ORIGINAL ARTICLE: GASTROENTEROLOGY
Virulence Factors and Antibiotic Resistance in Children With Helicobacter pylori Gastritis
Hamza Karabiber, Mukadder A. Selimoglu, yBaris Otlu, yOzge Yildirim, and zAli Ozer
ABSTRACT Objectives: There are limited data regarding the pattern of Helicobacter pylori (Hp) antibiotic resistance and virulence factors in children. Evaluation of prevalence of drug resistance and virulence-factor genotype in children with Hp gastritis and to investigate whether there is any relation between drug resistance and genotype were our aims in this study. Methods: Ninety-eight children with polymerase chain reaction–positive Hp gastritis were included. Antimicrobial susceptibility was tested by disc diffusion method and polymerase chain reaction assays were used for the determination of virulence factors. Results: The resistance rates to clarithromycin, metronidazole, and amoxicillin were 23.5%, 11.7%, and 3.9%, respectively. All strains carried vacA genotype, and 51%, 70.4%, 49%, 34.7%, and 25.5% were cagA-, cagE-, babA2-, iceA1-, and iceA2-positive, respectively. Of those 98 specimens, 81.6%, 19.4%, 38.8%, and 63.3% carried vacAs1, vacAs2, vacAm1, and vacAm2, respectively. Dominant vacA type was s1am2 (32.7%), followed by s1am1 (14.3%) and s2m2 (12.2%). Significant rates of clarithromycin resistance were observed in cagE-, iceA1-, babA2-, and vacAs1c-positive groups. In those with metronidazole resistance, vacAs1 and vacAs1c were more common (P < 0.05). Conclusions: The cagE-positive and vacA s1a/m2 genotypes, which are correlated with increased antibiotic resistance, were predominant in our population. In countries where Hp infection is prevalent, studies focusing on virulence factors and antibiotic susceptibility may provide anticipation of the prognosis and may be helpful to reduce morbidity and mortality. Key Words: antibiotic resistance, children, Helicobacter pylori, virulence
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H
elicobacter pylori (Hp) infection is a common chronic infection throughout the world, which sometimes leads to severe morbidity such as gastric carcinoma. Some genetic factors of both host and bacteria are the reasons why some individuals are asymptomatic, whereas others have severe pathology (1). Diagnosis and a successful treatment are the key points of the prevention of morbidity in Hp infection; however, antibiotic Received and accepted November 27, 2013. From the Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, the yDepartment of Clinical Microbiology, and the zDepartment of Public Health, Inonu University, Faculty of Medicine, Malatya, Turkey. Address correspondence and reprint requests to Dr Hamza Karabiber, Inonu Universitesi, Tip Fakultesi, Cocuk Sagligi ve Hastaliklari AD, 44280 Malatya, Turkey (e-mail: [email protected]). This study was funded by Inonu University Scientific Research Projects Unit with the project number of 2010/62. The authors report no conflicts of interest. Copyright # 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000273
resistance is an important obstacle for the achievement of this goal. The success of treatment is now compromised by the increase in antibiotic resistance of Hp, especially to clarithromycin and metronidazole (2). In a recent study by Megraud et al (2), which included 2204 patients (311 children), frequency of clarithromycin, metronidazole, and amoxicillin resistance was reported as 31.8%, 25.7%, and 0.3%, respectively. In countries where resistance rates are above 15% to 20%, the need for routine susceptibility test seems mandatory (3). Furthermore, because antibiotic resistance is an evolving process, it is also necessary to carry out point prevalence surveys on a regular basis to guide clinicians in therapeutic choice. Although there are many studies investigating antibiotic resistance and virulence factors in adults with Hp infection, pediatric studies are not satisfactory. We not only aimed to detect the prevalence of antibiotic resistance and virulence factors but also to determine the relation between Hp subtypes and antibiotic resistance in a pediatric population.
METHODS Ninety-eight children, who were 2 to 17 years old, with polymerase chain reaction (PCR)–proven Hp infection, of 159 children who underwent upper gastrointestinal endoscopy for dyspepsia, were included in this study. Endoscopic biopsy samples, which were taken from antrum and corpus, were placed into sterilized water containing tubes and immediately transferred to the laboratory for culture and PCR. Columbia Agar (Oxoid Ltd, Basingstoke, UK), containing Hp-selective supplement (Hp-selective supplement, Oxoid Ltd) and horse blood (Laked horse blood, Oxoid Ltd), was used as the culture medium. Microareophilic atmosphere (5% O2, 10% CO2, and 85% N2) that is needed for Hp growth was provided with a commercial kit (Genbag microaer, Biomerieux SA, Marcy l’Etoile, France). Samples were incubated at 378C for 72 hours. Horse blood–added Mueller-Hinton agar was used for antibiogram testing. Antibiotic susceptibility was evaluated by disc diffusion method. Susceptibility zone diameter values recommended by McNulty et al (4) were used as reference. BioRobot EZ1 system (Qiagen, Hilden, Germany) was used for bacterial DNA isolation. In-house PCR was performed based on predefined primer sequences (5,6). Phosphosphatase mutase (glmM) gene was selected as a target to determine Hp in gastric samples. For each gene region, combination of 25 mL of amplification mixture (12.5 mL TopTaq DNA PCR Master Mix, QIAGEN), 1 mL of each primer (10 pmol/mL, 8-mL H2O), and 2.5 mL of extraction product was processed using automated thermal cycler (Palm-cycler Corbett Research, Sydney, Australia) for amplification. After amplification, the product was electrophoresed in 1.5% to 3% agarose gel and stained within 400-mL of ultrapure water containing 5 mg/mL ethidium bromide and examined using gel imaging system (Gel Logic 2200 imaging system [1708 1280 pixels], Kodak, Rochester, NY). For detecting macrolide resistance, A2143G, A2144G, and A2143C mutations were investigated using the molecular beacon-real-time PCR method (Rotor-Gene 6000 real-time PCR
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Virulence Factors and Antibiotic Resistance in H pylori Gastritis
TABLE 1. Virulence factors of Helicobacter pylori–positive patients Virulence factors
Positive, n (%)
cagA cagE babA iceA1 iceA2 vacA S1 S1a S1b S1c S2 M1 M2
50 69 48 34 25 98 80 48 16 16 19 38 62
(51.0) (70.4) (49.0) (34.7) (25.5) (100) (81.6) (49.0) (16.3) (16.3) (19.4) (38.0) (62.0)
device, Corbett Research) (7,8). Mutations were confirmed using a PCR-based reverse hybridization test (GenoType HelicoDR, Hain Life Science, Nehren, Germany).
Statistical analyses were performed with SPSS 11.0 (SPSS Inc, Chicago, IL) using the x2, Fisher, and Student t tests. The study was funded by Inonu University Scientific Research Projects Unit (project no. 2010/62). The Inonu University ethics committee approved the study (2009/153).
RESULTS Antibiotic Resistance Of 98 children with PCR-positive Hp gastritis, 51 (52%) were also positive according to the culture results. Susceptibility of Hp to amoxicillin, clarithromycin, metronidazole, and tetracycline was studied in 51 children with positive culture results. Resistance rates were 23.5%, 11.7%, and 3.9% for clarithromycin, metronidazole, and amoxicillin, respectively. None of our patients had isolated tetracycline resistance, but 4 children (7.8%) had resistance to both amoxicillin and tetracycline. Among 98 children, we found 19.4% rate of resistance to macrolides according to a molecular test, which combines PCR and hybridization.
TABLE 2. Relation between virulence factors and antibiotic susceptibility Clarithromycin resistance Virulence factors cagA Negative Positive cagE Negative Positive babA2 Negative Positive iceA1 Negative Positive iceA2 Negative Positive vacAs1 Negative Positive vacAs1a Negative Positive vacAs1b Negative Positive vacAs1c Negative Positive vacAs2 Negative Positive vacAm1 Negative Positive vacAm2 Negative Positive
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Metronidazole resistance
Susceptible, n (%)
Resistant, n (%)
P
Susceptible, n (%)
101 (68.7) 46 (31.3)
8 (66.7) 4 (33.3)
1.000
106 (69.3) 47 (30.7)
88 (59.9) 59 (40.1)
2 (16.7) 10 (83.3)
0.004
106 (72.1) 41 (27.9)
5 (41.7) 7 (58.3)
119 (81.0) 28 (19)
Resistant, n (%)
P
3 (50) 3 (50)
0.380
89 (58.2) 64 (41.8)
1 (16.7) 5 (83.3)
0.086
0.027
109 (71.2) 44 (28.8)
2 (33.3) 4 (66.7)
0.068
6 (50) 6 (50)
0.012
121 (79.1) 32 (20.9)
4 (66.7) 2 (33.3)
0.609
124 (84.4) 23 (15.6)
10(83.3) 2 (16.7)
1.000
130 (85) 23 (15)
4 (66.7) 2 (33.3)
0.239
78 (53.1) 69 (46.9)
1 (8.3) 11 (91.7)
0.005
79 (51.6) 74 (48.4)
0 (0) 6 (100)
0.028
103 (70.1) 44 (29.9)
8 (66.7) 4 (33.3)
0.755
107 (69.9) 46 (30.1)
4 (66.7) 2 (33.3)
1.000
134 (91.2) 13 (8.8)
9 (89.9) 3 (10.1)
0.105
138 (90.2) 15 (9.8)
5 (83.3) 1 (16.7)
0.476
135 (91.8) 12 (8.2)
8 (66.7) 4 (33.3)
0.021
140 (91.5) 13 (8.5)
3 (50) 3 (50)
0.014
129 (87.8) 18 (12.2)
11 (91.7) 1 (8.3)
1.000
134 (87.6) 19 (12.4)
6 (100) 0(0)
1.000
113 (76.9) 34 (23.1)
8 (66.7) 4 (33.3)
0.483
117 (76.5) 36 (23.5)
4 (66.7) 2 (33.3)
0.630
93 (63.3) 54 (36.7)
4 (33.3) 8 (66.7)
0.062
95 (62.1) 58 (37.9)
2 (33.3) 4 (66.7)
0.210
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Virulence Factors Virulence factors such as cagA (51%), cagE (70.4%), babA (49%), iceA1 (34.7%), iceA2 (25.5%), and vacA (100%) subtypes were investigated in PCR-positive patients. Results are shown in Table 1. Although vacA was positive in all PCR-positive patients, the most common subtype of vacA was s1am2 (32.7%). In 3 patients, mutational multivacA (s1am1/m2, s1c/s2m2, and s2m1/m2) was detected.
Association Between Antibiotic Resistance and Virulence Factor Genotype Relation between antibiotic resistance and virulence factors was evaluated. CagE, babA2, iceA1, and vacAs1 were statistically more common in children with clarithromycin resistance (P < 0.05), whereas vacAs1c was more rare (P < 0.05). In children with metronidazole resistance, vacAs1 and vacAs1c were more common (P < 0.05). Relation between virulence factors and resistance to clarithromycin and metronidazole is shown in Table 2. Because amoxicillin and combined resistance (amoxicillin þ tetracycline) was detected in only 2 and 4 patients, respectively, no statistical significance could be found (P > 0.05).
DISCUSSION In recent years, increasing rates of resistance to amoxicillin, clarithromycin, and metronidazole, commonly used antibiotics for Hp infection, have led to significant treatment failures. Some factors such as age, sex, in-house infection, colonization density, gastric pH, as well as pre-exposure to antibiotics are thought to be responsible in resistance development (12–15). Although lack of standardization of susceptibility tests and interpretation may cause a significant difference in rates of antibiotic resistance, treatment failure is mostly attributed to clarithromycin resistance. It was reported that clarithromycin resistance doubled within 5 years, probably because of its wide usage for community-based pneumonia (16). For that reason, the Maastricht IV/Florence Consensus
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Report recommends first-line therapy including 1 proton pump inhibitor and amoxicillin and clarithromycin only in regions where clarithromycin and metronidazole resistance rates are below 15% to 20% and 40%, respectively (17). The largest pediatric study, investigating antibiotic resistance of Hp, revealed clarithromycin and metronidazole resistance rates as 20% and 23%, respectively (18). In the same study, combined resistance and amoxicillin resistance rates were 5.3% and 0.6%, respectively (18). Studies investigating antibiotic resistance rates in Hp infection in both adults and children are summarized in Table 3. The only pediatric study conducted in Turkey was by ¨ zc¸ay et al (34), which had a similar resistance rate for clarithroO mycin but a higher rate for metronidazole compared with our data. Furthermore, they did not report any amoxicillin resistance in 2004 (34). An adult study performed in 2000 from the same hospital we work at now revealed significantly higher and lower resistance rates for metronidazole and clarithromycin, respectively, compared with those we found (31). Resistance profile changes in time may be the reflection of increased clarithromycin usage for pneumonia but decreased metronidazole usage for diarrhea because of a gradual increase in hygienic conditions nationwide. The relation between virulence factors and antibiotic resistance is another important issue. Therefore, we investigated not only antibiotic susceptibility of Hp but also its virulence factors, vacA, cagA, cagE, babA, and iceA genes and vacA gene polymorphism. Godoy et al (41) found no relation at all. Likewise, Bag˘lan et al (42) found no relation between iceA genotypes and clarithromycin resistance; however, Boyanova et al (43) revealed that the iceA1 allele was 74.4% positive in those with susceptibility to clarithromycin, whereas it was 55.3% positive in those with resistance. It was also reported that treatment failure was related to vacAs1 and m1 positivity accompanied by cagA negativity (44,45). A meta-analysis (45) showed that eradication rates of Hp were 84% and 73%, respectively, in those with positive and negative cagA. A review by Sugimoto et al (46) revealed that treatment efficacy was lower in cagA-negative and vacAs2-positive patients and they concluded that treatment success is higher in those with
TABLE 3. Antibiotic resistance rates in Helicobacter pylori infection in both adults and children in different countries
Adults
Children
Adults in Turkey
Children in Turkey
610
Elviss et al (England 2004) (19) Boyanova et al (eastern Europe 2000) (20) Ben Mansour et al (Tunisia 2010) (21) Ahmad et al (Malaysia 2010) (22) Talebi Bezmin Abadi et al (Iraq 2010) (23) Alarcon et al (Argentina 2003) (24) Lopes et al (Portugal 2005) (25) Kalach et al (France 2005) (26) Koletzko et al (Europe 2006) (18) Rafeey et al (Iraq 2007) (27) Ben Mansour et al (Tunisia 2010) (21) Ve´csei et al (Austria 2010) (28) Garcia et al (Brazil 2010) (29) Zevit et al (Israel 2010) (30) Megraud et al (Europe 2012) (2) Kantarc¸eken et al (Malatya, Turkey 2000) (31) Can et al (Ankara, Turkey 2005) (32) Simsek et al (Ankara, Turkey 2005) (33) ¨ zc¸ay et al (Ankara, Turkey 2004) (34) O Our data (Malatya, Turkey 2011)
N
Clarithromycin resistance, %
Metronidazole resistance, %
Amoxiciline resistance, %
363 2340 225 777 132 96 109 377 1233 100 48 30 45 53 311 51 46 66 33 51
7 9.5 14.6 2.1 30 29.1 39.4 7.9 20 16 18.8 34.1 27 25 31.8 9.8 22.9 24.2 18.2 23.5
24 37.9 56.8 37.4 73.4 23.9 16.5 36.7 23 95 25 24.4 13 19 25.7 49 — — 36.4 11.7
0 0.9 0 0 6.8 0 0 0 0.6 59 — 0 4 0 0.3 — — — 0 3.9
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highly virulent strains. A positive correlation with metronidazole resistance and cagA negativity was reported, as well (57.6% vs 28.6%) (47). Elviss et al (19) demonstrated that strains susceptible to clarithromycin and metronidazole were associated with vacAs1m2 genotype. No correlation was detected with cagA and clarithromycin or metronidazole resistance in our study; however, clarithromycin resistance was more prevalent in cagE-positive patients compared with negative ones (83.3% vs 16.7%). Again, in babA2- and iceA1-positive cases, resistance to clarithromycin was higher. VacAs1-positive cases had 91.7% clarithromycin and 100% metronidazole resistance; however, metronidazole resistance was independent of CagE, babA, and iceA1 positivity. Although there are studies showing the relation between some genotypes (cagA, iceA, and vacA) and antibiotic resistance, there is no study for cagE and babA2 genotypes. It was speculated that in gastritis caused by virulent types, such as cagA, an efficient antibiotic delivery to gastric tissue may be provided because of dilated vessels within the severely inflamed tissue (45). Another explanation is the higher bacterial proliferation in cagA-positive cases, which is an advantage for antibiotics being effective because it is known that proliferating bacteria are more sensitive to antibiotics (9,45,48). Our data did not support the prediction that antibiotic susceptibility is higher in infections caused by virulent genotypes. In conclusion, we found clarithromycin, metronidazole, amoxicillin, and combined (amoxicillin and tetracycline) resistance as 23.5%, 11.7%, 3.9%, and 7.8%, respectively. CagE and VacAs1 were correlated with increased antibiotic resistance, whereas babAand iceA1-positive cases had less antibiotic resistance. In countries where Hp infection is prevalent, studies focusing on virulence factors and antibiotic susceptibility may provide anticipation of the prognosis and reduction in morbidity and mortality.
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44. Yakoob J, Jafri W, Abbas Z, et al. Risk factors associated with Helicobacter pylori infection treatment failure in a high prevalence area. Epidemiol Infect 2010;139:581–90. 45. Suzuki T, Matsuo K, Sawaki A, et al. Systematic review and metaanalysis: importance of CagA status for successful eradication of Helicobacter pylori infection. Aliment Pharmacol Ther 2006;24: 273–80. 46. Sugimoto M, Yamaoka Y. Virulence factor genotypes of Helicobacter pylori affect cure rates of eradication therapy. Arch Immunol Ther Exp (Warsz) 2009;57:45–56. 47. Taneike I, Nami A, O’Connor A, et al. Analysis of drug resistance and virulence-factor genotype of Irish Helicobacter pylori strains: is there any relationship between resistance to metronidazole and cagA status? Aliment Pharmacol Ther 2009;30:784–90. 48. Censini S, Lange C, Xiang Z, et al. CagA pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci U S A 1996;93:14648–53.
Chylous Ascites Phthisiologia, seu exercitationes de phthisi by Richard Morton (1637–1698) was published in 1689. Morton was the first to diagnose chylous ascites in a living child. He described a 2-year-old with cough, fever and dyspnea: . . .[At] the very beginning of the Fever his Belly began to be distended with a Dropsical swelling, which increased strangely every day; his Cough and shortness of Breath at the same time growing worse: All which Symptoms were at length accompanied with an Atrophy of the Parts, even to the degrees of a Maramus. But yet when his Body was a perfect Skeleton, and the Dropsie at a high Tide (which was very remarkable) he had a brisk and healthful look, and a lovely Countenance, without the least Tincture of a Yellowness, and a good, or rather greedy Appetite, and that to the very day he dyed. From whence I did rightly conjecture, and always told his Friends, as my Opinion, that his Dropsie was truly Chylous, caused by the Chyle flowing into the cavity of the Belly by the Lacteal Vessels, upon some rupture that had been made in them; which appeared very plainly from the Event. For in Tapping of the Child’s Belly, whilst he was yet alive, we took out several Pints of Milky Chyle... ‘‘Tapping’’ beginning with Haly Abbas (?900–994) was performed with a trocar, as illustrated in Clave Medico-Chirurgica Universal by Francisco del Hierra, Madrid, 1730.
—Contributed by Angel R. Colo´n, MD
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Virulence Factors and Antibiotic Resistance in Children With Helicobacter pylori Gastritis
Hamza Karabiber, Mukadder A. Selimoglu, yBaris Otlu, yOzge Yildirim, and zAli Ozer
ABSTRACT Objectives: There are limited data regarding the pattern of Helicobacter pylori (Hp) antibiotic resistance and virulence factors in children. Evaluation of prevalence of drug resistance and virulence-factor genotype in children with Hp gastritis and to investigate whether there is any relation between drug resistance and genotype were our aims in this study. Methods: Ninety-eight children with polymerase chain reaction–positive Hp gastritis were included. Antimicrobial susceptibility was tested by disc diffusion method and polymerase chain reaction assays were used for the determination of virulence factors. Results: The resistance rates to clarithromycin, metronidazole, and amoxicillin were 23.5%, 11.7%, and 3.9%, respectively. All strains carried vacA genotype, and 51%, 70.4%, 49%, 34.7%, and 25.5% were cagA-, cagE-, babA2-, iceA1-, and iceA2-positive, respectively. Of those 98 specimens, 81.6%, 19.4%, 38.8%, and 63.3% carried vacAs1, vacAs2, vacAm1, and vacAm2, respectively. Dominant vacA type was s1am2 (32.7%), followed by s1am1 (14.3%) and s2m2 (12.2%). Significant rates of clarithromycin resistance were observed in cagE-, iceA1-, babA2-, and vacAs1c-positive groups. In those with metronidazole resistance, vacAs1 and vacAs1c were more common (P < 0.05). Conclusions: The cagE-positive and vacA s1a/m2 genotypes, which are correlated with increased antibiotic resistance, were predominant in our population. In countries where Hp infection is prevalent, studies focusing on virulence factors and antibiotic susceptibility may provide anticipation of the prognosis and may be helpful to reduce morbidity and mortality. Key Words: antibiotic resistance, children, Helicobacter pylori, virulence
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H
elicobacter pylori (Hp) infection is a common chronic infection throughout the world, which sometimes leads to severe morbidity such as gastric carcinoma. Some genetic factors of both host and bacteria are the reasons why some individuals are asymptomatic, whereas others have severe pathology (1). Diagnosis and a successful treatment are the key points of the prevention of morbidity in Hp infection; however, antibiotic Received and accepted November 27, 2013. From the Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, the yDepartment of Clinical Microbiology, and the zDepartment of Public Health, Inonu University, Faculty of Medicine, Malatya, Turkey. Address correspondence and reprint requests to Dr Hamza Karabiber, Inonu Universitesi, Tip Fakultesi, Cocuk Sagligi ve Hastaliklari AD, 44280 Malatya, Turkey (e-mail: [email protected]). This study was funded by Inonu University Scientific Research Projects Unit with the project number of 2010/62. The authors report no conflicts of interest. Copyright # 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000273
resistance is an important obstacle for the achievement of this goal. The success of treatment is now compromised by the increase in antibiotic resistance of Hp, especially to clarithromycin and metronidazole (2). In a recent study by Megraud et al (2), which included 2204 patients (311 children), frequency of clarithromycin, metronidazole, and amoxicillin resistance was reported as 31.8%, 25.7%, and 0.3%, respectively. In countries where resistance rates are above 15% to 20%, the need for routine susceptibility test seems mandatory (3). Furthermore, because antibiotic resistance is an evolving process, it is also necessary to carry out point prevalence surveys on a regular basis to guide clinicians in therapeutic choice. Although there are many studies investigating antibiotic resistance and virulence factors in adults with Hp infection, pediatric studies are not satisfactory. We not only aimed to detect the prevalence of antibiotic resistance and virulence factors but also to determine the relation between Hp subtypes and antibiotic resistance in a pediatric population.
METHODS Ninety-eight children, who were 2 to 17 years old, with polymerase chain reaction (PCR)–proven Hp infection, of 159 children who underwent upper gastrointestinal endoscopy for dyspepsia, were included in this study. Endoscopic biopsy samples, which were taken from antrum and corpus, were placed into sterilized water containing tubes and immediately transferred to the laboratory for culture and PCR. Columbia Agar (Oxoid Ltd, Basingstoke, UK), containing Hp-selective supplement (Hp-selective supplement, Oxoid Ltd) and horse blood (Laked horse blood, Oxoid Ltd), was used as the culture medium. Microareophilic atmosphere (5% O2, 10% CO2, and 85% N2) that is needed for Hp growth was provided with a commercial kit (Genbag microaer, Biomerieux SA, Marcy l’Etoile, France). Samples were incubated at 378C for 72 hours. Horse blood–added Mueller-Hinton agar was used for antibiogram testing. Antibiotic susceptibility was evaluated by disc diffusion method. Susceptibility zone diameter values recommended by McNulty et al (4) were used as reference. BioRobot EZ1 system (Qiagen, Hilden, Germany) was used for bacterial DNA isolation. In-house PCR was performed based on predefined primer sequences (5,6). Phosphosphatase mutase (glmM) gene was selected as a target to determine Hp in gastric samples. For each gene region, combination of 25 mL of amplification mixture (12.5 mL TopTaq DNA PCR Master Mix, QIAGEN), 1 mL of each primer (10 pmol/mL, 8-mL H2O), and 2.5 mL of extraction product was processed using automated thermal cycler (Palm-cycler Corbett Research, Sydney, Australia) for amplification. After amplification, the product was electrophoresed in 1.5% to 3% agarose gel and stained within 400-mL of ultrapure water containing 5 mg/mL ethidium bromide and examined using gel imaging system (Gel Logic 2200 imaging system [1708 1280 pixels], Kodak, Rochester, NY). For detecting macrolide resistance, A2143G, A2144G, and A2143C mutations were investigated using the molecular beacon-real-time PCR method (Rotor-Gene 6000 real-time PCR
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Virulence Factors and Antibiotic Resistance in H pylori Gastritis
TABLE 1. Virulence factors of Helicobacter pylori–positive patients Virulence factors
Positive, n (%)
cagA cagE babA iceA1 iceA2 vacA S1 S1a S1b S1c S2 M1 M2
50 69 48 34 25 98 80 48 16 16 19 38 62
(51.0) (70.4) (49.0) (34.7) (25.5) (100) (81.6) (49.0) (16.3) (16.3) (19.4) (38.0) (62.0)
device, Corbett Research) (7,8). Mutations were confirmed using a PCR-based reverse hybridization test (GenoType HelicoDR, Hain Life Science, Nehren, Germany).
Statistical analyses were performed with SPSS 11.0 (SPSS Inc, Chicago, IL) using the x2, Fisher, and Student t tests. The study was funded by Inonu University Scientific Research Projects Unit (project no. 2010/62). The Inonu University ethics committee approved the study (2009/153).
RESULTS Antibiotic Resistance Of 98 children with PCR-positive Hp gastritis, 51 (52%) were also positive according to the culture results. Susceptibility of Hp to amoxicillin, clarithromycin, metronidazole, and tetracycline was studied in 51 children with positive culture results. Resistance rates were 23.5%, 11.7%, and 3.9% for clarithromycin, metronidazole, and amoxicillin, respectively. None of our patients had isolated tetracycline resistance, but 4 children (7.8%) had resistance to both amoxicillin and tetracycline. Among 98 children, we found 19.4% rate of resistance to macrolides according to a molecular test, which combines PCR and hybridization.
TABLE 2. Relation between virulence factors and antibiotic susceptibility Clarithromycin resistance Virulence factors cagA Negative Positive cagE Negative Positive babA2 Negative Positive iceA1 Negative Positive iceA2 Negative Positive vacAs1 Negative Positive vacAs1a Negative Positive vacAs1b Negative Positive vacAs1c Negative Positive vacAs2 Negative Positive vacAm1 Negative Positive vacAm2 Negative Positive
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Metronidazole resistance
Susceptible, n (%)
Resistant, n (%)
P
Susceptible, n (%)
101 (68.7) 46 (31.3)
8 (66.7) 4 (33.3)
1.000
106 (69.3) 47 (30.7)
88 (59.9) 59 (40.1)
2 (16.7) 10 (83.3)
0.004
106 (72.1) 41 (27.9)
5 (41.7) 7 (58.3)
119 (81.0) 28 (19)
Resistant, n (%)
P
3 (50) 3 (50)
0.380
89 (58.2) 64 (41.8)
1 (16.7) 5 (83.3)
0.086
0.027
109 (71.2) 44 (28.8)
2 (33.3) 4 (66.7)
0.068
6 (50) 6 (50)
0.012
121 (79.1) 32 (20.9)
4 (66.7) 2 (33.3)
0.609
124 (84.4) 23 (15.6)
10(83.3) 2 (16.7)
1.000
130 (85) 23 (15)
4 (66.7) 2 (33.3)
0.239
78 (53.1) 69 (46.9)
1 (8.3) 11 (91.7)
0.005
79 (51.6) 74 (48.4)
0 (0) 6 (100)
0.028
103 (70.1) 44 (29.9)
8 (66.7) 4 (33.3)
0.755
107 (69.9) 46 (30.1)
4 (66.7) 2 (33.3)
1.000
134 (91.2) 13 (8.8)
9 (89.9) 3 (10.1)
0.105
138 (90.2) 15 (9.8)
5 (83.3) 1 (16.7)
0.476
135 (91.8) 12 (8.2)
8 (66.7) 4 (33.3)
0.021
140 (91.5) 13 (8.5)
3 (50) 3 (50)
0.014
129 (87.8) 18 (12.2)
11 (91.7) 1 (8.3)
1.000
134 (87.6) 19 (12.4)
6 (100) 0(0)
1.000
113 (76.9) 34 (23.1)
8 (66.7) 4 (33.3)
0.483
117 (76.5) 36 (23.5)
4 (66.7) 2 (33.3)
0.630
93 (63.3) 54 (36.7)
4 (33.3) 8 (66.7)
0.062
95 (62.1) 58 (37.9)
2 (33.3) 4 (66.7)
0.210
609
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Karabiber et al
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Virulence Factors Virulence factors such as cagA (51%), cagE (70.4%), babA (49%), iceA1 (34.7%), iceA2 (25.5%), and vacA (100%) subtypes were investigated in PCR-positive patients. Results are shown in Table 1. Although vacA was positive in all PCR-positive patients, the most common subtype of vacA was s1am2 (32.7%). In 3 patients, mutational multivacA (s1am1/m2, s1c/s2m2, and s2m1/m2) was detected.
Association Between Antibiotic Resistance and Virulence Factor Genotype Relation between antibiotic resistance and virulence factors was evaluated. CagE, babA2, iceA1, and vacAs1 were statistically more common in children with clarithromycin resistance (P < 0.05), whereas vacAs1c was more rare (P < 0.05). In children with metronidazole resistance, vacAs1 and vacAs1c were more common (P < 0.05). Relation between virulence factors and resistance to clarithromycin and metronidazole is shown in Table 2. Because amoxicillin and combined resistance (amoxicillin þ tetracycline) was detected in only 2 and 4 patients, respectively, no statistical significance could be found (P > 0.05).
DISCUSSION In recent years, increasing rates of resistance to amoxicillin, clarithromycin, and metronidazole, commonly used antibiotics for Hp infection, have led to significant treatment failures. Some factors such as age, sex, in-house infection, colonization density, gastric pH, as well as pre-exposure to antibiotics are thought to be responsible in resistance development (12–15). Although lack of standardization of susceptibility tests and interpretation may cause a significant difference in rates of antibiotic resistance, treatment failure is mostly attributed to clarithromycin resistance. It was reported that clarithromycin resistance doubled within 5 years, probably because of its wide usage for community-based pneumonia (16). For that reason, the Maastricht IV/Florence Consensus
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Report recommends first-line therapy including 1 proton pump inhibitor and amoxicillin and clarithromycin only in regions where clarithromycin and metronidazole resistance rates are below 15% to 20% and 40%, respectively (17). The largest pediatric study, investigating antibiotic resistance of Hp, revealed clarithromycin and metronidazole resistance rates as 20% and 23%, respectively (18). In the same study, combined resistance and amoxicillin resistance rates were 5.3% and 0.6%, respectively (18). Studies investigating antibiotic resistance rates in Hp infection in both adults and children are summarized in Table 3. The only pediatric study conducted in Turkey was by ¨ zc¸ay et al (34), which had a similar resistance rate for clarithroO mycin but a higher rate for metronidazole compared with our data. Furthermore, they did not report any amoxicillin resistance in 2004 (34). An adult study performed in 2000 from the same hospital we work at now revealed significantly higher and lower resistance rates for metronidazole and clarithromycin, respectively, compared with those we found (31). Resistance profile changes in time may be the reflection of increased clarithromycin usage for pneumonia but decreased metronidazole usage for diarrhea because of a gradual increase in hygienic conditions nationwide. The relation between virulence factors and antibiotic resistance is another important issue. Therefore, we investigated not only antibiotic susceptibility of Hp but also its virulence factors, vacA, cagA, cagE, babA, and iceA genes and vacA gene polymorphism. Godoy et al (41) found no relation at all. Likewise, Bag˘lan et al (42) found no relation between iceA genotypes and clarithromycin resistance; however, Boyanova et al (43) revealed that the iceA1 allele was 74.4% positive in those with susceptibility to clarithromycin, whereas it was 55.3% positive in those with resistance. It was also reported that treatment failure was related to vacAs1 and m1 positivity accompanied by cagA negativity (44,45). A meta-analysis (45) showed that eradication rates of Hp were 84% and 73%, respectively, in those with positive and negative cagA. A review by Sugimoto et al (46) revealed that treatment efficacy was lower in cagA-negative and vacAs2-positive patients and they concluded that treatment success is higher in those with
TABLE 3. Antibiotic resistance rates in Helicobacter pylori infection in both adults and children in different countries
Adults
Children
Adults in Turkey
Children in Turkey
610
Elviss et al (England 2004) (19) Boyanova et al (eastern Europe 2000) (20) Ben Mansour et al (Tunisia 2010) (21) Ahmad et al (Malaysia 2010) (22) Talebi Bezmin Abadi et al (Iraq 2010) (23) Alarcon et al (Argentina 2003) (24) Lopes et al (Portugal 2005) (25) Kalach et al (France 2005) (26) Koletzko et al (Europe 2006) (18) Rafeey et al (Iraq 2007) (27) Ben Mansour et al (Tunisia 2010) (21) Ve´csei et al (Austria 2010) (28) Garcia et al (Brazil 2010) (29) Zevit et al (Israel 2010) (30) Megraud et al (Europe 2012) (2) Kantarc¸eken et al (Malatya, Turkey 2000) (31) Can et al (Ankara, Turkey 2005) (32) Simsek et al (Ankara, Turkey 2005) (33) ¨ zc¸ay et al (Ankara, Turkey 2004) (34) O Our data (Malatya, Turkey 2011)
N
Clarithromycin resistance, %
Metronidazole resistance, %
Amoxiciline resistance, %
363 2340 225 777 132 96 109 377 1233 100 48 30 45 53 311 51 46 66 33 51
7 9.5 14.6 2.1 30 29.1 39.4 7.9 20 16 18.8 34.1 27 25 31.8 9.8 22.9 24.2 18.2 23.5
24 37.9 56.8 37.4 73.4 23.9 16.5 36.7 23 95 25 24.4 13 19 25.7 49 — — 36.4 11.7
0 0.9 0 0 6.8 0 0 0 0.6 59 — 0 4 0 0.3 — — — 0 3.9
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highly virulent strains. A positive correlation with metronidazole resistance and cagA negativity was reported, as well (57.6% vs 28.6%) (47). Elviss et al (19) demonstrated that strains susceptible to clarithromycin and metronidazole were associated with vacAs1m2 genotype. No correlation was detected with cagA and clarithromycin or metronidazole resistance in our study; however, clarithromycin resistance was more prevalent in cagE-positive patients compared with negative ones (83.3% vs 16.7%). Again, in babA2- and iceA1-positive cases, resistance to clarithromycin was higher. VacAs1-positive cases had 91.7% clarithromycin and 100% metronidazole resistance; however, metronidazole resistance was independent of CagE, babA, and iceA1 positivity. Although there are studies showing the relation between some genotypes (cagA, iceA, and vacA) and antibiotic resistance, there is no study for cagE and babA2 genotypes. It was speculated that in gastritis caused by virulent types, such as cagA, an efficient antibiotic delivery to gastric tissue may be provided because of dilated vessels within the severely inflamed tissue (45). Another explanation is the higher bacterial proliferation in cagA-positive cases, which is an advantage for antibiotics being effective because it is known that proliferating bacteria are more sensitive to antibiotics (9,45,48). Our data did not support the prediction that antibiotic susceptibility is higher in infections caused by virulent genotypes. In conclusion, we found clarithromycin, metronidazole, amoxicillin, and combined (amoxicillin and tetracycline) resistance as 23.5%, 11.7%, 3.9%, and 7.8%, respectively. CagE and VacAs1 were correlated with increased antibiotic resistance, whereas babAand iceA1-positive cases had less antibiotic resistance. In countries where Hp infection is prevalent, studies focusing on virulence factors and antibiotic susceptibility may provide anticipation of the prognosis and reduction in morbidity and mortality.
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Chylous Ascites Phthisiologia, seu exercitationes de phthisi by Richard Morton (1637–1698) was published in 1689. Morton was the first to diagnose chylous ascites in a living child. He described a 2-year-old with cough, fever and dyspnea: . . .[At] the very beginning of the Fever his Belly began to be distended with a Dropsical swelling, which increased strangely every day; his Cough and shortness of Breath at the same time growing worse: All which Symptoms were at length accompanied with an Atrophy of the Parts, even to the degrees of a Maramus. But yet when his Body was a perfect Skeleton, and the Dropsie at a high Tide (which was very remarkable) he had a brisk and healthful look, and a lovely Countenance, without the least Tincture of a Yellowness, and a good, or rather greedy Appetite, and that to the very day he dyed. From whence I did rightly conjecture, and always told his Friends, as my Opinion, that his Dropsie was truly Chylous, caused by the Chyle flowing into the cavity of the Belly by the Lacteal Vessels, upon some rupture that had been made in them; which appeared very plainly from the Event. For in Tapping of the Child’s Belly, whilst he was yet alive, we took out several Pints of Milky Chyle... ‘‘Tapping’’ beginning with Haly Abbas (?900–994) was performed with a trocar, as illustrated in Clave Medico-Chirurgica Universal by Francisco del Hierra, Madrid, 1730.
—Contributed by Angel R. Colo´n, MD
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