**Quantification of bacterial density**

Quantification of different genotypes was directly performed on 159 DNA samples with defined genotype by using TaqMan real-time-PCR assay. The bacterial density by this technique could be evaluated for 159 *H. pylori*-positive patients and ranged from 1.53×104 to 5.89×1012. In order to evaluate relationship between bacterial concentration and point mutations, samples divided in 9 groups between 1×104 to 6×1012, including 104, 105, 106, 107, 108, 109,1010, 1011 and 1012 groups had 6, 11, 87, 59, 9, 2, 2, 2 and 2 samples respectively (Table 7 and 8).


Table 9. Distribution of 5 different genotypes based on their concentrations

### **Statistical analysis**

Chi-square analysis revealed more relationship between gastritis and age group 30-40 (*p*=0.007), NUD and 40-50 (*p*=0.001), early satiety and 10-20 (*p*=0.05), flatulence and 10-20

Clarithromycin Resistance and *23S rRNA* Mutations in *Helicobacter pylori* 119

mutation may be important since clarithromycin MICs are associated with the type of *23S*

Several studies which done by Pina [34], Marais [28] and Russmann [36] in Europe revealed that A2144G, A2143G and A2143C were more prevalent in Europe respectively. However, the current study showed that A2143G, A2142G, A2143C and A2144G were more prevalent mutation in Iran respectively. Our data showed that wild type strains more related to 106 to 107 bacteria in gastric biopsy specimens of patients. Also, strains with clarithromycin resistance mutation assessed in this study are significantly related to 106 in gastric biopsy which are equal to density of wild type strain. This data revealed that these mutated strains has a same growth rate to wild type, preferentially produced in populations and selected by

The authors offer their thanks and appreciation to the Islamic Azad University, Jahrom and

[1] Chisholm SA, Owen RJ, Teare EL, and Saverymuttu S. 2001. PCR-Based Diagnosis of

[2] Lottspeich C, Schwarzer A, Panthel K, Koletzko S, and Russmann H. 2007. Evaluation of

[3] Kusters JG, van Vliet AHM, and Kuipers EJ. 2006. Pathogenesis of *Helicobacter pylori* 

[4] Correa P, Haenszel W, Cuello C, Tannenbaum S, and Archer M. 1975. A model for gastric

[5] Cirak MY, Akyön Y, and Mégraud F. 2007. Diagnosis of *Helicobacter pylori*. *Helicobacter*. 12

[6] Godoy APO, Ribeiro ML, Benvengo YHB, Vitiello L, Miranda MCB, Mendonça S and

[7] Goldman RC, Zakula D, Flamm R, Beyer J, and Capobianvo J. 1994. Tight Binding of

[8] Liu Z, Shen J, Zhang L, Shen L, Li Q, Zhang B, Zhou J, Gu L, Feng G, Ma J, You WC and

[9] Yilmaz Ö and Demiray E. Clinical role and importance of fl uorescence in situ

*pylori* Ribosomes. *Antimicrob Agents Chemother*. 38(7):1496-1500.

Available: http://www.biomedcentral.com/1471-2180/8/81.

Pedrazzoli Jr J. 2003. Analysis of antimicrobial susceptibility and virulence factors in *Helicobacter pylori* clinical isolates. *BMC Gastroenterology*. 3(20): Available:

Clarithromycin, Its 14-(R)-Hydroxy Metabolite, and Erythromycin to *Helicobacter* 

Deng D. 2008. Prevalence of A2143G mutation of *H. pylori-23S rRNA* in Chinese subjects with and without clarithromycin use history. *BMC Microbiology*, 8(81):

hybridization method in diagnosis of *H. pylori* infection and determination of

Symptomatic Children*. J Clin Microbiol*. 45(6): 1718–1722.

Infection. *Clin Microbiol Reviews*. 19(3): 449–490.

http://www.biomedcentral.com/1471-230X/3/20.

cancer epidemiology. *Lancet*. ii:58–59.

*Helicobacter pylori* Infection and Real-Time Determination of Clarithromycin Resistance Directly from Human Gastric Biopsy Samples. *J Clin Microbiol*. 39(4):

the Novel *Helicobacter pylori* ClariRes Real-Time PCR Assay for Detection and Clarithromycin Susceptibility Testing of *H. pylori* in Stool Specimens from

natural selection force and they can exist and distributed in *H. pylori* population

shahrekord branch for their executive protection of this project.

*rRNA* mutation in *H. pylori* [11].

**10. Acknowledgment** 

1217–1220.

(Suppl. 1): 4–9.

**11. References** 

(*p*=0.038), vomiting and 20-30 (*p*=0.001), pain and 50-60 (*p*=0.003), heaviness after meal and 50-60 (*p*=0.018), early satiety and 60-70 (*p*=0.05).

Also statistical analysis revealed more relationship between wild type and 106 (*p*=0.00) and 107 (*p*=0.00), A2144G and 106 (*p*=0.033), A2143G and 106 (*p*=0.005), A2143C and 106 (*p*=0.005) and A2142G and 106 (*p*=0.015) (Table 9) [61].

Also, results showed relation ships between Ages 10-20 and early satiety (*p*=0.05) and flatulence (*p*=0.038), 20-30 and vomiting (*p*=0.001), 50-60 and pain (*p*=0.003) and heaviness after meal (*p*=0.018) and 50-60 and early satiety (*p*=0.05).

## **9. Conclusion**

In conclusion, we have developed a TaqMan real-time PCR assay that permits accurate, fast, and cost-effective detection of *H. pylori* directly from gastric biopsy specimens as well as detection of clarithromycin resistance. This PCR technique is a good candidate for automated real-time PCR methods allowing simple and rapid detection of *H. pylori* and its resistance to clarithromycin by clinical laboratories which do not practice *H. pylori* culture. Identification of sensitive and resistant strains by using PCR-RFLP method was based on the determination of resistant strains. Therefore, other resistant strains, which mutations cannot be identified by PCR-RFLP method, are considered as sensitive. But, Identification of sensitive and resistant strains by using real-time PCR method was based on the determination of sensitive strains. In the first step, determination of sensitive, resistance and mix of sensitive and resistance strains were done and in the next step, determination of mutations types in resistance and mix strains were done, that leading to accurate diagnosis of the resistant and sensitive strains.

Based on the patterns of competitive growth as well as the individual growth of different clarithromycin resistance mutant strains, Wang and colleagues conclude that the order of preference of competitive accumulation is A2142G A2143G A2142C A2143C (A2142T). If the same is true in vivo, once an A-to-G transient mutation occurs (spontaneously or drug induced), the other types of mutation that exist in the same environment, if any, are likely to be overgrown after a period of time. A-to-C or A-to-T mutants could be isolated only when an A-to-G mutant has not appeared at that particular gastric niche. Their results provide a rational explanation for the mutation pattern observed in clinical isolates [13]. These results are confirmed by Van der Ende and coworkers and discussed that the growth rates of *H. pylori* isolates with the A21423G, A21423C, or A21433G mutation did not differ from that of the wild type, but *H. pylori* isolates with other *23S rDNA* mutations grew more slowly [11].

Based on two opinions, we used real-time PCR for simultaneous identification of *H. pylori*, Clarithromycin resistance point mutations and direct quantification of gastric mucosal density in order to answer to this question; why some clarithromycin resistance point mutations are more prevalent than other mutations. At first; Wang described an additional possible mechanism yet to be identified, by which the A-to-G mutations are preferentially produced in *H. pylori*, may also contribute to the observed predominance of A-to-G mutations [13]. Second; Van der Ende [11] described that; If only a single colony from the primary *H. pylori* populations is used to test for clarithromycin susceptibility, the results can be misinterpreted. Assessment of *23S rRNA* mutations in *H. pylori* directly from biopsy specimens by molecular biological techniques has the advantage that infection with a mixed *H. pylori* population is easily detected. In addition, knowledge of the type of *23S rRNA*

mutation may be important since clarithromycin MICs are associated with the type of *23S rRNA* mutation in *H. pylori* [11].

Several studies which done by Pina [34], Marais [28] and Russmann [36] in Europe revealed that A2144G, A2143G and A2143C were more prevalent in Europe respectively. However, the current study showed that A2143G, A2142G, A2143C and A2144G were more prevalent mutation in Iran respectively. Our data showed that wild type strains more related to 106 to 107 bacteria in gastric biopsy specimens of patients. Also, strains with clarithromycin resistance mutation assessed in this study are significantly related to 106 in gastric biopsy which are equal to density of wild type strain. This data revealed that these mutated strains has a same growth rate to wild type, preferentially produced in populations and selected by natural selection force and they can exist and distributed in *H. pylori* population

#### **10. Acknowledgment**

The authors offer their thanks and appreciation to the Islamic Azad University, Jahrom and shahrekord branch for their executive protection of this project.

#### **11. References**

118 Gastrointestinal Endoscopy

(*p*=0.038), vomiting and 20-30 (*p*=0.001), pain and 50-60 (*p*=0.003), heaviness after meal and

Also statistical analysis revealed more relationship between wild type and 106 (*p*=0.00) and 107 (*p*=0.00), A2144G and 106 (*p*=0.033), A2143G and 106 (*p*=0.005), A2143C and 106 (*p*=0.005)

Also, results showed relation ships between Ages 10-20 and early satiety (*p*=0.05) and flatulence (*p*=0.038), 20-30 and vomiting (*p*=0.001), 50-60 and pain (*p*=0.003) and heaviness

In conclusion, we have developed a TaqMan real-time PCR assay that permits accurate, fast, and cost-effective detection of *H. pylori* directly from gastric biopsy specimens as well as detection of clarithromycin resistance. This PCR technique is a good candidate for automated real-time PCR methods allowing simple and rapid detection of *H. pylori* and its resistance to clarithromycin by clinical laboratories which do not practice *H. pylori* culture. Identification of sensitive and resistant strains by using PCR-RFLP method was based on the determination of resistant strains. Therefore, other resistant strains, which mutations cannot be identified by PCR-RFLP method, are considered as sensitive. But, Identification of sensitive and resistant strains by using real-time PCR method was based on the determination of sensitive strains. In the first step, determination of sensitive, resistance and mix of sensitive and resistance strains were done and in the next step, determination of mutations types in resistance and mix strains were done, that leading to accurate diagnosis

Based on the patterns of competitive growth as well as the individual growth of different clarithromycin resistance mutant strains, Wang and colleagues conclude that the order of preference of competitive accumulation is A2142G A2143G A2142C A2143C (A2142T). If the same is true in vivo, once an A-to-G transient mutation occurs (spontaneously or drug induced), the other types of mutation that exist in the same environment, if any, are likely to be overgrown after a period of time. A-to-C or A-to-T mutants could be isolated only when an A-to-G mutant has not appeared at that particular gastric niche. Their results provide a rational explanation for the mutation pattern observed in clinical isolates [13]. These results are confirmed by Van der Ende and coworkers and discussed that the growth rates of *H. pylori* isolates with the A21423G, A21423C, or A21433G mutation did not differ from that of the wild type, but *H. pylori* isolates with other *23S rDNA* mutations grew more slowly [11]. Based on two opinions, we used real-time PCR for simultaneous identification of *H. pylori*, Clarithromycin resistance point mutations and direct quantification of gastric mucosal density in order to answer to this question; why some clarithromycin resistance point mutations are more prevalent than other mutations. At first; Wang described an additional possible mechanism yet to be identified, by which the A-to-G mutations are preferentially produced in *H. pylori*, may also contribute to the observed predominance of A-to-G mutations [13]. Second; Van der Ende [11] described that; If only a single colony from the primary *H. pylori* populations is used to test for clarithromycin susceptibility, the results can be misinterpreted. Assessment of *23S rRNA* mutations in *H. pylori* directly from biopsy specimens by molecular biological techniques has the advantage that infection with a mixed *H. pylori* population is easily detected. In addition, knowledge of the type of *23S rRNA*

50-60 (*p*=0.018), early satiety and 60-70 (*p*=0.05).

and A2142G and 106 (*p*=0.015) (Table 9) [61].

of the resistant and sensitive strains.

**9. Conclusion** 

after meal (*p*=0.018) and 50-60 and early satiety (*p*=0.05).


Clarithromycin Resistance and *23S rRNA* Mutations in *Helicobacter pylori* 121

[21] Kargar M, Baghernejad M, and Doosti A. 2010. Role of *NADPH-insensitive* 

[22] De Francesco V, Margiotta M, Zullo A, Hassan C, Valle ND, Burattini O, Cea U,

[23] Pan ZJ, Su WW, Tytgat GNJ, Dankert J, and Van Der Ende A. 2002. Assessment of

[25] Falsafi T, Mobasheri F, Nariman F, and Najafi M. 2004. Susceptibilities to Different

[26] Kargar M, Baghernejad M, Doosti A, and Ghodsi M. "Evaluation and prevalence of

[27] Kato S, Fujimura S, Udagawa H, Shimizu T, Maisawa S, Ozawa K, and Iinuma K. 2002.

[28] Marais A, Monteiro L, Occhialini A, Pina M, Lamouliatte H, Mégraud F. 1999. Direct

[29] Matsuoka M, Yoshida Y, Hayakawa K, Fukuchi S, Sugano K. 1999. Simultaneous

[31] Washington WJ, Stephen A, William J, Koneman E, Procop G, Scheckenberger P, (2006).

[32] Pina M, Occhialini A, Monteiro L, Doermann HP, and Megraud F. 1998. Detection of

[34] Kalach N, Benhamou PH, Campeotto F, Bergeret M, Dupont C, and Raymond J. 2001.

by Hybridization in Liquid Phase. *J Clin Microbiol*. 36(11): 3285–3290. [33] Kalach N, Bergeret M, Benhamou PH, Dupont C, and Raymond J. 2001. High Levels of

in patients with no history of clarithromycin exposure. *Gut*. 45:503–507. [30] Taylor DE, Ge Z, and Purych D, Lo T, and Hiratsuka K. 1997. Cloning and Sequence

Medical Center of Tehran, Iran. *J Clin Microbiol*. 42(1): 387–389.

*DARU*. 18(2):137-140.

Analysis*. J Clin Microbiol*. 42(8): 3901–3903.

medicine, Tehran, Iran, 2008 December 20-22:

*Microbiol*. 40(2): 649–653.

*Agents Ch*. 41(12): 2621–2628.

Lippincott Williams & Wilkins. 765-773.

Children*. J Clin Microbiol*. 39(1): 394–397.

*Antimicrob Agents Chemother*. 45(7): 2134–2135.

23:429–435.

*nitroreductase* gene to metronidazole resistance of *Helicobacter pylori* strains.

Stoppino G, Amoruso A, Stella F, Morini S, Panella C, and Ierardi E. 2006. Primary clarithromycin resistance in Italy assessed on *Helicobacter pylori* DNA sequences by TaqMan real-time polymerase chain reaction. *Aliment Pharm Ther*.

Clarithromycin-Resistant *Helicobacter pylori* among Patients in Shanghai and Guangzhou, China, by Primer-Mismatch PCR. *J Clin Microbiol*. 40(1): 259–261. [24] Taneike I, Suzuki K, Nakagawa S, and Yamamoto T. 2004. Intrafamilial Spread of the

Same Clarithromycin-Resistant *Helicobacter pylori* Infection Confirmed by Molecular

Antibiotics of *Helicobacter pylori* Strains Isolated from Patients at the Pediatric

clarithromycin resistance point mutations in *Helicobacter pylori* in Shahrekord," in Proceedings of the 17th Iranian congress on infectious disease and tropical

Antibiotic Resistance of *Helicobacter pylori* Strains in Japanese Children*. J Clin* 

detection of *Helicobacter pylori* resistance to macrolides by a polymerase chain reaction/DNA enzyme immunoassay in gastric biopsy specimens. *Gut*. 44:463–467.

colonisation of *Helicobacter pylori* with and without mutations in the *23S rRNA* gene

Analysis of Two Copies of a *23S rRNA* Gene from *Helicobacter pylori* and Association of Clarithromycin Resistance with *23S rRNA* Mutations. *Antimicrob* 

Color atlas and textbook of diagnostic microbiology. 6th ed. Philadelphia:

Point Mutations Associated with Resistance of *Helicobacter pylori* to Clarithromycin

Resistance to Metronidazole and Clarithromycin in *Helicobacter pylori* Strains in

Clarithromycin Resistance and Eradication of *Helicobacter pylori* in Children*.* 

clarithromycin resistance in *H pylori* eradication therapy. *World J Gastroenterol* 2007;13(5):671-675.


[10] Lascols C, Lamarque D, Costa JM, Copie-Bergman C, Glaunec JML, Deforges L, Soussy

[11] Van Der Eede A, Van Doorn LJ, Rooijakkers S, Feller M, Tytgat GNJ, and Dankert J.

[12] Fontana C, Favaro M, Minelli S, Criscuolo AA, Pietroiusti A, Galante A, and Favalli C.

[13] Wang GE and Taylor DE. 1998. Site-Specific Mutations in the 23S rRNA Gene of

Streptogramin B Antibiotics. *Antimicrob Agents Chemother*. 42(8): 1952–1958. [14] Pfister P, Corti N, Hobbie S, Bruell C, Zarivach R, Yonath A, and Bottger EC. 2005. 23S

[16] Oleastro M, Menard A, Santos A, Lamouliatte H, Monteiro L, Barthelemy P, and

[17] Chisholm SA, Owen RJ, Teare EL, and Saverymuttu S. 2001. PCR-Based Diagnosis of

[18] Trebesius K, Panthel K, Strobel S, Vogt K, Faller G, Kirchner T, Kist M, Heesemann J,

[20] Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, Yao JDC, Wengenack

macrolide resistance mutation 2058A→G. *PNAS*. 102(14): 5180–5185. [15] Woo HY, Park DI, Park H, Kim MK, Kim DH, Kim IS, and Kim YJ. 2009. Dual-Priming

2007;13(5):671-675.

2648–2651.

46(12): 3765–3769.

*Helicobacter*. 14:22–28.

*Microbiol*. 41(1): 397–402.

1217–1220.

2326.

19(1): 165–256.

*J Clin Microbiol*. 41(10): 4573–4577.

clarithromycin resistance in *H pylori* eradication therapy. *World J Gastroenterol*

CJ, Petit JC, Delchier JC, and Tankovic J. 2003. Fast and Accurate Quantitative Detection of *Helicobacter pylori* and Identification of Clarithromycin Resistance Mutations in *H*. *pylori* Isolates from Gastric Biopsy Specimens by Real-Time PCR.

2001. Clarithromycin-Susceptible and -Resistant *Helicobacter pylori* Isolates with Identical Randomly Amplified Polymorphic DNA-PCR Genotypes Cultured from Single Gastric Biopsy Specimens Prior to Antibiotic Therapy. *J Clin Microbiol*. 39(7):

2002. New Site of Modification of *23S rRNA* Associated with Clarithromycin Resistance of *Helicobacter pylori* Clinical Isolates. *Antimicrob Agents Chemother*.

*Helicobacter pylori* Confer Two Types of Resistance to Macrolide-Lincosamide-

rRNA base pair 2057–2611 determines ketolide susceptibility and fitness cost of the

Oligonucleotide-Based Multiplex PCR for the Detection of *Helicobacter pylori* and Determination of Clarithromycin Resistance with Gastric Biopsy Specimens.

Me´graud F. 2003. Real-Time PCR Assay for Rapid and Accurate Detection of Point Mutations Conferring Resistance to Clarithromycin in *Helicobacter pylori*. *J Clin* 

*Helicobacter pylori* Infection and Real-Time Determination of Clarithromycin Resistance Directly from Human Gastric Biopsy Samples. *J Clin Microbiol*. 39(4):

and Haas R. 2000. Rapid and specific detection of *Helicobacter pylori* macrolide resistance in gastric tissue by fluorescent in situ hybridization. *Gut*. 46:608–614. [19] Burucoa C, Garnier M, Silvain C, and Fauchere JL. Quadruplex Real-Time PCR Assay

Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to *Helicobacter pylori*. *J Clin Microbiol* 2008; 46(7):2320–

NL, Rosenblatt JE, Cockerill FR, and Smith TF. 2006. Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. *Clin Microbiol Reviews*.


Clarithromycin Resistance and *23S rRNA* Mutations in *Helicobacter pylori* 123

[46] Sun QJ, Liang X, Zheng Q, Gu WQ, Liu WZ, Xiao SD, Lu H. 2010. Resistance of

[47] Maeda S, Yoshida H, Ogura K, Kanai F, Shiratori Y and Omata M. 1998. *Helicobacter* 

[48] Maeda S, Yoshida H, Matsunaga H, Ogura K, Kawamata O, Shiratori Y, and Omata M.

[49] Matsumura M, YOKO Hikiba Y, KEIJI Ogura K, GOICHI Togo G, Tsukud I, Ushikawa

[50] Kato S, Fujimura S, Udagawa H, Shimizu T, Maisawa S, Ozawa K, and Iinuma K. 2002.

[51] Ahmad N, Zakaria WR, Abdullah SA, Mohamed R. 2009. Characterization of

[52] Kumala W, and Rani A. 2006. Pattern Of *Helicobacter pylori* Isolate Resistance To

[53] Lee JH, Shin JH, Roe IH, Sohn SG, Lee JH, Kang GH, Lee HK, Jeong BC, and Lee SH.

[54] Kargar M, Baghernejad M, Doosti A, Ghorbani-Dalini S. 2011. Clarithromycin resistance

[55] Ghorbani-Dalini S. 2010. Direct detection and identification of Clarithromycin resistance

[56] Farshad S, Alborzi A, Japoni A, Ranjbar R, Hosseini-Asl K, Badiee P, Amin-Shahidi

[57] Ben Mansour K, Burucoa C, Zribi M, Masmoudi A, Karoui S, Kallel L, Chouaib S, Matri

[58] Llanes R, Soria C, Nagashima S, Kobayashi N, Gala A, Guzmán D, Feliciano O, Valdés

http://www.ann-clinmicrob.com/content/9/1/22.

*J Health Popul Nutr*. 28(2):124-129.

Infected Adults. *Antimicrob Agents Chemother*. 49(4): 1600–1603.

Homoduplex Formation Assay. *J Clin Microbiol*. 38(1): 210–214.

with clarithromycin resistance. *Gut*. 43:317–321.

the Bacterium. *J Clin Microbiol*. 39(2): 691–695.

16(40): 5118-5121.

*Microbiol*. 40(2): 649–653.

2011. In Press

University.

5751.

*J Gastroenterol*. 15(25): 3161-3165.

*J Trop Med Public Health*. 37(5):970-974.

*Helicobacter pylori* to antibiotics from 2000 to 2009 in Shanghai. *World J Gastroenterol*.

*pylori* specific nested PCR assay for the detection of *23S rRNA* mutation associated

2000. Detection of Clarithromycin-Resistant *Helicobacter pylori* Strains by a Preferential

K, Shiratori Y, and Omata M. 2001, Rapid Detection of Mutations in the *23S rRNA* Gene of *Helicobacter pylori* That Confers Resistance to Clarithromycin Treatment to

Antibiotic Resistance of *Helicobacter pylori* Strains in Japanese Children*. J Clin* 

clarithromycin resistance in Malaysian isolates of *Helicobacter pylori*. *World* 

Fluoroquinolones, Amoxicilin, Clarithromycin and Metronidazoles. *Southeast Asian* 

2005. Impact of Clarithromycin Resistance on Eradication of *Helicobacter pylori* in

and *23S rRNA* Mutations in *Helicobacter pylori* isolates from Iran. *Afr J Microbiol Res*.

point mutations in *Helicobacter pylori* strains isolated from biopsy specimens by Real-Time PCR assay. Department of Biology, Jahrom Branch, Islamic Azad

M, Hosseini M. 2010.Antimicrobial susceptibility of Helicobacter pylori strains isolated from patients in Shiraz, Southern Iran. *World J Gastroenterol*. 16(45): 5746-

S, Fekih M, Zarrouk S, Labbene M, Boubaker J, Cheikh I, Ben Hriz M, Siala N, Ayadi A, Filali A, Ben Mami N, Najjar T, Maherzi A, Sfar MT, Fendri C. 2010. Primary resistance to clarithromycin, metronidazole and amoxicillin of *Helicobacter pylori* isolated from Tunisian patients with peptic ulcers and gastritis: a prospective multicentre study. *Annals of Clin Microbiol and Antimicrob*. 2010, 9:22. Available:

L, Gutiérrez O, Fernández H, Llop A, and Wada A. 2010. Phenotypic and Genetic Characterization of Antimicrobial Profiles of *Helicobacter pylori* Strains in Cuba.


[35] Cambau E, Allerheiligen V, Coulon C, Corbel C, Lascols C, Deforges L, Soussy CJ,

[36] Russmann H, Adler K, Haas R, Gebert B, Koletzko S, and Heesemann J. 2001. Rapid

[37] Koletzko S, Richy F, Bontems P, Crone J, Kalach N, Monteiro ML, Gottrand F, Celinska-

[38] Fontana C, Favaro M, Pietroiusti A, Pistoia ES, Galante A, and Favalli C. 2003. Detection

[39] Ryan KA, Van Doorn LJ, Moran AP, Glennon M, Smith T, and Maher M. 2001.

[40] Agudo S, Perez-Perez G, Alarcon T, and Lopez-Brea M. 2010. High Prevalence of

[42] Torres J, Camorling-Ponce M , Perez-Perez G , Garza AMD, Dehesa M, Gonzalez-

[43] Vasquez A, Valdez Y, Gilman RH, Mcdonald JJ, Westblom TU, Berg D, Mayta H,

[44] Alarcon T, Vega AE, Domingo D, Martínez MJ, and Lopez-Brea M. 2003. Clarithromycin

[45] Stone GG, Shortridge D, Versalovich J, Beyer J, Flamm RK, Graham DY, Ghoneim AT,

with Resistance in Madrid, Spain*. J Clin Microbiol*. 48(10): 3703–3707. [41] Magalhães PP, Queiroz DMM, Barbosa DVC, Rocha GA, Mendes EN, Santos A, Corrêa

from children living in Europe. *Gut*. 55:1711–1716.

*Microbiol*. 47(11): 3600–3607.

4142–4144.

41(8): 3636–3640.

2677–2680.

*Microbiol*. 39(5): 1978–1980.

*Agents Chemother*. 46(6): 2021–2023.

Format. *J Clin Microbiol*. 34(5): 1232–1234.

Polymorphism Analysis. *J Clin Microbiol*. 41(1): 486–488.

*pylori*. *Antimicrob Agents Chemother*. 41(3): 712–714.

Delchier JC, and Megraud F. 2009. Evaluation of a New Test, GenoType HelicoDR, for Molecular Detection of Antibiotic Resistance in *Helicobacter pylori*. *J Clin* 

and Accurate Determination of Genotypic Clarithromycin Resistance in Cultured *Helicobacter pylori* by Fluorescent in Situ Hybridization*. J Clin Microbiol*. 39(11):

Cedro D, Roma-Giannikou E, Orderda G, Kolacek S, Urruzuno P, Mart´nez-Go´mez MJ, Casswall T, Ashorn M, Bodanszky H, Me´graud F. 2006. Prospective multicentre study on antibiotic resistance of Helicobacter pylori strains obtained

of Clarithromycin-Resistant *Helicobacter pylori* in Stool Samples. *J Clin Microbiol*.

Evaluation of Clarithromycin Resistance and *cagA* and *vacA* Genotyping of *Helicobacter pylori* Strains from the West of Ireland Using Line Probe Assays*. J Clin* 

Clarithromycin-Resistant *Helicobacter pylori* Strains and Risk Factors Associated

PRV, Rocha AMC, Teixeira LM, and de Oliveira CA. 2002. *Helicobacter pylori*  Primary Resistance to Metronidazole and Clarithromycin in Brazil. *Antimicrob* 

Valencia G, and Munoz O. 2001. Increasing Multidrug Resistance in *Helicobacter pylori* Strains Isolated from Children and Adults in Mexico. *J Clin Microbiol*. 39(7):

Gutierrez V, and The Gastrointestinal Phisiology Working Group Of Universidad Peruana Cayetano Heredia and The John Hopkins University. 1996. Metronidazole and Clarithromycin Resistance in *Helicobacter pylori* Determined by Measuring MICs of Antimicrobial Agents in Color Indicator Egg Yolk Agar in a Miniwell

Resistance among *Helicobacter pylori* Strains Isolated from Children: Prevalence and Study of Mechanism of Resistance by PCR-Restriction Fragment Length

and Tanaka SK. 1997. A PCR-Oligonucleotide Ligation Assay To Determine the Prevalence of *23S rRNA* Gene Mutations in Clarithromycin-Resistant *Helicobacter* 


**10** 

Vui Heng Chong

*Brunei Darussalam* 

**Heterotopic Gastric Mucosal** 

**Patch of the Proximal Esophagus** 

*Division of Gastroenterology and Hepatology, Department of Medicine,* 

*Raja Isteri Pengiran Anak Saleha (RIPAS) Hospital, Bandar Seri Begawan BA 1710,* 

Heterotopic gastric mucosa (HGM) is abnormally placed gastric mucosa outside of the stomach and can be found almost anywhere within the gastrointestinal tract (von Rahden et al., 2004). HGM is most commonly found in the esophagus. The most widely known HGM in the gastrointestinal tract is in the Meckel's diverticulum. However, HGM is also found in the other part of the gastrointestinal tract including the tongue (Melato & Ferlito, 1975; Ortiz, 1982; Surana, 1993), duodenum (Kibria et al., 2009; Mann, 2000), gallbladder (Hayama et al., 2010; Popkharitoy et al., 2008), jejunum (Boybeyi et al., 2008; Nowak & Deppisch, 1998), ileum (Chan et al., 1999; Erez et al., 1991), rectum (Garmendia et al., 2007; Vieth et al., 2005) and anus (Steele et al., 2004). Interestingly, HGM of the umbilicus, a part of remnant

HGM patch (HGMP) of the proximal esophagus, also referred to as cervical inlet patch (CIP) is typically found in the proximal esophagus. It can also be found in the other part of the esophagus (Borhan-Manesh & Franum, 1991; Katsanos et al., 2010). On endoscopy, HGMP/CIP is clearly distinct from the esophageal squamous mucosa. HGMP/CIP is widely considered to be congenital in nature. However, it has also been proposed to be an acquired condition (Avidan et al., 2001; Meining & Baubouj, 2010). In clinical practice, HGMP/CIP is an under-recognized condition. The incidence reported in the literature varies with lower estimates in the earlier endoscopic studies (von Rahden et al., 2004). Later studies reported higher incidence (Ohara, 2010). The highest incidence was reported in an autopsy study (von Rahden et al., 2004). Use of newer endoscopic modalities has been

HGMP/CIP is largely asymptomatic and is incidental findings during endoscopy evaluations for other gastrointestinal complaints. Commonly reported symptoms are those symptoms complex referred to as extra-esophageal manifestations of gastro-esophagael reflux disorders. Other common upper aero-digestive disorders have also been linked to HGMP/CIP. Despite the benign nature of HGMP/CIP, serious and important complications have been reported (von Rahden et al., 2004). Furthermore, associations with higher frequencies of laryngopharyngeal malignancies have also been reported (Basseri et al., 2009). A clinico-pathologic classification has been proposed which categorized HGMP/CIP into five distinct groups based on clinical, endoscopic and histological findings (von Rahden et

alimentary tract has also been report (Heo et al., 2010).

reported to increase the pick up rates of HGMP/CIP.

**1. Introduction** 

