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Journal of Clinical Microbiology, June 2007, p. 1718-1722, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00103-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Evaluation of the Novel Helicobacter pylori ClariRes Real-Time PCR Assay for Detection and Clarithromycin Susceptibility Testing of H. pylori in Stool Specimens from Symptomatic Children{triangledown}

Christian Lottspeich,1 Andrea Schwarzer,2 Klaus Panthel,1 Sibylle Koletzko,2 and Holger Rüssmann1*

Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie,1 Dr. v. Haunersches Kinderspital, Ludwig-Maximilians-Universität München, Munich, Germany2

Received 15 January 2007/ Returned for modification 2 March 2007/ Accepted 18 March 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of the present study was to evaluate the Helicobacter pylori ClariRes assay (Ingenetix, Vienna, Austria) for the detection of H. pylori infection and the simultaneous clarithromycin susceptibility testing of the H. pylori isolates in stool samples from 100 symptomatic children. The results obtained by this novel biprobe real-time PCR method were directly compared with the results obtained from histological examination of gastric biopsy specimens, culturing, the [13C]urea breath test, and a monoclonal antibody-based stool antigen enzyme immunoassay (EIA). Fecal specimens from all 54 children who were shown to be noninfected by "gold standard" tests gave true-negative PCR results (specificity, 100%). Of the remaining 46 individuals with a positive H. pylori status, 29 were found to be positive by real-time PCR (sensitivity, 63%). For these 29 cases, the H. pylori ClariRes assay confirmed all results from phenotypic clarithromycin susceptibility testing by Etest. In summary, this investigation demonstrates that detection of Helicobacter DNA in stool samples by real-time PCR is a difficult task and that this method cannot replace the stool antigen EIA (sensitivity, 95.7%) for the accurate diagnosis of H. pylori infection in children.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The gram-negative bacterium Helicobacter pylori colonizes the human gastric mucosa and potentially induces chronic gastritis and peptic ulcer disease (2). In addition, H. pylori plays a role in the etiology of gastric cancer and cancer of the mucosa-associated lymphoid tissue (2, 3, 24). To date, various diagnostic assays for the assessment of an H. pylori infection are available (33). The "gold standard" is the histological detection and culturing of the pathogen, which require gastric biopsy specimens obtained by invasive gastroduodenoscopy (17). In the last decade, noninvasive approaches, such as serological methods, the [13C]urea breath test (UBT), and fecal H. pylori antigen or DNA detection, helped to improve the evaluation of the patient's H. pylori infection status (12, 17). Most serological tests are not appropriate for pediatric patients due to their low sensitivities for those younger than 12 years of age (23). The UBT is a well-established noninvasive diagnostic tool and gives excellent performance for both adults and children, but its specificity decreases for infants and young children (8, 10). In addition, the performance of UBT with infants and young children requires trained staff for air sampling with a face mask, and the test also requires expensive instruments, such as an isotope ratio mass spectrometer or an infrared isotope ratio spectrometer (9). Enzyme immunoassays (EIAs) for the identification of H. pylori antigens in fecal specimens circumvent these difficulties. EIAs based on monoclonal antibodies have shown consistent excellent results, with very high sensitivities and specificities for both adults and children (11, 14, 16, 34). A major disadvantage of all the noninvasive tests described above is their inability to provide information on the susceptibility or resistance of H. pylori to antibiotics. To eradicate H. pylori and to cure the peptic ulcer disease caused by this pathogen, a 1-week triple therapy is recommended (17). The triple therapy comprises a proton pump inhibitor in combination with two antibiotics, including amoxicillin, clarithromycin, or metronidazole (17). In many cases, the macrolide drug clarithromycin is the key component of these combination therapies, since the occurrence of macrolide resistance in H. pylori is the most important cause of treatment failure (5, 7, 19). In clinical H. pylori isolates, resistance to clarithromycin is caused predominantly by three distinct point mutations within the peptidyltransferase region of the 23S rRNA (A2142G, A2143G, and A2142C) (25, 29, 31, 32). Successful detection of these mutations in cultured strains or gastric biopsy specimens has been described by the use of fluorescent in situ hybridization (26), PCR-restriction fragment length polymorphism (21), reverse hybridization line probe assay (30), PCR and EIA of DNA (18), and several real-time PCR methods (1, 13, 22). In addition, protocols for the identification of H. pylori DNA in human feces (15, 20), which thus eliminate the need for the invasive endoscopy procedure, have been elaborated. Recently, a novel biprobe real-time PCR protocol for the detection of H. pylori infection and simultaneous clarithromycin susceptibility testing was evaluated in a clinical study with 92 adult patients (28). With respect to the detection of H. pylori infection, PCR showed a sensitivity and specificity of 98% with stool samples. All clarithromycin-susceptible strains were identified in fecal specimens (specificity, 100%), whereas the sensitivity of the assay for the detection of macrolide-resistant H. pylori isolates was lower (73%). This attractive real-time PCR method is now commercially available (H. pylori ClariRes assay; Ingenetix, Vienna, Austria). The aim of this study was to directly compare the practicality and reliability of this novel assay with those of the gold standard invasive and noninvasive tests with fecal specimens from 100 pediatric patients.

(This study was conducted by C. Lottspeich in partial fulfillment of the requirements for a Ph.D. from the Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig-Maximilians-University, Munich, Germany.)


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients. For the evaluation of the H. pylori ClariRes assay, stool specimens from 100 children (mean age, 9.96 years; age range, 0.3 to 18 years) with abdominal symptoms were frozen at the time of upper gastrointestinal endoscopy. None of the patients had been treated for H. pylori infection in the past. Children were excluded from the study if they had taken antibiotics or acid-suppressive drugs (proton pump inhibitors, H2-receptor antagonists, antacids, or bismuth preparations) within 4 weeks prior to testing or if the H. pylori infection status was not clearly defined, as described below. The study protocol was approved by the ethical committee of the Ludwig-Maximilians-University.

Upper gastrointestinal endoscopy. All 100 children underwent gastroduodenoscopy. Altogether, a minimum of four gastric biopsy specimens were obtained from each child during the endoscopic procedure. Two biopsy specimens of the antrum and two of the corpus were formalin fixed, stained with modified Giemsa or hematoxylin-eosin, and viewed for the presence of H. pylori by a pathologist, who was blinded to the results of the other tests performed.

For 87 of the 100 children, another antral biopsy specimen was placed directly into a transport medium (Portagerm pylori; Biomerieux, Marcy l'Etoile, France) and was sent within 4 h to the microbiology laboratory for culture growth of H. pylori.

Culturing of H. pylori. The biopsy specimens were cut into small pieces and homogenized in a petri dish with a sterile scalpel. To culture H. pylori, the biopsy specimens were smeared on the surfaces of Columbia agar plates supplemented with 5% sheep erythrocytes (Becton Dickinson, Heidelberg, Germany) and Schaedler agar plates supplemented with 5% sheep erythrocytes and vitamin K1 (Becton Dickinson). The inoculated, vented plates were placed in an anaerobic jar together with a GENbox Microaer paper sachet (Biomerieux) to generate a microaerophilic environment (oxygen concentration, 7 to 10%; CO2 concentration, 20%) and incubated for 5 to 10 days. H. pylori microorganisms were identified on the basis of characteristic colony morphology; typical appearance on Gram staining; and positive urease, oxidase, and catalase tests.

Clarithromycin susceptibility testing. For clarithromycin susceptibility testing of H. pylori by Etest, colonies from the Columbia or the Schaedler agar plates were suspended in broth and carefully homogenized to minimize aeration. The inoculum suspension was prepared to a McFarland 3 turbidity standard. After a sterile swab was dipped into the inoculum, the entire surfaces of Mueller-Hinton agar plates supplemented with 5% sheep erythrocytes were swabbed in three directions. Before the Etest strips (AB Biodisk, Solna, Sweden) were applied onto the agar surface with a sterile forceps, the moisture was allowed to be absorbed for 5 min. The inoculated plates were incubated in a microaerophilic environment at 37°C for 2 days, and the MIC for each strain was determined. H. pylori isolates were considered to be resistant when the MICs of clarithromycin were >1 µg/ml (27). Each clarithromycin susceptibility test was repeated twice.

UBT. The UBT was performed with 56 of the 100 children as described previously (8, 10). Briefly, after a fasting period of at least 4 h, a baseline breath value was obtained by using a breath bag or, for very young children, a face mask. The children drank 150 ml of apple juice (pH 3.4). Thereafter, they received 20 ml of juice containing 75 mg 13C-labeled urea, and then they drank 30 ml of pure apple juice to flush the tracer from the mouth. Children <3 years old ingested only a total of 80 to 100 ml apple juice. Another breath sample was obtained 30 min after tracer application. The exhaled air was transferred into 10-ml Vacutainer tubes. The breath samples were analyzed by isotope ratio mass spectrometry. The results were considered positive when a change over the baseline value of ≥5{per thousand} was obtained.

EIA for detection of H. pylori antigen in fecal samples. The parents were asked to bring a stool sample from their child at the time of endoscopy. All 100 samples were stored frozen at –20°C until they were tested. An Amplified IDEIA Hp StAR assay (DakoCytomation, Cambridge, United Kingdom) was performed according to the manufacturer's recommendations. This sandwich-type EIA uses dual amplification technology and coating with a monoclonal antibody directed against the catalase of H. pylori. After the color change at the end of the test, the intensity was determined spectrophotometrically with a wavelength of 450 nm and a reference wavelength of between 620 and 650 nm. The absorbance was expressed as an optical density (OD) value. In accordance with the manufacturer's guidelines, an OD value of <0.150 was defined as a negative test result and an OD value of ≥0.150 was defined as a positive test result.

Definition of H. pylori infection status. For the definition of a positive H. pylori infection status, we used the results of histological examination, microbiological culture, UBT, and stool antigen EIA. A child was considered positive for H. pylori infection when at least three of these four tests gave positive results. A negative H. pylori infection status was considered if two of the four tests performed gave concordant negative results.

Real-time PCR with stool specimens. The novel commercially available H. pylori ClariRes assay (Ingenetix) was used for the detection of H. pylori infection and the simultaneous clarithromycin susceptibility testing of stool specimens from children. The assay was performed according to the manufacturer's recommendations. Briefly, DNA was extracted from the stool samples (0.2 g) by using a QIAamp DNA stool minikit (QIAGEN, Hilden, Germany). The 20-µl PCR mixture for H. pylori-specific 23S rRNA gene amplification and melting peak analysis contained 2 µl of LightCycler FastStart DNA Master SYBR green I (Roche Molecular Biochemicals, Mannheim, Germany), 2.4 µl of 4 mM MgCl2, 12.1 µl deionized water, 0.5 µl H. pylori ClariRes assay solution (Ingenetix), 1 µl of freshly diluted internal control (Ingenetix), and 2 µl of the DNA extract. A more detailed description of the real-time PCR has been published by Schabereiter-Gurtner et al. (28). The data and the melting curves were analyzed with Roche LightCycler software (version 3.5.3). Samples were considered H. pylori positive upon determination of a biprobe-specific melting curve.

Statistical analysis. The specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) were calculated by use of the chi-square test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Determination of H. pylori infection status. By use of the criteria adopted for this study, 46 of the 100 (46%) children tested were positive for H. pylori (Table 1). Of these patients, 38 gave concordant positive results (group I) by all four methods (histology, culture, UBT, and stool antigen EIA). For another five children (group II), the UBT was not performed. However, H. pylori was concordantly identified by the remaining three methods. Discordant positive results were recorded for three patients. Either stool EIA (group III) or culture (group IV) gave negative results. The H. pylori infection status was negative for 54 (54%) children with concordant negative test results (groups V to VIII) obtained by at least two of the four methods performed (Table 1).


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  		TABLE 1. Determination of H. pylori infection status in 100 children

 
Detection and clarithromycin susceptibility testing of the H. pylori isolates in stool specimens by H. pylori ClariRes assay. As demonstrated in Table 2, of 46 children positive for H. pylori infection (groups I to IV), 29 were found to be positive by the real-time PCR. The remaining 54 patients (groups V to VIII) were not infected with H. pylori, and all of their stool samples were negative, as determined by the H. pylori ClariRes assay.


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  		TABLE 2. Detection and clarithromycin susceptibility testing of H. pylori in stool specimens by H. pylori ClariRes assay

 
Of the 45 culture-positive patients (groups I to III), 39 were infected with a clarithromycin-susceptible strain and 6 (13.3%) were infected with a clarithromycin-resistant strain, as determined by Etest (Table 2). Of these six cases, the resistant genotype in stool samples from four cases (groups I and II) was confirmed by real-time PCR. 23S rRNA PCR and melting curve analysis clearly revealed melting peaks of 54°C for isolates with the A2142G and A2143G mutations (data not shown). In the other two cases (groups I and III) infected with a resistant strain, the H. pylori ClariRes assay gave false-negative results. To exclude the possibility that the respective Helicobacter strains contained point mutations that were not detected by the H. pylori ClariRes assay (e.g., A2115G or G2141A) (31), we applied the fluorescent in situ hybridization method to cultured H. pylori strains (27). Both strains harbored the A2143G point mutation (data not shown).

The sensitivity, specificity, and predictive values of the H. pylori ClariRes assay are given in Table 3 for all 100 stool specimens. In our study, this real-time PCR assay showed a sensitivity of 63% (NPV, 76.1%) and a specificity of 100% (PPV, 100%).


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  		TABLE 3. Performance of the H. pylori ClariRes assay for detection and clarithromycin susceptibility testing of H. pylori in 100 stool specimens from children

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The majority of patients with dyspeptic symptoms initially visit primary care physicians. As recommended by the Maastricht 2-2000 Consensus Report (17), a "test-and-treat" approach should be offered to adult patients under the age of 45 years (the age cutoff may differ locally, according to the mean age of gastric cancer onset) presenting to a primary care physician with persistent dyspepsia; however, this approach should not be used with those with predominantly gastroesophageal reflux disease symptoms, nonsteroidal anti-inflammatory drug users, and those with alarm symptoms (e.g., unexplained weight loss or anemia). In contrast to endoscopy and UBT, stool antigen EIAs have the advantage that they do not require the patient to fast before coming to the physician's office or outpatient department. Thus, as a noninvasive method, stool tests gain importance in clinical practice for the accurate diagnosis of H. pylori infection in adults and especially in children. However, the use of first-line treatment regimens without information on the infecting isolate's susceptibility to clarithromycin may result in an increasing number of treatment failures since the rate of primary macrolide resistance in clinical isolates of H. pylori was reported to be up to 20% (6, 11).

In the past, the development of real-time PCR protocols on the basis of biprobes (1, 4) or hybridization probes (13, 22) for the detection of point mutations in the 23S rRNA gene associated with resistance to clarithromycin has significantly improved macrolide susceptibility testing of cultured H. pylori isolates and the direct testing of the susceptibilities of isolates in Helicobacter-positive gastric biopsy specimens. The first data on the applicability of real-time PCR protocols for H. pylori detection and clarithromycin susceptibility testing in stool specimens were published by Schabereiter-Gurtner et al. (28). For these diagnostic purposes, this novel biprobe-based 23S rRNA gene real-time PCR assay (H. pylori ClariRes assay; Ingenetix), in combination with melting curve analysis, was found to be a highly accurate noninvasive method that could be applied to stool samples from adult patients (28). Our laboratory was interested in evaluating the H. pylori ClariRes assay by the use of fecal specimens from 100 symptomatic children. In contrast to the study published by Schabereiter-Gurtner and colleagues (28), we directly compared the results of the real-time PCR assay not only with the results of histology and culturing but also with the results of UBT and the stool antigen EIA.

In our study population, 46% of the children tested with invasive and noninvasive gold standard tests were positive for H. pylori. Of these 46 individuals, 29 were found to be positive by the H. pylori ClariRes assay. Thus, the sensitivity (63%) of the PCR for the detection of H. pylori in stool samples from children was significantly lower than the sensitivity (98%) reported with stool specimens from adult patients (28). The noninvasive stool antigen test based on a monoclonal antibody directed against the catalase from Helicobacter gave a positive result for 44 of 46 children with positive H. pylori infection status (sensitivity, 95.7%), which confirms the accuracy of the EIA demonstrated in earlier reports (11, 14, 16, 34). The question arises of why the H. pylori ClariRes assay revealed false-negative results with the stool samples from 17 patients. Interestingly, among these 17 individuals, 15 were H. pylori positive, as determined by the stool antigen EIA, arguing that at least protein components derived from Helicobacter were present in the respective fecal samples. In contrast to the EIA, the H. pylori ClariRes assay is performed with the 23S rRNA gene, which must be extracted from the stool sample by using a QIAamp DNA stool minikit. The DNA concentration after elution ranged from 185 to 250 ng/µl (data not shown). Thus, the amounts of DNA were comparable in all 100 samples tested. However, a lack of intact DNA could have occurred in individual stool specimens because frozen instead of fresh samples were used, thus contributing to fewer positive real-time PCR results. The presence of PCR inhibitors can be excluded as a reason for the false-negative results because an internal amplification control is provided with the H. pylori ClariRes assay kit. In addition, we tried to improve the robustness of the PCR by adding to the PCR mixture bovine serum albumin at a final concentration of 0.1 µg/µl (data not shown). However, this procedure failed to increase the sensitivity of the real-time PCR method. Differences in the gastrointestinal tracts of children and those of adults (e.g., the different compositions of the fecal microbiota and the shorter gastrointestinal passage time in children) might also have contributed to the low sensitivity of the H. pylori ClariRes assay in our study.

All 29 cases found to be positive by the H. pylori ClariRes assay were also culture positive. It is important to emphasize that the PCR assay confirmed all results from phenotypic clarithromycin susceptibility testing by Etest. Melting curve analysis revealed unequivocal melting peaks that were easy to interpret. Thus, in the case of positive H. pylori ClariRes assay results, accurate genotyping of macrolide susceptibility or resistance was provided.

Taken together, the H. pylori ClariRes assay with feces from children positive for H. pylori infection revealed an excellent specificity (100%) but a poor sensitivity (63%). Our study demonstrates that the detection of Helicobacter DNA in stool samples by real-time PCR is a difficult task and that this method cannot replace the sensitive stool antigen EIA for the accurate diagnosis of H. pylori infection in children.


   ACKNOWLEDGMENTS
 
We thank the members of the Pathological Institute (chairs, U. Löhrs and T. Kirchner), LMU Munich, for the histological examination of the gastric biopsy specimens and H. Demmelmair (Dr. v. Haunersches Kinderspital, LMU Munich) for the performance of UBTs.


   FOOTNOTES
 
* Corresponding author. Mailing address: Max von Pettenkofer-Institute, Ludwig-Maximilians-University, Pettenkoferstr. 9a, 80336 Munich, Germany. Phone: 0049-89-51605280. Fax: 0049-89-51605223. E-mail: ruessmann{at}mvp.uni-muenchen.de Back

{triangledown} Published ahead of print on 28 March 2007. Back


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Clinical Microbiology, June 2007, p. 1718-1722, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00103-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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