate strain diversity has revealed substantial sequence heterogeneity between strains from unrelated individuals [7] and that most infected individuals in developed countries harbor just one single H. pylori strain [7–9]. Mixed infections, while less common, have also been reported [8,9] and were found to relatively common in patients with gastro-duodenal symptoms in India [10]. However, the frequency of mixed H. pylori infections in populations with variable risk of H. pylori infection has not been investigated, and potential competition among strains during mixed infections has not been assessed. The diversity within the bacterial population in single infected hosts has been documented. Molecular
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fingerprinting studies using RAPD [11] with multiple single-colony isolates or cultures obtained from separate biopsy samples from single patients revealed that strains isolated from single patients were closely related, but there were subtle differences in the majority of the isolates. The variants have even been referred to as quasispecies of a single strain [12]. The most studied virulence factors of H. pylori are the cagA oncogene and toxin-encoding vacA gene. VacA induces vacuolation in infected cells [13], and CagA is associated with rearrangements of the actin cytoskeleton leading to phenotypic changes called the scattering or “hummingbird” phenotype [14,15]. VacA has two variable parts: The s (signal peptide) region that exists as allele s1 or s2 and the m (middle) region that occurs as m1 or m2 allelic type. All possible combinations of these regions (s1/m1, s1/m2, s2/m1, and s2/m2) have been reported in clinical isolates of H. pylori, and all, with exception of the s2/m1, alleles are common [16,17]. The allelic combinations s1/m1, s1/m2, and s2/m2 correspond with high, medium, and low toxin activity, respectively. In studies investigating the clinical relevance to vacA genotypes, the s1 type vacA allele was found to be associated with the presence of peptic ulcers [16]. However, m1VacA is cytotoxic to HeLa cells, and m2VacA induces vacuolization in primary cultured human gastric cells and in nongastric cell lines such as RK-13, but lacks cytotoxic activity in HeLa cells [18]. We hypothesize that the co-colonization of H. pylori permits the horizontal transfer of genetic material between strains, which may facilitate the emergence of new variants/subpopulations that have a selective advantage in a given human host over the primary infecting strain(s). To address this hypothesis, we aimed to characterize the genetic diversity in the H. pylori populations colonizing the stomachs of single human hosts from a population with a high rate of infection, in symptomatic and asymptomatic children and adults in Dhaka.
Materials and Methods Study Population This study was conducted at the International Center for Diarrhoeal Diseases Research, Bangladesh (ICDDR, B) field surveillance site at Nandipara, a periurban community near Dhaka, Bangladesh, during July 2005 to November 2007. Details of study population are as previously described [19]. The study was approved ICDDR, B ethical review committee (ERC) (Approval # 2005-010). Since 1985, Clinical Services Divisions (CSD), ICDDR, B, maintained a household listing of Nandipara and a database of 3000 population and used to provide routine
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clinical services through outpatient clinic. We used the database and infrastructure of CSD, ICDDR, B, for selection and enrollment of patients during the study period. One hundred and twelve families from Nandipara community were randomly selected for the study; the selected families including the family head were (i) invited to visit the outpatient clinics at Nandipara, (ii) approached to participate in the study, and (iii) screened for inclusion/exclusion criteria (exclusion criteria includes household with less than three children; concomitant disease; regular NSAID or corticosteroid use; treatment with antibiotics or proton-pump inhibitors for any household members in last 6 weeks; and pregnancy). Of the 112 families approached, fifty-five families (312 family members) were eligible for enrollment and agreed to provide a fecal sample for stool antigen testing. All family members of the selected families were screened for H. pylori infection by stool antigen test, and 279 subjects from 44 families were positive for stool antigen test and were included in the study (Figure S1). Parents and their children who tested positive were invited to admit to ICDDR, B research unit for preparation for upper GI endoscopy for biopsy or nasogastric intubation for gastric juice collection. Informed consent (parental consent in case of children below 18 years) was obtained from all subjects. Families were compensated for wage lose and transportation. All H. pylori stool antigen-positive subjects (irrespective of participation to upper GI endoscopy) were offered treatment with standard triple therapy.
Stool Antigen Test Stool samples were collected from each household and stored at 20 °C before analyzed for H. pylori antigen using HPSA immunoassay test (FemtoLab H. pylori Cnx, DakoCytomation Ltd, Cambridge, UK). Values greater than or equal to 0.150 and less than 0.150 were defined as positive and negative, respectively, according to the instructions of the manufacturers (the sensitivity and specificity of the test were 78.9 and 87.0%, respectively) [20].
Clinical Information Collection, Endoscopy and Culture of Biopsy Specimen and Gastric Juice Collection All subjects consented for endoscopic examination or for gastric juice collection were examined by a gastroenterologist, and information on clinical symptoms (gastrointestinal) was collected. During endoscopic examination, the gastroenterologist observed and recorded the gastric abnormalities identified. Subjects
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who reported epigastric or abdominal pain as well as acid eructation or heartburn were considered as symptomatic; otherwise, subjects were considered as asymptomatic. The procedure for endoscopy to collect biopsy specimen and gastric juice sample as well as their culture was performed as previously described [6]. Total smear from the primary plates (pooled isolates) and ten randomly selected single colonies from each biopsy specimen were subcultured on plates. Culture from each isolate (single colony and smear from primary plate) was stored at 86 °C in 0.5 mL of brucella broth with 15% glycerol until further study.
DNA Extraction and RAPD Chromosomal DNA was extracted by the CTAB (hexadecyl-trimethyl ammonium bromide) extraction method [21] from confluent brain–heart infusion agar plate cultures of 2010 single colonies and 128 total smear culture. All DNA extracted was quantified, and a suspension of equal concentration of DNA was prepared for RAPD analysis. The presence of single or mixed infection was determined by RAPD analysis of 2010 single colony isolated from 201 specimens from 128 patients (20 isolates from 73 patients, 10 isolates from 31 patients, and 10 isolates from 24 gastric juice samples). The purpose of the RAPD analysis was to examine whether the representative isolates from a patient are identical or not. RAPD was carried out in a final volume of 25 lL containing 10 ng template DNA, 100 pmol primer, 50 mmol/L MgCl2, 1 U of Taq DNA polymerase (Invitrogen, GibcoBRL, Carlsbad, CA, USA), 2.5 mmol/L deoxynucleoside triphosphates (TaKaRa dNTP Mixture, Takara Bio Inc., Otsu, Shiga, Japan). For the analysis of the RAPD fingerprinting, random primer 1281 (50 -AACGCGCAAC-30 ) (Invitrogen) was used for amplification of H. pylori DNA. A DNA Engineâ Peltier Thermal Cycler (BIORAD, Hercules, California, USA) was used for amplification. The cycling program was initiated with a denaturation step of 94 °C for 5 minutes followed by 45 cycles of 94 °C for 1 minutes, 36 °C for 90 seconds, 72 °C for 2 minutes, followed by a final incubation at 72 °C for 10 minutes. Ten microliter aliquots of product were electrophoresed in 2% agarose gels containing 0.5 lg/mL of ethidium bromide in the gel while using a 0.59 Tris-acetate-EDTA running buffer. The 100-bp DNA ladder (Invitrogen, Gibco BRL) was used as a size marker in all gels.
Multiplex PCR To explore the relationship between virulence factors and types of infection (single or mixed), we performed
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multiplex PCR analysis to identify the cagA, vacA m1 and m2, and s1 and s2 alleles in each 128 pool DNA samples. DNA extracted from pooled culture was analyzed for genotyping of the H. pylori by multiplex PCR using the protocol describe earlier [22]. Amplification was performed in a 25 ll of final volume consisting of 100-ng template DNA, 2.5 pmol of primers VAG-F and VAG-R, 25 pmol of primers VA1-F and VA1-R, 10 pmol of primers cag5c-F and cag3c-R as described earlier [22], 2.5 mmol/L of deoxynucleoside triphosphate (TaKaRa dNTP Mixture, TAKARA BIO INC., Otsu, Shiga, Japan), 1.5 U of Taq DNA polymerase (Invitrogen, GibcoBRL), and 50 mmol/L of MgCl2 in standard PCR buffer (Takara, Otsu, Shiga, Japan). Products were amplified under the following conditions: 3 minutes at 94 °C for initial denaturation followed by 35 cycles of 1 minutes at 94 °C, 1 minutes at 55 °C, and 1 minutes at 72 °C, with a final round of 10 minutes at 72 °C, in a DNA Engineâ Peltier Thermal Cycler (BIORAD).
Statistical Analysis The gel image of RAPD amplicons and multiplex PCR were analyzed by Kodak Digital ScienceTM 1D Image Analysis Software. The RAPD band pattern was analyzed by cluster analyses by WinSTAT software (R. Fitch Software, Staufen, Germany), and dendrograms were designed following the single-linkage method. In a cluster, the fingerprints with distances lower than or equal to 5 were considered as related and distances greater than 5 as unrelated. Single cluster was considered as single infection, and multiple clusters were recorded as mixed infection. The cagA/vacA status of H. pylori was assessed visually. To estimate the association between disease presentation, virulence factors, and nature of infection (single vs mixed), adjusted odds ratios (ORs) and 95% confidence intervals (95% CI) were calculated using unconditional logistic regression. Odds ratio (OR) was also adjusted for the confounding factors age and gender. The level of statistical significance was set at 0.05, and all tests were two-tailed. All analyses were performed with SPSS for Windows (version 17.0 SPSS Inc, Chicago, IL, USA).
Results Of the 279 individuals in 44 families, 174 (125 adult and 49 children) were available for endoscopic examination. The key reasons for nonparticipation were refusal to undergo an endoscopy or being unavailable on the selected days for the procedure. The characteristics of the enrolled subjects (gender, clinical presentation and source of H. pylori isolated, etc.), samples, and
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Table 1 The characteristics of 174 enrolled subjects; 275 samples and 201 isolates enrolled and analyzed in the study Variable
Number (%)
Gender (n = 174) Male 92 (52.9) Female 82 (47.1) Clinical presentation (n = 174) Symptomatic 83 (47.7) Asymptomatic 91 (52.3) H. pylori isolate origin (n = 275) Antrum 94/117 (80.3) Body 83/109 (76.1) Gastric juice 24/49 (48.9) Helicobacter status (n = 174) Positive 128 (73.6) Negative 46 (26.4) Type of infection in patients and variant/quasispecies of strains present in a single patient (n = 128) Single infection 51/128 (39.8) Mixed infection 77/128 (60.2) 2 strains 36/128 (28.1) 3 strains 26/128 (20.3) 4 strains 10/128 (7.8) 5 strains 5/128 (3.9)
isolates are summarized in Table 1. Approximately half of the enrolled subjects were males and the rest were females. Two-third of the enrolled subjects were culture positive for H. pylori and of those approximately 47.7% (83/174) were symptomatic and 52.3% (91/174) were asymptomatic. A total of 275 biopsies (117 from antrum, 109 from body, and 49 gastric juice specimens) were collected from these subjects. Among the 174 subjects and 275 biopsies/samples, 128 (73.5%) subjects and 201 (73%) samples (80.3%) (94/117) were culture positive for H. pylori, respectively. These positive samples originated from the antrum (83/109 positive, 76.15%), the body (83/109 positive, 76.1%), and the gastric juice (24/49 positive, 48.9%).
Identification of single or mixed infection The type of H. pylori infection (single or mixed infection) among 2010 single-colony isolates from 128 patients was analyzed (Fig. 1) by RADP. Dendrogram analysis of RAPD pattern revealed that 39.8% (51/128) had single infection and 60.2% (77/128) had mixed infection. 32.0% (41/128) patients had mixed infection with more than two types of strains (Table 1). Further the association between type of infection (single or mixed infection) and clinical presentation (symptomatic and asymptomatic) were analyzed, and the prevalence of mixed infection was significantly high in symptom-
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atic patients (53% vs 36.3%, P-value .005). The prevalence of mixed infection was also significantly higher in females compared to males (69.5% vs 52.2%) (p < .048, odds ratio 2.088; 95% CI 1.008–4.325) (Table 2). However, no differences were observed when patients were stratified by age groups (data not shown).
Virulence Factor and Types of Infection To explore the relationship between type of infection (single or mixed) and the presence and absence of cagA pathogenicity island and allelic combination of vacA, signal peptide (s) and middle region (m), pooled isolates from 128 subjects were analyzed by multiplex PCR (Fig. 2). Entirely, 75 of 128 pooled isolates (58.6%) were positive for cagA gene, and approximately twothird of patients with mixed infection were cagA positive compared to one-third in single infection (Table 3). The effect of mixed infection on the prevalence of cagA and vac genotype was further analyzed by multivariate logistic regression, and the odds ratio was adjusted for confounding factors age and sex. A high prevalence of H. pylori cagA gene in the subjects with mixed infection (OR 1.926, 95% CI, 0.829–4.472). Moreover, the prevalence of H. pylori vacA m1 (OR 1.514; 95% CI, 0.662– 3.466) was found moderately higher, and vacA s1 (OR 3.708; 95% CI, 1.317–10.441) allele was found significantly higher in subjects with mixed infection (Table S1). Not only that, vacA m1s1 (OR 4.546; 95% CI, 1.517–13.624), m1s2, and m2s1 allele combination (OR 11.449; 95% CI 1.020–128.545) was higher and m2s2 allele combination was lower in the subjects with mixed infection compared with the subjects infected by single strain of H. pylori (Table S1). There are no major differences observed between the adjusted odds ratios and the crude odds ratios.
Discussion The data presented here indicated a high prevalence of mixed infection in the study subjects from the lower socioeconomic area in Bangladesh. In addition, we found that the prevalence of mixed H. pylori infection was higher in females than males. We suggest that the high prevalence of mixed infection in the population of this area is driven by a higher rate of H. pylori transmission as a result of poor sanitation and fecal contamination of water. We additionally identified a significant relationship between mixed H. pylori infections and symptomatic infection (p < .05). This finding is broadly in keeping with a recent RAPD-based study which demonstrated multiple infections in 30 clinical patients with gastro-duodenal problems; however, the authors
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Table 2 The relationship between disease presentation (symptomatic and asymptomatic) and gender and nature of infection (infection with single or mixed strains of Helicobacter pylori) among 128 patients that were culture positive for H. pylori
Disease presentation Asymptomatic infection Symptomatic infection Gender Male Female
Single infection (%)
Mixed infection (%)
Odds ratio (95% CI)
P-value
35 (38.5) 16 (19.3)
33 (36.3) 44 (53.0)
1 2.917 (1.386–6.138)
.005
33 (47.8) 18 (30.5)
36 (52.2) 41 (69.5)
1 2.088 (1.008–4.325)
.048
Figure 1 Image displaying examples of cluster analyses among the fingerprints of Helicobacter pylori isolates from the subjects infected with single strain and mixed strains. The relationship between different isolates of H. pylori from a single sample was determined by RAPD image analysis by WinSTAT. The distances are measured by single-linkage clustering method. Isolates from patient 26B fall under two clusters and are labeled as mixed infection, whereas all isolates from patient 25A fall under one cluster and are labeled as single infection.
did not compare this population with asymptomatic subjects [10]. We predict that a high mixed infection rate is a risk factor for the development of disease symptoms and that strain of mixing of H. pylori facilitates the circulation of virulence genes. A high degree of genetic variation among H. pylori isolates is well established, yet the mechanism by which this variation is generated is not well understood. Persistent infection occurs as a result of colonization of founder H. pylori strain in the gastric mucosa [23]. As a founder strain spreads throughout the stomach, it encounters different selective pressures. Thus, to
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survive and to meet these challenges, H. pylori is able to generate genetic diversity within the population [24] and to adapt to this changing environment. Kersulyte et al. found two different H. pylori strains from a single Lithuanian patient. Analyzing the RAPD band pattern, the subtle differences in five isolates of single H. pylori strain were observed and they suggested this as genetic variants of the original strain that had arisen during chronic infection. It was also demonstrated that recombination had occurred between both the strains on at least six occasions, thus accounting for the observed heterogeneity [25].
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Table 3 The cagA and vacA status of the study subjects and their distribution in subjects infected by single and mixed strain of Helicobacter pylori Status
Single infection (%)
Mixed infection (%)
cagA (+ve) cagA (ve) vacA m1 vacA m2 vacA m1 & m2 vacA m negative vacA s1 vacA s2 vacA s1 & s2 VacA s negative vacA genotype vacA m1s1 vacA m1s2 vacA m2s1 vacA m2s2 vacA s1 vacA s2 vacA m1s1 & m2s2 vacA negative
25 26 25 8 1 17 33 16 1 1
(49.0) (51.0) (49.0) (15.7) (2.0) (33.3) (64.7) (31.3) (2.0) (2.0)
50 27 45 9 1 22 66 9 1 1
(64.9) (35.1) (58.4) (11.7) (1.3) (28.6) (85.7) (11.7) (1.3) (1.3)
25 0 2 6 6 10 1 1
(49.0)
43 2 5 4 18 3 1 1
(55.8) (2.6) (6.5) (5.2) (23.4) (3.9) (1.3) (1.3)
(3.9) (11.8) (11.8) (19.6) (2.0) (2.0)
The high degree of genomic diversity among H. pylori strains in one individual, which can be due to mechanisms such as free recombination [26] or coinfection with mixed H. pylori strains [27,28], has been confirmed by AFLP analysis [29]. In the present study, we found that some of the subjects are infected with mixed strains, have similar band pattern with either emergence of new bands or lack of some bands. This
indicates that there may be some kind of recombination occurred in H. pylori for the adaptation in the gastric hostile environment. We found high mixed infection rate in our study population. We speculate that mixed infection was generated by frequent recombination; this is supported by the observation that patients can be simultaneously colonized with strains that differ in their randomly amplified polymorphic DNA pattern [30,31]. Even if most individuals were infected with H. pylori early in childhood and harbored their individual strain thereafter, occasional mixed colonization and transformation would lead to extensive genetic rearrangements through time. H. pylori sequences have a uniquely high homoplasy ratio [26], an indirect measure of genetic shuffling. These observations are thought to result from horizontal genetic exchange during mixed colonization by unrelated strains. H. pylori is characterized by the highly unusual combination of high import frequency and low import size. Recombination is so frequent that appreciable fractions of the entire genome are exchanged during the colonization of a single human, resulting in a highly flexible genome content and frequent shuffling of sequence polymorphisms throughout the local gene pool [32]. For fingerprinting of the H. pylori DNA, we used one primer (1281); Akopyanz et al. [7] showed that each of 64 independent H. pylori isolates was distinguishable even with a single RAPD primer. We performed genotyping of the pool DNA sample of H. pylori for virulence marker cagA, vacA m1, m2, s1, and s2 to understand their relation with mixed infection status of the study subjects. The prevalence of
Figure 2 Typical example of gel-based analyses of vacA/cagA, status. Multiplex PCR of the Helicobacter pylori DNA from 128 smears from primary culture was to be amplified for vacA m1, vacA m2, vacA s1 and vacA s2 alleles and cagA gene. The bands in lane 1 is designated positive for vacA s1, cagA and vacA m1. A 100 bp DNA marker is used as size marker (M).
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cagA gene and the vacA virulence allele m1 and s1 was higher in the subjects with mixed infection. High prevalence of virulence properties, that is, m1s1 and m2s1 allele combination was observed from subjects with mixed infection, whereas the lower virulence property of m2s2 allele combination is higher in the subjects with single infection. There is an unexpectedly high prevalence of intermediate virulence factors (i.e., m1s2 and m2s1) is found in the subjects with mixed infection even higher than the high virulence factors (i.e., m1s1) which may be the error due to the small sample size of the isolate of intermediate virulence. However, we do not have any direct evidence for the emergence of virulent genes due to the presence of mixed strains of H. pylori. As a developing country, it is expected that Bangladeshi population will have high prevalence of H. pylori infection. But in this study we have found a high prevalence of mixed infection of H. pylori in a lower socioeconomic area of Bangladesh. This may have occurred due to lower socioeconomic condition, poor hygiene, intrafamilial transmission of H. pylori, mostly from mother to child [6] and chronic colonization. There is a high prevalence of H. pylori with virulence marker, that is, cagA and vacA in our study subjects. Our study presents a scenario in which hosts infected by mixed strains of H. pylori are highly prone to symptoms associated with gastric acidity, and the H. pylori strains are highly virulent. It might be possible that chronic co-colonization of mixed strains of H. pylori facilitates the virulence gene transfer horizontally and contributes in symptoms development. One of the limitations of our study is the population from which the samples were collected (population from low socioeconomic class) and the number of subjects analyzed. Further studies are necessary to understand the risk factors and behavioral determinants responsible for mixed infection of H. pylori and their relationship with virulence gene expression.
Acknowledgements and Disclosures This research was conducted in the International Centre for Diarrhoeal Diseases Research, Bangladesh (ICDDR, B), which is supported by countries and agencies that share its concern for the health problems of developing countries. This program is entirely supported by SIDA/SAREC (Grant no. GR-00384, Research Protocol Number 2005-010). ICDDR, B, acknowledges with gratitude the commitment of SIDA to the Centre’s research efforts. The authors thank Dr. Habiba Yeasmin, Dr. Shamima Sultana and the field staffs of Nandipara Clinic of ICDDR, B for their help in the study. The authors also thank Dr. Dipak kumar mitra and Gowranga Kumar Paul for their help in statistical analyses and Dr. Steven Baker for reviewing of the manuscript.
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Competing interests: the authors declare no competing interests.
References 1 Kauser F, Khan AA, Hussain MA, et al. The cag pathogenicity island of Helicobacter pylori is disrupted in the majority of patient isolates from different human populations. J Clin Microbiol 2004;42: 5302–8. 2 Brenner H, Weyermann M, Rothenbacher D. Clustering of Helicobacter pylori infection in couples: differences between high- and low-prevalence population groups. Ann Epidemiol 2006;16:516–20. 3 Drumm B, Perez-Perez GI, Blaser MJ, Sherman PM. Intrafamilial clustering of Helicobacter pylori infection. N Engl J Med 1990;322:359–63. 4 Kivi M, Johansson AL, Reilly M, Tindberg Y. Helicobacter pylori status in family members as risk factors for infection in children. Epidemiol Infect 2005;133:645–52. 5 Kivi M, Tindberg Y, Sorberg M, Casswall TH, Befrits R, Hellstrom PM, et al. Concordance of Helicobacter pylori strains within families. J Clin Microbiol 2003;41:5604–8. 6 Nahar S, Kibria KM, Hossain ME, Sultana J, Sarker SA, Engstrand L, et al. Evidence of intra-familial transmission of Helicobacter pylori by PCR-based RAPD fingerprinting in Bangladesh. Eur J Clin Microbiol Infect Dis 2009;28(7):767– 73. 7 Akopyanz N, Bukanov NO, Westblom TU, Kresovich S, Berg DE. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res 1992;11:5137–42. 8 Fujimoto S, Marshall B, Blaser MJ. PCR-based restriction fragment length polymorphism typing of Helicobacter pylori. J Clin Microbiol 1994;32:331–4. 9 Owen RJ, Bell GD, Desai M, Moreno M, Gant PW, Jones PH, et al. Biotype and molecular fingerprints of metronidazoleresistant strains of Helicobacter pylori from antral gastric mucosa. J Med Microbiol 1993;38:6–12. 10 Patra R, Chattopadhyay S, De R, Ghosh P, Ganguly M, Chowdhury A, et al. Multiple infection and microdiversity among Helicobacter pylori isolates in a single host in India. PLoS ONE 2012;7:e43370. 11 Wong BC, Wang WH, Berg DE, Fung FM, Wong KW, Wong WM, et al. High prevalence of mixed infections by Helicobacter pylori in Hong Kong: metronidazole sensitivity and overall genotype. Aliment Pharmacol Ther 2001;15:493–503. 12 Kuipers EJ, Israel DA, Kusters JG, Gerrits MM, Weel J, van Der Ende A, et al. Quasispecies development of Helicobacter pylori observed in paired isolates obtained years apart from the same host. J Infect Dis 2000;181:273–82. 13 Reyrat JM, Pelicic V, Papini E, Montecucco C, Rappuoli R, Telford JL. Towards deciphering the Helicobacter pylori cytotoxin. Mol Microbiol 1999;34:197–204. 14 Backert S, Moese S, Selbach M, Brinkmann V, Meyer TF. Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells. Mol Microbiol 2001;42:631–44. 15 Segal ED, Cha J, Lo J, Falkow S, Tompkins LS. Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori. Proc Natl Acad Sci USA 1999;96:14559–64.
403
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16 Atherton JC, Cao P, Peek RM Jr, Tummuru MK, Blaser MJ, Cover TL. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration. J Biol Chem 1995;270:17771– 7. 17 van Doorn LJ, Figueiredo C, Sanna R, Plaisier A, Schneeberger P, de Boer W, et al. Clinical relevance of the cagA, vacA, and iceA status of Helicobacter pylori. Gastroenterology 1998;115:58– 66. 18 Pagliaccia C, de Bernard M, Lupetti P, Ji X, Burroni D, Cover TL, et al. The m2 form of the Helicobacter pylori cytotoxin has cell type-specific vacuolating activity. Proc Natl Acad Sci USA 1998;95:10212–7. 19 Sarker SA, Mahalanabis D, Hildebrand P, Rahaman MM, Bardhan PK, Fuchs G, et al. Helicobacter pylori: prevalence, transmission, and serum pepsinogen II concentrations in children of a poor periurban community in Bangladesh. Clin Infect Dis 1997;25:990–5. 20 Korkmaz H, Kesli R, Karabagli P, Terzi Y. Comparison of the diagnostic accuracy of five different stool antigen tests for the diagnosis of Helicobacter pylori infection. Helicobacter 2013;18:384–91. 21 Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds). Current protocols in molecular biology. New York, NY: Greene Publishing and Wiley-Interscience, 1993. 22 Chattopadhyay S, Patra R, Ramamurthy T, Chowdhury A, Santra A, Dhali GK, et al. Multiplex PCR assay for rapid detection and genotyping of Helicobacter pylori directly from biopsy specimens. J Clin Microbiol 2004;42:2821–4. 23 Marshall DG, Dundon WG, Beesley SM, Smyth CJ. Helicobacter pylori–a conundrum of genetic diversity. Microbiology 1998;144 (Pt.11):2925–39. 24 Taddei F, Matic I, Godelle B, Radman M. To be a mutator, or how pathogenic and commensal bacteria can evolve rapidly. Trends Microbiol 1997;5:427–8; discussion 8–9. 25 Kersulyte D, Chalkauskas H, Berg DE. Emergence of recombinant strains of Helicobacter pylori during human infection. Mol Microbiol 1999;31:31–43. 26 Suerbaum S, Smith JM, Bapumia K, Morelli G, Smith NH, Kunstmann E, et al. Free recombination within Helicobacter pylori. Proc Natl Acad Sci USA 1998;95:12619–24.
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27 Cellini L, Di Campli E, Di Candia M, Marzio L. Molecular fingerprinting of Helicobacter pylori strains from duodenal ulcer patients. Lett Appl Microbiol 2003;36:222–6. 28 Prewett EJ, Bickley J, Owen RJ, Pounder RE. DNA patterns of Helicobacter pylori isolated from gastric antrum, body, and duodenum. Gastroenterology 1992;102:829–33. 29 Cellini L, Grande R, Di Campli E, Di Bartolomeo S, Capodicasa S, Marzio L. Analysis of genetic variability, antimicrobial susceptibility and virulence markers in Helicobacter pylori identified in Central Italy. Scand J Gastroenterol 2006;41:280– 7. 30 Taylor NS, Fox JG, Akopyants NS, Berg DE, Thompson N, Shames B, et al. Long-term colonization with single and multiple strains of Helicobacter pylori assessed by DNA fingerprinting. J Clin Microbiol 1995;33:918–23. 31 Berg DE, Gilman RH, Lelwala-Guruge J, Srivastava K, Valdez Y, Watanabe J, et al. Helicobacter pylori populations in Peruvian patients. Clin Infect Dis 1997;25:996–1002. 32 Falush D, Kraft C, Taylor NS, Correa P, Fox JG, Achtman M, et al. Recombination and mutation during long-term gastric colonization by Helicobacter pylori: estimates of clock rates, recombination size, and minimal age. Proc Natl Acad Sci USA 2001;98:15056–61.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 The approaches for population selection and analysis of study. Table S1 Odd ratios for the effect of mixed infection of H. pylori on the prevalence of cagA and vacA genotype of 128 pooled isolates. Single infection was considered as reference category. To understand the effect of mixed infection in the prevalence of virulence genotype, the variables were grouped into two or three classes, high (highly virulent), intermediate (moderately virulent) and low (less or avirulent).
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The Prevalence of Mixed Helicobacter pylori Infections in Symptomatic and Asymptomatic Subjects in Dhaka, Bangladesh Khandoker Mohammad K. Kibria,* Md Enayet Hossain,† Jinath Sultana,‡ Shafiqul A. Sarker,† Pradip Kumar Bardhan,† Motiur Rahman§ and Shamsun Nahar† *Mawlana Bhashani Science and Technology University, Shantosh, Tangail, Bangladesh, †International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh, ‡North-South University, Dhaka, Bangladesh, §Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam
Keywords Helicobacter pylori, Bangladesh, mixed infection, cagA, vacA. Reprint requests to: Shamsun Nahar, Enteric Microbiology Laboratory, Laboratory Sciences Division, ICDDR, B GPO Box-128, Dhaka 1000, Bangladesh. E-mail: [email protected]
Abstract Background: Helicobacter pylori is a highly genetically diverse bacterial species, which can persist in the gastric environment for decades. Recent studies have shown that single infections predominate in developed countries, whereas mixed infections are more prevalent in developing countries. Mixed infections of this bacterium may be important for adaptation to the hostile gastric environment and may facilitate dyspeptic symptoms. Materials and Methods: To calculate the prevalence of mixed infections in symptomatic and asymptomatic subjects, 2010 H. pylori isolates collected from 83 symptomatic and 91 asymptomatic subjects from Dhaka, Bangladesh, were analyzed by (i) random amplified polymorphic DNA fingerprinting (RAPD) and (ii) multiplex PCR amplification for cagA and vacA virulence gene alleles. Results: The overall prevalence of mixed H. pylori infection was 60.15% (77/128), indicating substantial co-colonization in this population. We additionally found that symptomatic subjects (53%) had a significantly higher rate of mixed infection than asymptomatic individuals (36.3%) (p = .016) and that the prevalence of the cagA and vacA and vacA m1/s1 and vacA m2/s1 alleles were higher in subjects with mixed infection. Conclusion: Our findings suggest that an increased diversity of the H. pylori strains in the gastric environment may contribute to the development of disease symptoms.
Helicobacter pylori is one of the most genetically diverse known bacterial species. Colonization of the stomach with H. pylori is extremely common in humans, and although generally asymptomatic, infection can cause abdominal pain, dyspepsia, peptic ulcers, and gastric cancer. Infection rates of H. pylori within human populations vary from between 20 and 30% in developed countries to between 80 and 90% in developing countries [1]. The population structure of H. pylori is atypical with respect to many bacterial species as a consequence of vertical transmission of the bacteria within families, predominantly from parents to child, or between children via oral–oral, gastric–oral, or fecal–oral transmission [2–5]. Our recent work, in Dhaka, Bangladesh, added to these date and indicated that mother to child transmission of H. pylori occurs mainly in the early childhood [6].
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The natural habitat of H. pylori is deep within the gastric mucus, an ecological niche where there are no other bacterial species. The use of molecular fingerprinting techniques, such as RAPD-PCR, to investigate strain diversity has revealed substantial sequence heterogeneity between strains from unrelated individuals [7] and that most infected individuals in developed countries harbor just one single H. pylori strain [7–9]. Mixed infections, while less common, have also been reported [8,9] and were found to relatively common in patients with gastro-duodenal symptoms in India [10]. However, the frequency of mixed H. pylori infections in populations with variable risk of H. pylori infection has not been investigated, and potential competition among strains during mixed infections has not been assessed. The diversity within the bacterial population in single infected hosts has been documented. Molecular
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fingerprinting studies using RAPD [11] with multiple single-colony isolates or cultures obtained from separate biopsy samples from single patients revealed that strains isolated from single patients were closely related, but there were subtle differences in the majority of the isolates. The variants have even been referred to as quasispecies of a single strain [12]. The most studied virulence factors of H. pylori are the cagA oncogene and toxin-encoding vacA gene. VacA induces vacuolation in infected cells [13], and CagA is associated with rearrangements of the actin cytoskeleton leading to phenotypic changes called the scattering or “hummingbird” phenotype [14,15]. VacA has two variable parts: The s (signal peptide) region that exists as allele s1 or s2 and the m (middle) region that occurs as m1 or m2 allelic type. All possible combinations of these regions (s1/m1, s1/m2, s2/m1, and s2/m2) have been reported in clinical isolates of H. pylori, and all, with exception of the s2/m1, alleles are common [16,17]. The allelic combinations s1/m1, s1/m2, and s2/m2 correspond with high, medium, and low toxin activity, respectively. In studies investigating the clinical relevance to vacA genotypes, the s1 type vacA allele was found to be associated with the presence of peptic ulcers [16]. However, m1VacA is cytotoxic to HeLa cells, and m2VacA induces vacuolization in primary cultured human gastric cells and in nongastric cell lines such as RK-13, but lacks cytotoxic activity in HeLa cells [18]. We hypothesize that the co-colonization of H. pylori permits the horizontal transfer of genetic material between strains, which may facilitate the emergence of new variants/subpopulations that have a selective advantage in a given human host over the primary infecting strain(s). To address this hypothesis, we aimed to characterize the genetic diversity in the H. pylori populations colonizing the stomachs of single human hosts from a population with a high rate of infection, in symptomatic and asymptomatic children and adults in Dhaka.
Materials and Methods Study Population This study was conducted at the International Center for Diarrhoeal Diseases Research, Bangladesh (ICDDR, B) field surveillance site at Nandipara, a periurban community near Dhaka, Bangladesh, during July 2005 to November 2007. Details of study population are as previously described [19]. The study was approved ICDDR, B ethical review committee (ERC) (Approval # 2005-010). Since 1985, Clinical Services Divisions (CSD), ICDDR, B, maintained a household listing of Nandipara and a database of 3000 population and used to provide routine
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clinical services through outpatient clinic. We used the database and infrastructure of CSD, ICDDR, B, for selection and enrollment of patients during the study period. One hundred and twelve families from Nandipara community were randomly selected for the study; the selected families including the family head were (i) invited to visit the outpatient clinics at Nandipara, (ii) approached to participate in the study, and (iii) screened for inclusion/exclusion criteria (exclusion criteria includes household with less than three children; concomitant disease; regular NSAID or corticosteroid use; treatment with antibiotics or proton-pump inhibitors for any household members in last 6 weeks; and pregnancy). Of the 112 families approached, fifty-five families (312 family members) were eligible for enrollment and agreed to provide a fecal sample for stool antigen testing. All family members of the selected families were screened for H. pylori infection by stool antigen test, and 279 subjects from 44 families were positive for stool antigen test and were included in the study (Figure S1). Parents and their children who tested positive were invited to admit to ICDDR, B research unit for preparation for upper GI endoscopy for biopsy or nasogastric intubation for gastric juice collection. Informed consent (parental consent in case of children below 18 years) was obtained from all subjects. Families were compensated for wage lose and transportation. All H. pylori stool antigen-positive subjects (irrespective of participation to upper GI endoscopy) were offered treatment with standard triple therapy.
Stool Antigen Test Stool samples were collected from each household and stored at 20 °C before analyzed for H. pylori antigen using HPSA immunoassay test (FemtoLab H. pylori Cnx, DakoCytomation Ltd, Cambridge, UK). Values greater than or equal to 0.150 and less than 0.150 were defined as positive and negative, respectively, according to the instructions of the manufacturers (the sensitivity and specificity of the test were 78.9 and 87.0%, respectively) [20].
Clinical Information Collection, Endoscopy and Culture of Biopsy Specimen and Gastric Juice Collection All subjects consented for endoscopic examination or for gastric juice collection were examined by a gastroenterologist, and information on clinical symptoms (gastrointestinal) was collected. During endoscopic examination, the gastroenterologist observed and recorded the gastric abnormalities identified. Subjects
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who reported epigastric or abdominal pain as well as acid eructation or heartburn were considered as symptomatic; otherwise, subjects were considered as asymptomatic. The procedure for endoscopy to collect biopsy specimen and gastric juice sample as well as their culture was performed as previously described [6]. Total smear from the primary plates (pooled isolates) and ten randomly selected single colonies from each biopsy specimen were subcultured on plates. Culture from each isolate (single colony and smear from primary plate) was stored at 86 °C in 0.5 mL of brucella broth with 15% glycerol until further study.
DNA Extraction and RAPD Chromosomal DNA was extracted by the CTAB (hexadecyl-trimethyl ammonium bromide) extraction method [21] from confluent brain–heart infusion agar plate cultures of 2010 single colonies and 128 total smear culture. All DNA extracted was quantified, and a suspension of equal concentration of DNA was prepared for RAPD analysis. The presence of single or mixed infection was determined by RAPD analysis of 2010 single colony isolated from 201 specimens from 128 patients (20 isolates from 73 patients, 10 isolates from 31 patients, and 10 isolates from 24 gastric juice samples). The purpose of the RAPD analysis was to examine whether the representative isolates from a patient are identical or not. RAPD was carried out in a final volume of 25 lL containing 10 ng template DNA, 100 pmol primer, 50 mmol/L MgCl2, 1 U of Taq DNA polymerase (Invitrogen, GibcoBRL, Carlsbad, CA, USA), 2.5 mmol/L deoxynucleoside triphosphates (TaKaRa dNTP Mixture, Takara Bio Inc., Otsu, Shiga, Japan). For the analysis of the RAPD fingerprinting, random primer 1281 (50 -AACGCGCAAC-30 ) (Invitrogen) was used for amplification of H. pylori DNA. A DNA Engineâ Peltier Thermal Cycler (BIORAD, Hercules, California, USA) was used for amplification. The cycling program was initiated with a denaturation step of 94 °C for 5 minutes followed by 45 cycles of 94 °C for 1 minutes, 36 °C for 90 seconds, 72 °C for 2 minutes, followed by a final incubation at 72 °C for 10 minutes. Ten microliter aliquots of product were electrophoresed in 2% agarose gels containing 0.5 lg/mL of ethidium bromide in the gel while using a 0.59 Tris-acetate-EDTA running buffer. The 100-bp DNA ladder (Invitrogen, Gibco BRL) was used as a size marker in all gels.
Multiplex PCR To explore the relationship between virulence factors and types of infection (single or mixed), we performed
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multiplex PCR analysis to identify the cagA, vacA m1 and m2, and s1 and s2 alleles in each 128 pool DNA samples. DNA extracted from pooled culture was analyzed for genotyping of the H. pylori by multiplex PCR using the protocol describe earlier [22]. Amplification was performed in a 25 ll of final volume consisting of 100-ng template DNA, 2.5 pmol of primers VAG-F and VAG-R, 25 pmol of primers VA1-F and VA1-R, 10 pmol of primers cag5c-F and cag3c-R as described earlier [22], 2.5 mmol/L of deoxynucleoside triphosphate (TaKaRa dNTP Mixture, TAKARA BIO INC., Otsu, Shiga, Japan), 1.5 U of Taq DNA polymerase (Invitrogen, GibcoBRL), and 50 mmol/L of MgCl2 in standard PCR buffer (Takara, Otsu, Shiga, Japan). Products were amplified under the following conditions: 3 minutes at 94 °C for initial denaturation followed by 35 cycles of 1 minutes at 94 °C, 1 minutes at 55 °C, and 1 minutes at 72 °C, with a final round of 10 minutes at 72 °C, in a DNA Engineâ Peltier Thermal Cycler (BIORAD).
Statistical Analysis The gel image of RAPD amplicons and multiplex PCR were analyzed by Kodak Digital ScienceTM 1D Image Analysis Software. The RAPD band pattern was analyzed by cluster analyses by WinSTAT software (R. Fitch Software, Staufen, Germany), and dendrograms were designed following the single-linkage method. In a cluster, the fingerprints with distances lower than or equal to 5 were considered as related and distances greater than 5 as unrelated. Single cluster was considered as single infection, and multiple clusters were recorded as mixed infection. The cagA/vacA status of H. pylori was assessed visually. To estimate the association between disease presentation, virulence factors, and nature of infection (single vs mixed), adjusted odds ratios (ORs) and 95% confidence intervals (95% CI) were calculated using unconditional logistic regression. Odds ratio (OR) was also adjusted for the confounding factors age and gender. The level of statistical significance was set at 0.05, and all tests were two-tailed. All analyses were performed with SPSS for Windows (version 17.0 SPSS Inc, Chicago, IL, USA).
Results Of the 279 individuals in 44 families, 174 (125 adult and 49 children) were available for endoscopic examination. The key reasons for nonparticipation were refusal to undergo an endoscopy or being unavailable on the selected days for the procedure. The characteristics of the enrolled subjects (gender, clinical presentation and source of H. pylori isolated, etc.), samples, and
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Table 1 The characteristics of 174 enrolled subjects; 275 samples and 201 isolates enrolled and analyzed in the study Variable
Number (%)
Gender (n = 174) Male 92 (52.9) Female 82 (47.1) Clinical presentation (n = 174) Symptomatic 83 (47.7) Asymptomatic 91 (52.3) H. pylori isolate origin (n = 275) Antrum 94/117 (80.3) Body 83/109 (76.1) Gastric juice 24/49 (48.9) Helicobacter status (n = 174) Positive 128 (73.6) Negative 46 (26.4) Type of infection in patients and variant/quasispecies of strains present in a single patient (n = 128) Single infection 51/128 (39.8) Mixed infection 77/128 (60.2) 2 strains 36/128 (28.1) 3 strains 26/128 (20.3) 4 strains 10/128 (7.8) 5 strains 5/128 (3.9)
isolates are summarized in Table 1. Approximately half of the enrolled subjects were males and the rest were females. Two-third of the enrolled subjects were culture positive for H. pylori and of those approximately 47.7% (83/174) were symptomatic and 52.3% (91/174) were asymptomatic. A total of 275 biopsies (117 from antrum, 109 from body, and 49 gastric juice specimens) were collected from these subjects. Among the 174 subjects and 275 biopsies/samples, 128 (73.5%) subjects and 201 (73%) samples (80.3%) (94/117) were culture positive for H. pylori, respectively. These positive samples originated from the antrum (83/109 positive, 76.15%), the body (83/109 positive, 76.1%), and the gastric juice (24/49 positive, 48.9%).
Identification of single or mixed infection The type of H. pylori infection (single or mixed infection) among 2010 single-colony isolates from 128 patients was analyzed (Fig. 1) by RADP. Dendrogram analysis of RAPD pattern revealed that 39.8% (51/128) had single infection and 60.2% (77/128) had mixed infection. 32.0% (41/128) patients had mixed infection with more than two types of strains (Table 1). Further the association between type of infection (single or mixed infection) and clinical presentation (symptomatic and asymptomatic) were analyzed, and the prevalence of mixed infection was significantly high in symptom-
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atic patients (53% vs 36.3%, P-value .005). The prevalence of mixed infection was also significantly higher in females compared to males (69.5% vs 52.2%) (p < .048, odds ratio 2.088; 95% CI 1.008–4.325) (Table 2). However, no differences were observed when patients were stratified by age groups (data not shown).
Virulence Factor and Types of Infection To explore the relationship between type of infection (single or mixed) and the presence and absence of cagA pathogenicity island and allelic combination of vacA, signal peptide (s) and middle region (m), pooled isolates from 128 subjects were analyzed by multiplex PCR (Fig. 2). Entirely, 75 of 128 pooled isolates (58.6%) were positive for cagA gene, and approximately twothird of patients with mixed infection were cagA positive compared to one-third in single infection (Table 3). The effect of mixed infection on the prevalence of cagA and vac genotype was further analyzed by multivariate logistic regression, and the odds ratio was adjusted for confounding factors age and sex. A high prevalence of H. pylori cagA gene in the subjects with mixed infection (OR 1.926, 95% CI, 0.829–4.472). Moreover, the prevalence of H. pylori vacA m1 (OR 1.514; 95% CI, 0.662– 3.466) was found moderately higher, and vacA s1 (OR 3.708; 95% CI, 1.317–10.441) allele was found significantly higher in subjects with mixed infection (Table S1). Not only that, vacA m1s1 (OR 4.546; 95% CI, 1.517–13.624), m1s2, and m2s1 allele combination (OR 11.449; 95% CI 1.020–128.545) was higher and m2s2 allele combination was lower in the subjects with mixed infection compared with the subjects infected by single strain of H. pylori (Table S1). There are no major differences observed between the adjusted odds ratios and the crude odds ratios.
Discussion The data presented here indicated a high prevalence of mixed infection in the study subjects from the lower socioeconomic area in Bangladesh. In addition, we found that the prevalence of mixed H. pylori infection was higher in females than males. We suggest that the high prevalence of mixed infection in the population of this area is driven by a higher rate of H. pylori transmission as a result of poor sanitation and fecal contamination of water. We additionally identified a significant relationship between mixed H. pylori infections and symptomatic infection (p < .05). This finding is broadly in keeping with a recent RAPD-based study which demonstrated multiple infections in 30 clinical patients with gastro-duodenal problems; however, the authors
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Table 2 The relationship between disease presentation (symptomatic and asymptomatic) and gender and nature of infection (infection with single or mixed strains of Helicobacter pylori) among 128 patients that were culture positive for H. pylori
Disease presentation Asymptomatic infection Symptomatic infection Gender Male Female
Single infection (%)
Mixed infection (%)
Odds ratio (95% CI)
P-value
35 (38.5) 16 (19.3)
33 (36.3) 44 (53.0)
1 2.917 (1.386–6.138)
.005
33 (47.8) 18 (30.5)
36 (52.2) 41 (69.5)
1 2.088 (1.008–4.325)
.048
Figure 1 Image displaying examples of cluster analyses among the fingerprints of Helicobacter pylori isolates from the subjects infected with single strain and mixed strains. The relationship between different isolates of H. pylori from a single sample was determined by RAPD image analysis by WinSTAT. The distances are measured by single-linkage clustering method. Isolates from patient 26B fall under two clusters and are labeled as mixed infection, whereas all isolates from patient 25A fall under one cluster and are labeled as single infection.
did not compare this population with asymptomatic subjects [10]. We predict that a high mixed infection rate is a risk factor for the development of disease symptoms and that strain of mixing of H. pylori facilitates the circulation of virulence genes. A high degree of genetic variation among H. pylori isolates is well established, yet the mechanism by which this variation is generated is not well understood. Persistent infection occurs as a result of colonization of founder H. pylori strain in the gastric mucosa [23]. As a founder strain spreads throughout the stomach, it encounters different selective pressures. Thus, to
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survive and to meet these challenges, H. pylori is able to generate genetic diversity within the population [24] and to adapt to this changing environment. Kersulyte et al. found two different H. pylori strains from a single Lithuanian patient. Analyzing the RAPD band pattern, the subtle differences in five isolates of single H. pylori strain were observed and they suggested this as genetic variants of the original strain that had arisen during chronic infection. It was also demonstrated that recombination had occurred between both the strains on at least six occasions, thus accounting for the observed heterogeneity [25].
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Table 3 The cagA and vacA status of the study subjects and their distribution in subjects infected by single and mixed strain of Helicobacter pylori Status
Single infection (%)
Mixed infection (%)
cagA (+ve) cagA (ve) vacA m1 vacA m2 vacA m1 & m2 vacA m negative vacA s1 vacA s2 vacA s1 & s2 VacA s negative vacA genotype vacA m1s1 vacA m1s2 vacA m2s1 vacA m2s2 vacA s1 vacA s2 vacA m1s1 & m2s2 vacA negative
25 26 25 8 1 17 33 16 1 1
(49.0) (51.0) (49.0) (15.7) (2.0) (33.3) (64.7) (31.3) (2.0) (2.0)
50 27 45 9 1 22 66 9 1 1
(64.9) (35.1) (58.4) (11.7) (1.3) (28.6) (85.7) (11.7) (1.3) (1.3)
25 0 2 6 6 10 1 1
(49.0)
43 2 5 4 18 3 1 1
(55.8) (2.6) (6.5) (5.2) (23.4) (3.9) (1.3) (1.3)
(3.9) (11.8) (11.8) (19.6) (2.0) (2.0)
The high degree of genomic diversity among H. pylori strains in one individual, which can be due to mechanisms such as free recombination [26] or coinfection with mixed H. pylori strains [27,28], has been confirmed by AFLP analysis [29]. In the present study, we found that some of the subjects are infected with mixed strains, have similar band pattern with either emergence of new bands or lack of some bands. This
indicates that there may be some kind of recombination occurred in H. pylori for the adaptation in the gastric hostile environment. We found high mixed infection rate in our study population. We speculate that mixed infection was generated by frequent recombination; this is supported by the observation that patients can be simultaneously colonized with strains that differ in their randomly amplified polymorphic DNA pattern [30,31]. Even if most individuals were infected with H. pylori early in childhood and harbored their individual strain thereafter, occasional mixed colonization and transformation would lead to extensive genetic rearrangements through time. H. pylori sequences have a uniquely high homoplasy ratio [26], an indirect measure of genetic shuffling. These observations are thought to result from horizontal genetic exchange during mixed colonization by unrelated strains. H. pylori is characterized by the highly unusual combination of high import frequency and low import size. Recombination is so frequent that appreciable fractions of the entire genome are exchanged during the colonization of a single human, resulting in a highly flexible genome content and frequent shuffling of sequence polymorphisms throughout the local gene pool [32]. For fingerprinting of the H. pylori DNA, we used one primer (1281); Akopyanz et al. [7] showed that each of 64 independent H. pylori isolates was distinguishable even with a single RAPD primer. We performed genotyping of the pool DNA sample of H. pylori for virulence marker cagA, vacA m1, m2, s1, and s2 to understand their relation with mixed infection status of the study subjects. The prevalence of
Figure 2 Typical example of gel-based analyses of vacA/cagA, status. Multiplex PCR of the Helicobacter pylori DNA from 128 smears from primary culture was to be amplified for vacA m1, vacA m2, vacA s1 and vacA s2 alleles and cagA gene. The bands in lane 1 is designated positive for vacA s1, cagA and vacA m1. A 100 bp DNA marker is used as size marker (M).
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cagA gene and the vacA virulence allele m1 and s1 was higher in the subjects with mixed infection. High prevalence of virulence properties, that is, m1s1 and m2s1 allele combination was observed from subjects with mixed infection, whereas the lower virulence property of m2s2 allele combination is higher in the subjects with single infection. There is an unexpectedly high prevalence of intermediate virulence factors (i.e., m1s2 and m2s1) is found in the subjects with mixed infection even higher than the high virulence factors (i.e., m1s1) which may be the error due to the small sample size of the isolate of intermediate virulence. However, we do not have any direct evidence for the emergence of virulent genes due to the presence of mixed strains of H. pylori. As a developing country, it is expected that Bangladeshi population will have high prevalence of H. pylori infection. But in this study we have found a high prevalence of mixed infection of H. pylori in a lower socioeconomic area of Bangladesh. This may have occurred due to lower socioeconomic condition, poor hygiene, intrafamilial transmission of H. pylori, mostly from mother to child [6] and chronic colonization. There is a high prevalence of H. pylori with virulence marker, that is, cagA and vacA in our study subjects. Our study presents a scenario in which hosts infected by mixed strains of H. pylori are highly prone to symptoms associated with gastric acidity, and the H. pylori strains are highly virulent. It might be possible that chronic co-colonization of mixed strains of H. pylori facilitates the virulence gene transfer horizontally and contributes in symptoms development. One of the limitations of our study is the population from which the samples were collected (population from low socioeconomic class) and the number of subjects analyzed. Further studies are necessary to understand the risk factors and behavioral determinants responsible for mixed infection of H. pylori and their relationship with virulence gene expression.
Acknowledgements and Disclosures This research was conducted in the International Centre for Diarrhoeal Diseases Research, Bangladesh (ICDDR, B), which is supported by countries and agencies that share its concern for the health problems of developing countries. This program is entirely supported by SIDA/SAREC (Grant no. GR-00384, Research Protocol Number 2005-010). ICDDR, B, acknowledges with gratitude the commitment of SIDA to the Centre’s research efforts. The authors thank Dr. Habiba Yeasmin, Dr. Shamima Sultana and the field staffs of Nandipara Clinic of ICDDR, B for their help in the study. The authors also thank Dr. Dipak kumar mitra and Gowranga Kumar Paul for their help in statistical analyses and Dr. Steven Baker for reviewing of the manuscript.
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Competing interests: the authors declare no competing interests.
References 1 Kauser F, Khan AA, Hussain MA, et al. The cag pathogenicity island of Helicobacter pylori is disrupted in the majority of patient isolates from different human populations. J Clin Microbiol 2004;42: 5302–8. 2 Brenner H, Weyermann M, Rothenbacher D. Clustering of Helicobacter pylori infection in couples: differences between high- and low-prevalence population groups. Ann Epidemiol 2006;16:516–20. 3 Drumm B, Perez-Perez GI, Blaser MJ, Sherman PM. Intrafamilial clustering of Helicobacter pylori infection. N Engl J Med 1990;322:359–63. 4 Kivi M, Johansson AL, Reilly M, Tindberg Y. Helicobacter pylori status in family members as risk factors for infection in children. Epidemiol Infect 2005;133:645–52. 5 Kivi M, Tindberg Y, Sorberg M, Casswall TH, Befrits R, Hellstrom PM, et al. Concordance of Helicobacter pylori strains within families. J Clin Microbiol 2003;41:5604–8. 6 Nahar S, Kibria KM, Hossain ME, Sultana J, Sarker SA, Engstrand L, et al. Evidence of intra-familial transmission of Helicobacter pylori by PCR-based RAPD fingerprinting in Bangladesh. Eur J Clin Microbiol Infect Dis 2009;28(7):767– 73. 7 Akopyanz N, Bukanov NO, Westblom TU, Kresovich S, Berg DE. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res 1992;11:5137–42. 8 Fujimoto S, Marshall B, Blaser MJ. PCR-based restriction fragment length polymorphism typing of Helicobacter pylori. J Clin Microbiol 1994;32:331–4. 9 Owen RJ, Bell GD, Desai M, Moreno M, Gant PW, Jones PH, et al. Biotype and molecular fingerprints of metronidazoleresistant strains of Helicobacter pylori from antral gastric mucosa. J Med Microbiol 1993;38:6–12. 10 Patra R, Chattopadhyay S, De R, Ghosh P, Ganguly M, Chowdhury A, et al. Multiple infection and microdiversity among Helicobacter pylori isolates in a single host in India. PLoS ONE 2012;7:e43370. 11 Wong BC, Wang WH, Berg DE, Fung FM, Wong KW, Wong WM, et al. High prevalence of mixed infections by Helicobacter pylori in Hong Kong: metronidazole sensitivity and overall genotype. Aliment Pharmacol Ther 2001;15:493–503. 12 Kuipers EJ, Israel DA, Kusters JG, Gerrits MM, Weel J, van Der Ende A, et al. Quasispecies development of Helicobacter pylori observed in paired isolates obtained years apart from the same host. J Infect Dis 2000;181:273–82. 13 Reyrat JM, Pelicic V, Papini E, Montecucco C, Rappuoli R, Telford JL. Towards deciphering the Helicobacter pylori cytotoxin. Mol Microbiol 1999;34:197–204. 14 Backert S, Moese S, Selbach M, Brinkmann V, Meyer TF. Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells. Mol Microbiol 2001;42:631–44. 15 Segal ED, Cha J, Lo J, Falkow S, Tompkins LS. Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori. Proc Natl Acad Sci USA 1999;96:14559–64.
403
Mixed H. pylori Infections in Bangladesh
16 Atherton JC, Cao P, Peek RM Jr, Tummuru MK, Blaser MJ, Cover TL. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration. J Biol Chem 1995;270:17771– 7. 17 van Doorn LJ, Figueiredo C, Sanna R, Plaisier A, Schneeberger P, de Boer W, et al. Clinical relevance of the cagA, vacA, and iceA status of Helicobacter pylori. Gastroenterology 1998;115:58– 66. 18 Pagliaccia C, de Bernard M, Lupetti P, Ji X, Burroni D, Cover TL, et al. The m2 form of the Helicobacter pylori cytotoxin has cell type-specific vacuolating activity. Proc Natl Acad Sci USA 1998;95:10212–7. 19 Sarker SA, Mahalanabis D, Hildebrand P, Rahaman MM, Bardhan PK, Fuchs G, et al. Helicobacter pylori: prevalence, transmission, and serum pepsinogen II concentrations in children of a poor periurban community in Bangladesh. Clin Infect Dis 1997;25:990–5. 20 Korkmaz H, Kesli R, Karabagli P, Terzi Y. Comparison of the diagnostic accuracy of five different stool antigen tests for the diagnosis of Helicobacter pylori infection. Helicobacter 2013;18:384–91. 21 Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds). Current protocols in molecular biology. New York, NY: Greene Publishing and Wiley-Interscience, 1993. 22 Chattopadhyay S, Patra R, Ramamurthy T, Chowdhury A, Santra A, Dhali GK, et al. Multiplex PCR assay for rapid detection and genotyping of Helicobacter pylori directly from biopsy specimens. J Clin Microbiol 2004;42:2821–4. 23 Marshall DG, Dundon WG, Beesley SM, Smyth CJ. Helicobacter pylori–a conundrum of genetic diversity. Microbiology 1998;144 (Pt.11):2925–39. 24 Taddei F, Matic I, Godelle B, Radman M. To be a mutator, or how pathogenic and commensal bacteria can evolve rapidly. Trends Microbiol 1997;5:427–8; discussion 8–9. 25 Kersulyte D, Chalkauskas H, Berg DE. Emergence of recombinant strains of Helicobacter pylori during human infection. Mol Microbiol 1999;31:31–43. 26 Suerbaum S, Smith JM, Bapumia K, Morelli G, Smith NH, Kunstmann E, et al. Free recombination within Helicobacter pylori. Proc Natl Acad Sci USA 1998;95:12619–24.
404
Kibria et al.
27 Cellini L, Di Campli E, Di Candia M, Marzio L. Molecular fingerprinting of Helicobacter pylori strains from duodenal ulcer patients. Lett Appl Microbiol 2003;36:222–6. 28 Prewett EJ, Bickley J, Owen RJ, Pounder RE. DNA patterns of Helicobacter pylori isolated from gastric antrum, body, and duodenum. Gastroenterology 1992;102:829–33. 29 Cellini L, Grande R, Di Campli E, Di Bartolomeo S, Capodicasa S, Marzio L. Analysis of genetic variability, antimicrobial susceptibility and virulence markers in Helicobacter pylori identified in Central Italy. Scand J Gastroenterol 2006;41:280– 7. 30 Taylor NS, Fox JG, Akopyants NS, Berg DE, Thompson N, Shames B, et al. Long-term colonization with single and multiple strains of Helicobacter pylori assessed by DNA fingerprinting. J Clin Microbiol 1995;33:918–23. 31 Berg DE, Gilman RH, Lelwala-Guruge J, Srivastava K, Valdez Y, Watanabe J, et al. Helicobacter pylori populations in Peruvian patients. Clin Infect Dis 1997;25:996–1002. 32 Falush D, Kraft C, Taylor NS, Correa P, Fox JG, Achtman M, et al. Recombination and mutation during long-term gastric colonization by Helicobacter pylori: estimates of clock rates, recombination size, and minimal age. Proc Natl Acad Sci USA 2001;98:15056–61.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 The approaches for population selection and analysis of study. Table S1 Odd ratios for the effect of mixed infection of H. pylori on the prevalence of cagA and vacA genotype of 128 pooled isolates. Single infection was considered as reference category. To understand the effect of mixed infection in the prevalence of virulence genotype, the variables were grouped into two or three classes, high (highly virulent), intermediate (moderately virulent) and low (less or avirulent).
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