Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: J Dig Dis. 2012 Jan;13(1):54–59. doi: 10.1111/j.1751-2980.2011.00549.x

Detection of clarithromycin resistance in Helicobacter pylori following noncryogenic storage of rapid urease tests for 30 days

Yuan LI 1, Emiko RIMBARA 1, Selvi THIRUMURTHI 1,*, Alba TRESPALACIOS 1, Rita REDDY 1, Saman SABOUNCHI 1, Taraq Assed ATTUMI 1, David Yates GRAHAM 1
PMCID: PMC3245639  NIHMSID: NIHMS335937  PMID: 22188917

Abstract

Objective

Traditional Helicobacter pylori (H. pylori) eradication therapy has been undermined by increasing antimicrobial, especially clarithromycin, resistance. Susceptibility testing in most areas is difficult or unavailable. We assessed whether gastric biopsies stored at room temperature in a rapid urease test were suitable for H. pylori clarithromycin susceptibility testing.

Methods

After 30 days of storage at room temperature, DNA was extracted from a gastric biopsy present within a rapid urease test (Hpfast). H. pylori status and clarithromycin susceptibility were evaluated used H. pylori-specific PCR for ureA, vacA, and allele-specific primer-polymerase chain reaction of the 23S rRNA genes. The PCR results were compared with histology, RUT, and culture. H. pylori positive was defined as RUT and either culture or histology positive; H. pylori negative as RUT, culture and histology negative.

Results

Samples from 31 subjects were evaluated; 11 were H. pylori positive including 9 by culture; 8 of which had allele-specific primer-PCR results from the RUT specimen for the detection of mutations of the 23S rRNA gene. When both tests were available, culture and PCR results were concordant in 8/8 (100%). Fifteen of the 20 histology, RUT and culture negative cases had all 3 PCR’s negative. In one all 3 were positive; in 3 only the 23S rRNA was positive and in 1 only ureA was positive.

Conclusion

Gastric biopsy specimens stored within the gel of an RUT for 30 days can be used to for molecular testing confirm the diagnosis of H. pylori infection and test for clarithromycin susceptibility.

Keywords: Susceptibility tests, clarithromycin resistance, polymerase chain reaction, noncryogenic storage, rapid urease tests

Introduction

Helicobacter pylori (H. pylori) is a major human pathogen etiologically involved the pathogenesis of gastritis, peptic ulcer disease, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma1. H. pylori eradication therapy is recommended whenever an H. pylori infection is detected2. The most successful H. pylori eradication regimens contain an antisecretory agent and two or more antimicrobial agents. Clarithromycin is one of the most widely used antimicrobials in H. pylori eradication regimens. However, clarithromycin resistance has been increasing worldwide and currently clarithromycin resistance is the major cause of treatment failure3–5. The decision regarding which H. pylori treatment regimen to use differs from most other common infections in that culture and susceptibility testing is often not available and clinicians are required to make empiric choices in terms of a treatment regimen. Regularly updated data regarding local and regional patterns of resistance would be especially useful to allow clinicians to choose among eradication regimens but such data are rarely available.

One approach to obtaining susceptibility data despite lack of culture facilities is to use molecular methods. Clarithromycin susceptibility is especially amenable to such a strategy as clarithromycin resistance is most commonly caused by point mutations of the 23S ribosomal ribonucleic acid (rRNA) gene, the main component of the 50S subunit. The most common mutations associated with resistance are at position 2142/43 (A2142 to G/C/T; A2143 to G/C) in the peptidyltransferase region from the V domain. Mutations result in prevention of clarithromycin binding to 50S ribosomal subunit such that the drug becomes ineffective6. The major mutations are transitions from A to G or T to C and transversion of A to T or G to C from adenine to guanine at positions 2142 and 2143 of the 23S rRNA gene (A2142G and A2143G)7. Many methods to detect these mutations are available including direct sequence, polymerase chain reaction (PCR), restriction fragment length polymorphism 8, real-time PCR assays9, microelectronic chip array 10 and fluorescent in situ hybridization 11.

Molecular techniques to assess clarithromycin resistance often use gastric mucosal biopsies collected specifically for that purpose. However, biopsy samples originally taken for rapid urease testing have also been utilized 12. In developing countries endoscopy is often readily available and thus collection of biopsies is not a limiting factor. However, subsequent molecular testing at off site facilities requires the ability to reliably freeze, store and ship frozen samples and this often proves difficult or impossible.

The aim of this study was to determine whether gastric mucosal biopsy samples used for rapid urease testing and stored for 30 days at room temperature in situ in the RUT gel could also be used successfully for molecular testing to both confirm the presence of H. pylori infection and to determine clarithromycin susceptibility.

It was hypothesized that if this were possible, it should theoretically be possible to easily and reliably determine the prevalence of clarithromycin resistance despite the lack of an adequate cryogenic storage capability which would allow susceptibility information to be provided to clinicians and also used to help interpret the outcome of clinical trials.

Materials and methods

Patients and H. pylori infection diagnosis

Patients who underwent upper endoscopy at the Ben Taub General Hospital, Houston, Texas between April and July, 2010 were enrolled. After informed consent was obtained, 3 gastric biopsy biopsies were obtained at the incisura: one for the RUT, one for histology and one for culture. Additional biopsies were taken from the gastric antrum and body for histology. Biopsy forceps were large capacity style with needle (Boston Scientific Radial Jaw 4, Natick, MA, USA). The biopsy for the rapid urease test (RUT) (Hpfast; CheckMed systems, Camp Hill, PA, USA) was placed in the RUT gel which was then placed in an RUT warmer at 37C (Helicoview; GI Supply, Camp Hill, PA, USA) for up to 24 hours. The sample was interpreted after 24 hours and the RUT test containing the biopsy was then stored in at room temperature for 30 days after which the biopsy was recovered from the gel and the deoxyribonucleic acid (DNA) was extracted (see below).

The specimen for culture was immediately placed in H. pylori transport media and frozen at −70°C until cultured 13. Biopsies for histology were sent to the clinical laboratory for staining with hematoxylin and eosin and for immunohistochemical stains for H. pylori.

The study was approved by the institutional review board at Baylor College of Medicine and all subjects signed informed consent prior to entry.

Culture and clarithromycin acceptability testing

Mucosal biopsies were crushed between frosted ends of sterilized slides and the homogenized tissue suspension was inoculated on non-selective and H. pylori special peptone agar (HPSPA) selective plates consisting of a horse blood agar (HBA) plate (nonselective medium) or a HBA plate containing 10 μg/mL nalidixic acid, 5 μg/mL trimethoprim, 3 μg/mL, vancomycin and 2 μg/mL amphotericin B (selective medium). Plates were incubated under microaerophilic conditions at 37°C for up to 14 days as previously described 41. H. pylori isolates were defined as Gram-negative spiral-shaped bacilli that were oxidase, catalase, and urease positive. Fresh growth from brain heart infusion plates containing 7% horse blood was harvested in 6 mL of saline and adjusted to turbidity equivalent to a No. 2 McFarland turbidity standard. The suspended inoculum was applied uniformly on the entire surface of Mueller Hinton Agar plates containing 5% sheep blood were covered with 0.3–0.5 mL of each suspended inoculum to produce a lawn of bacterial growth. A clarithromycin E-test strip (AB Biodisk, Solna, Sweden) was applied to the surface of plate and after incubation in microaerophilic conditions at 37°C for 72 hours; results were read according to the manufacturer’s guide. The minimal inhibitory concentration (MIC) was recorded as the lowest concentration that inhibited visible growth of organisms. For clarithromycin, the cut-off for resistance was an MIC of >1 mg/L.

DNA extraction from the gastric biopsy samples

DNA was extracted from gastric biopsy using QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacture’s instruction. The extracted DNA was stored at −70°C until use.

PCR for the ureA, vacA gene and allele-specific primer-PCR for the 23S rRNA gene

PCR for the ureA gene of H. pylori was performed using GoTaq polymerase (Promega, WI, U.S.A.) and the primers ureA_197F (5′-AACCGGATGATGTGATGGAT-3′) and ureA_413R (5′-GGTCTGTCGCCAACATTTTT-3′) reported by Kim et al 15. PCR condition for the ureA gene is performed as follows: Denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 20 sec, and extension at 72°C for 30 sec, with final extension 72°C for 5 min.

PCR for the vacA gene was performed using GoTaq polymerase (Promega, WI, USA) and the primers vacA_8U21F (5′-TACAACAAACACACCGCAAAA-3′) and vacA_131L20AR (5′-TGTAGCGATACCCCCAACAA-3′) reported by Ayala et al 16. PCR condition for the vacA gene is performed as follows: Denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 20 s, and extension at 72°C for 30 s, with final extension 72°C for 5 min. Allele-specific primer-PCR for the detection of the 23S rRNA gene mutations, A2142G and A2143G, were performed according to the method reported by Furuta et al. 17. KOD Xtreme™ Hot start DNA polymerase kit (Toyobo, Osaka, Japan) was used for PCR System. The total volume of PCR system was 10 μL which included approximately 0.1 μg of DNA, 0.2 U KOD Xtreme Hot Start DNA Polymerase, 4 μmol dNTPs (each), 5 μL 2X Xtreme Buffer and 0.3 nmol/L of primers FP-1 (5′-TCGAAGGTTAAGAGGATGCGTCAGTC-3 ′), R P-1(5′-GACTCCATAAGAGCCAAAGCCCTTAC-3′), RP2142G(5′-AGTAAAGGTCCACGGGGTATTCC-3′), and FP2143G(5′-CCGCGGCAAGACAGAGA -3′). allele-specific primer-PCR was performed as follows: denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 98°C for 10 s, annealing at 65°C for 30 s, and extension at 68°C for 20 s, with final extension 72°C for 2 min. The 320bp fragment indicates the presence of wild-type H. pylori 23S rRNA gene, while 238 bp and 118 bp fragments indicates the presence of a mutation A2142G and A2143G, respectively, which cause clarithromycin resistance. All PCR products were subjected to electrophoresis on a 2% agarose gel along with a 100bp DNA marker.

Analyses

Those performing the PCR, culture, and histology were blinded to the histological, RUT, and culture results until all analyses were completed. For primary analysis we defined a positive test as a positive RUT and one other test (either culture or histology). The requirement for a positive RUT was based on the premise that positive results of both the RUT and the molecular testing requires that the specimen utilized contain adequate numbers of bacterial cells (i.e., a positive RUT would guard against the problem that in vivo the infection may exhibit patchiness in terms of presence and intensity of the infection). For example, a single biopsy from an H. pylori infected patient might be from an area of intestinal metaplasia which would contain few if any H. pylori leading to a falsely negative result. H. pylori negative was defined as all three tests negative. To better understand the limitations of this approach, we separately examined cases in which the RUT was negative and both culture and histology were positive.

Results

Thirty-one subjects (21 women, 10 men, average age 55.2 years) were entered including 11 (33.3%) with active H. pylori infections. Per protocol two positive tests were required to define an active H. pylori infection and 8 (72.7%) had positive culture and histology, two had positive RUT and histology (18.1%), and one had positive RUT and culture (9%).

In 9 of the 11 (82%) H. pylori positive cases the PCR for ureA, vacA, and the H. pylori 23S rRNA gene were also positive (Table 1) and all three of the 3 PCR’s were negative in two cases (Table 1). Fifteen of the 20 H. pylori negative cases (75%) were also negative by all three PCR’s. Three of the samples in which the RUT, culture, and histology were negative had negative PCR for both the ureA and vacA genes but the PCR for the 23S rRNA gene was positive. In addition, one case negative by RUT, histology and culture was positive for ureA, vacA, and the 23S rRNA gene. We believe this likely represents the presence of a true positive where the density of H. pylori was below the level required for detection using traditional less sensitive methods. The remaining three cases in which only the 23S rRNA gene was positive were judged to most likely represent false positive results possibly related to the presence of cross reacting organisms 18.

Table 1.

Results of testing for H. pylori and susceptibility from the 31 subjects entered.

RUT Histology Culture MIC mg/L ureA vacA 23S rRNA genotype
positive positive positive <0.016 positive positive positive wt
positive positive positive 12 positive positive positive 2142G
positive positive positive <0.016 positive positive positive wt
positive positive positive 0.016 positive positive positive wt
positive positive positive <0.016 positive positive positive wt
positive positive positive <0.016 positive positive positive wt
positive positive positive <0.016 positive positive positive wt
positive positive positive 0.032 negative negative negative -
positive positive negative - positive positive positive wt
positive positive negative - positive positive positive wt
positive negative positive 3 negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - positive negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative - negative negative negative -
negative negative negative negative negative positive A2143G
negative negative negative positive positive positive wt
negative negative negative negative negative positive wt
negative negative negative negative negative positive A2143G
Open in a new tab

RUT, rapid urease test; MIC, minimum inhibitory concentration; wt, wild type; A2142G, mutation from adenine to guanine at position 2142; A2143G, mutation from adenine to guanine at position 2143.

The results of clarithromycin susceptibility testing of the 9 H. pylori isolates were compared with the results of allele-specific primer-PCR obtained from the biopsies that had been stored at room temperature for 30 days in the RUT gel. Two isolates were clarithromycin resistant (MIC = 12 mg/L and 3 mg/L) and one had a negative PCR; the other allele-specific primer-PCR of the biopsy sample showed an A2142G mutation consistent with the presence of resistance. Thus, the results of susceptibility testing in which the two methods were both positive were in concordance in 8/8 (100%). In two cases the culture was positive but the PCR was negative. Follow up of the two cases negative by PCR (one susceptible and one resistant by culture) was done. The susceptible strain was recultured from the original gastric biopsy material and the PCR confirmed a susceptible genotype (wild type). The other strain was not recoverable from the stock and thus PCR could not be done.

Discussion

Rapid urease testing is a convenient and inexpensive way to diagnosis H. pylori infection and is used worldwide clinically and for research. After interpretation of the RUT, the biopsy specimen utilized for RUT testing is typically discarded. Here, we asked whether it might be possible to store the RUT device with the sample remaining in the gel noncryogenetically (at room temperature) and still utilize the sample to confirm H. pylori status, and more importantly, to test whether the organism was susceptible to clarithromycin. Our results confirmed the hypothesis that gastric biopsies obtained for rapid urease testing could be stored in the RUT get at room temperature and still be used for molecular testing for the presence of the infection and for clarithromycin susceptibility. This technique could be especially useful to examine the effect of clarithromycin resistance clinically and in clinical trials, particularly trials done in areas where H. pylori infections were prevalent but lacking in advanced medical facilities that offered culture or molecular susceptibility testing. Simply mailing the RUT sample to an off-site facility would theoretically allow susceptibility testing for regions that lack reliable electrical supplies and cold storage facilities. We eagerly await testing of this hypothesis.

There are several caveats regarding the study and the data. First, the study design was based on the premise that a positive RUT result would increase the likelihood that the specimen would contain a sufficient number of H. pylori (i.e., DNA) to allow clarithromycin susceptibility testing using molecular methods. To reduce the chance of false positive results from unrelated organisms (e.g., oral flora) that might be present, we utilized PCR assays for both the ureA and vacA genes to confirm the presence of H. pylori in the specimen 19–21.

Two RUT’s and one other test positive samples were negative for all three PCR’s despite being RUT positive. Of interest 4 of the 20 (20%) presumably H. pylori negative cases had positive PCR results for the presence of the H. pylori 23S rRNA gene and in 3 neither the ureA nor the vacA genes were present (i.e. the confirmatory molecular test for the presence of H. pylori was negative) which is most consistent with notion that the PCR result for the H. pylori 23S rRNA gene was a false positive result and confirming the importance of testing for the presence of H. pylori by the presence of a positive RUT, PCR or both. Finally, it is unknown whether gel type RUT tests other than the one we used would provide similar or identical results.

This pilot study showed that gastric biopsy specimens positive by RUT using the Hpfast gel-type RUT, and presumably other gel type RUT tests, can be used to evaluate clarithromycin susceptibility using molecular testing. Subsequent studies are underway to refine the methods including evaluating other methods of storing the biopsy sample, the effect of the size of the biopsy, whether more than one biopsy would reduce false negative results, whether the samples can be mailed, and the effect of higher and varying temperature (i.e., simulating tropical regions).

In conclusion, we showed that gastric biopsy specimens in an RUT gel can be used to confirm the diagnosis of H. pylori infection and to test clarithromycin susceptibility despite having been stored at room temperature for 30 days. This method could potentially be used for genotyping of pathogenetic genes of H. pylori and should prove useful for monitoring the susceptibility of H. pylori to antimicrobial agents.

Acknowledgments

Dr. Graham is supported in part by the Office of Research and Development Medical Research Service Department of Veterans Affairs, Public Health Service grant DK56338 which funds the Texas Medical Center Digestive Diseases Center, DK067366 and CA116845. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the VA or NIH. Dr. Graham is a unpaid consultant for Novartis in relation to vaccine development for treatment or prevention of H. pylori infection. Dr. Graham is a also a paid consultant for Otsuka Pharmaceuticals regarding diagnostic testing until has received royalties on the Baylor College of Medicine patent covering materials related to 13C-urea breath test. The other authors have nothing to declare.

References

  • 1.Graham DY, Sung JY. Helicobacter pylori. In: Feldman M, Friedman LS, Brandt LJ, editors. Sleisenger & Fordtran’s Gastrointestinal and liver disease. Pathophysiology, diagnosis, management. Philadelphia: WB Saunders Co; 2006. pp. 1049–1066. [Google Scholar]
  • 2.Malfertheiner P, Megraud F, O’Morain C, et al. Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report. Gut. 2007;56:772–781. doi: 10.1136/gut.2006.101634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Horiki N, Omata F, Uemura M, et al. Annual change of primary resistance to clarithromycin among Helicobacter pylori isolates from 1996 through 2008 in Japan. Helicobacter. 2009;14:86–90. doi: 10.1111/j.1523-5378.2009.00714.x. [DOI] [PubMed] [Google Scholar]
  • 4.Megraud F. H pylori antibiotic resistance: prevalence, importance, and advances in testing. Gut. 2004;53:1374–1384. doi: 10.1136/gut.2003.022111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Graham DY, Fischbach L. Helicobacter pylori treatment in the era of increasing antibiotic resistance. Gut. 2010;59:1143–1153. doi: 10.1136/gut.2009.192757. [DOI] [PubMed] [Google Scholar]
  • 6.Taylor DE, Ge Z, Purych D, Lo T, Hiratsuka K. Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations. Antimicrob Agents Chemother. 1997;41:2621–2628. doi: 10.1128/aac.41.12.2621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Versalovic J, Shortridge D, Kibler K, et al. Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori. Antimicrob Agents Chemother. 1996;40:477–480. doi: 10.1128/aac.40.2.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Alarcon T, Vega AE, Domingo D, Martinez MJ, Lopez-Brea M. Clarithromycin resistance among Helicobacter pylori strains isolated from children: Prevalence and study of mechanism of resistance by PCR- restriction fragment length polymorphism analysis. J Clin Microbiol. 2003;41:486–499. doi: 10.1128/JCM.41.1.486-488.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Schabereiter-Gurtner C, Hirschl AM, Dragosics B, et al. Novel real-time PCR assay for detection of Helicobacter pylori infection and simultaneous clarithromycin susceptibility testing of stool and biopsy specimens. J Clin Microbiol. 2004;42:4512–4518. doi: 10.1128/JCM.42.10.4512-4518.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Trebesius K, Panthel K, Strobel S, et al. Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric tissue by fluorescent in situ hybridisation. Gut. 2000;46:608–614. doi: 10.1136/gut.46.5.608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Xing JZ, Clarke C, Zhu L, Gabos S. Development of a microelectronic chip array for high-throughput genotyping of Helicobacter species and screening for antimicrobial resistance. J Biomol Screen. 2005 Apr;10(3):235–45. doi: 10.1177/1087057104273781. [DOI] [PubMed] [Google Scholar]
  • 12.Furuta T, Sagehashi Y, Shirai N, et al. Influence of CYP2C19 polymorphism and Helicobacter pylori genotype determined from gastric tissue samples on response to triple therapy for H. pylori infection. Clin Gastroenterol Hepatol. 2005;3:564–573. doi: 10.1016/s1542-3565(04)00779-7. [DOI] [PubMed] [Google Scholar]
  • 13.Han SW, Flamm R, Hachem CY, et al. Transport and storage of Helicobacter pylori from gastric mucosal biopsies and clinical isolates. Eur J Clin Microbiol Infect Dis. 1995;14:349–352. doi: 10.1007/BF02116531. [DOI] [PubMed] [Google Scholar]
  • 14.Graham DY, Hoffman J, El-Zimaity HM, Graham DP, Osato M. Twice a day quadruple therapy (bismuth subsalicylate, tetracycline, metronidazole plus lansoprazole) for treatment of Helicobacter pylori infection. Aliment Pharmacol Ther. 1997;11:935–938. doi: 10.1046/j.1365-2036.1997.00219.x. [DOI] [PubMed] [Google Scholar]
  • 15.Kim N, Marcus EA, Wen Y, et al. Genes of Helicobacter pylori regulated by attachment to AGS cells. Infect Immun. 2004;72:2358–2368. doi: 10.1128/IAI.72.4.2358-2368.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ayala G, Chihu L, Perales G, et al. Quantitation of H. pylori cytotoxin mRNA by real-time RT-PCR shows a wide expression range that does not correlate with promoter sequences. Microb Pathog. 2004;37:163–167. doi: 10.1016/j.micpath.2004.06.003. [DOI] [PubMed] [Google Scholar]
  • 17.Furuta T, Soya Y, Sugimoto M, et al. Modified allele-specific primer-polymerase chain reaction method for analysis of susceptibility of Helicobacter pylori strains to clarithromycin. J Gastroenterol Hepatol. 2007;22:1810–1815. doi: 10.1111/j.1440-1746.2007.04919.x. [DOI] [PubMed] [Google Scholar]
  • 18.Sugimoto M, Wu JY, Abudayyeh S, et al. Caution regarding PCR detection of Helicobacter pylori in clinical or environmental samples. J Clin Microbiol. 2009 doi: 10.1128/JCM.01563-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Clayton CL, Kleanthous H, Coates PJ, Morgan DD, Tabaqchali S. Sensitive detection of Helicobacter pylori by using polymerase chain reaction. J Clin Microbiol. 1992;30:192–200. doi: 10.1128/jcm.30.1.192-200.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Fabre R, Sobhani I, Laurent-Puig P, et al. Polymerase chain reaction assay for the detection of Helicobacter pylori in gastric biopsy specimens: comparison with culture, rapid urease test, and histopathological tests. Gut. 1994;35:905–908. doi: 10.1136/gut.35.7.905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.de Martel C, Plummer M, van Doorn LJ, et al. Comparison of polymerase chain reaction and histopathology for the detection of Helicobacter pylori in gastric biopsies. Int J Cancer. 2010;126:1992–1996. doi: 10.1002/ijc.24898. [DOI] [PubMed] [Google Scholar]

RESOURCES