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Original Articles: Gastroenterology

Usefulness of Gastric Biopsy–Based Real-Time Polymerase Chain Reaction for the Diagnosis of Helicobacter pylori Infection in Children

Kalach, Nicolas*; Gosset, Pierre; Dehecq, Eric; Decoster, Anne; Spyckerelle, Claire*; Papadopolos, Stephan; Dupont, Christophe§; Raymond, Josette||

Author Information
Journal of Pediatric Gastroenterology and Nutrition: September 2015 - Volume 61 - Issue 3 - p 307-312
doi: 10.1097/MPG.0000000000000787
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Abstract

What Is Known

  • In adults, quantitative real-time polymerase chain reaction–based method is a most reliable method for H pylori diagnosis than the other routine procedures.

What Is New

  • In children, the reliability of the H pylori quantitative real-time polymerase chain reaction–based method is confirmed.
  • H pylori quantitative real-time polymerase chain reaction–based method allows detecting low bacterial load.
  • The bacterial load is correlated to the intensity of gastritis.

The numerous diagnostic tests available for the detection of Helicobacter pylori infection are direct, invasive (culture, histology, rapid urease test [RUT]), or noninvasive (serology, 13C-Urea breath test13 [C-UBT], and stool antigen test) (1–3). There is, however, no single “reference method” to detect H pylori infection reliably and accurately (1–3). Bacterial culture of gastric biopsy has 100% specificity, but sensitivity is low (1–3). Histology and RUT provided excellent diagnostic accuracy, but the detection of H pylori decreased in the presence of bleeding peptic ulcers or gastric atrophy (4,5). Therefore, concordant results of at least 2 tests are recommended to define the H pylori infection status in children (3). PCR-based methods have been shown to be the most reliable method for the diagnosis of H pylori infection in adults (6–9) and children (10). Furthermore, in adults, quantitative PCR-based methods have shown that they can detect infection, in case of low numbers of H pylori organisms and minimal gastric inflammation in which histologic-gastric biopsies are negative (7,10).

The aim of our study was to assess usefulness of gastric biopsy–based quantitative real-time polymerase chain reaction (qPCR) for the detection of H pylori infection and the identification of clarithromycin-resistant strains in children.

METHODS

From January 2010 to December 2012, 403 unselected consecutive children (215 girls, 188 boys) with clinical gastritis were enrolled. Parental informed consent was obtained. The protocol was approved by the bioethics’ committee of the French Minister of research and registered under the number DC-2013-1969.

Biopsies were taken in both gastric antrum (n = 4) and corpus (n = 4) during an upper tract gastrointestinal (GI) endoscopy. Children who had already received antibiotics, anti-acid or nonsteroidal anti-inflammatory medications during the 4 previous weeks were excluded.

Histology

Two antral and 2 corpus specimens were formalin fixed and paraffin embedded, and a simple hematoxylin and eosin stains were used for the identification and the grading of the intensity of histologic inflammation, and in order to improve the detection of H pylori, the Giemsa staining was used to identify the presence of H pylori and to grade its intensity according to updated Sydney classification (11). The grade of inflammation, activity, atrophy, metaplasia, and H pylori were established by the visual analog scale given in the updated Sydney classification (11).

Inflammation

Intensity of histological gastric inflammation, that is, histological gastritis, was estimated according to mononuclear cells and lymphocytes, which are as follows: absent (grade 0), few (grade 1, mild), moderate number (grade 2, moderate), or high number (grade 3, marked).

Activity

The degree of histological gastritis activity was estimated according to neutrophils infiltration, that is, absent (grade 0), few (grade 1, mild), moderate number (grade 2, moderate), or high number (grade 3, marked). Gastric glandular atrophy and intestinal metaplasia were assessed as absent (grade 0), mild (grade 1), moderate (grade 2), or marked (grade 3).

H pylori

Intensity of infection with H pylori was classified as absent (grade 0), mild or rare (grade 1), moderate (grade 2), or marked (grade 3). Further histological analysis was done based on the presence or absence of the follicular gastritis.

Rut

One antral and 1 corpus biopsy specimens were used for RUT as previously described (12).

Culture

One antral and 1 corpus biopsy specimens were used for culture and qPCR detection. Gastric biopsies were homogenized using a glass Griffith tube, and H pylori was cultured according to the previously described method (12). Colonies obtained from the primary culture were harvested together to be tested for antimicrobial susceptibility.

Clarithromycin resistance was detected by antimicrobial susceptibility testing according to the “E-test” method (AB Biodisk, Solna, Sweden) using Mueller-Hinton agar (Oxoïd, Dardilly, France) supplemented with 10% horse blood and incubated in microaerophilic conditions for 3 days. Strains were considered as resistant when the minimum inhibitory concentration levels were ≥1 mg/L (13). Strains were stored at −80° C in 3 mL meat-liver media (Mérieux, Marcy l’Etoile, France) added with 0.3 mL of glycerol. Amoxicillin, metronidazole (MET), tetracycline, rifampicin, and ciprofloxacin susceptibilities were also determined by “E-test” method (AB Biodisk). The strains were considered amoxicillin, MET, and ciprofloxacin resistant with minimum inhibitory concentrations ≥1, 8, 1, and 4 mg/L (12).

PCR

A qPCR assay was used directly on DNA obtained from gastric biopsies to detect both the presence of H pylori and the point mutations conferring resistance. DNA was isolated by using a QIAamp DNA mini kit (Qiagen SA, Courtaboeuf, France). A 267-bp fragment of the 23S rRNA gene of H pylori was amplified using primers HPYS and HPYA as described (14). Using the LightCycler thermocycler (Roche Diagnostics, Neuilly sur Seine, France), the PCR and hybridization reactions were carried out in glass capillaries in a volume of 20 μL containing 3 μL of template DNA, 1.6 μL of MgCl2 (25 mmol/L), 0.4 μL of forward and reverse primers (20 μmol/L each), 0.2 μL of sensor and anchorprobes (20 μmol/L each), and 2 μL of FastStart DNA Master Hybridization Probes (Roche Diagnostics). PCR amplification comprised an initial denaturation cycle at 95°C for 10 minutes, followed by 50 amplification cycles (with a temperature transition rate of 20°C/s) consisting of 95°C for 0 second, annealing at 60°C for 10 seconds, and extension at 72°C for 17 seconds. After amplification a melting step was performed, consisting of 95°C for 0 second, cooling to 45°C for 30 seconds (with a temperature transition rate of 20°C/s), and finally a slow rise in the temperature to 85°C at a rate of 0.1°C/s with continuous acquisition of fluorescence decline. The sensitivity and specificity of the real-time PCR were 98.4% and 94.1%, respectively. (14). Each run included positive and negative controls, the former being prepared from 10−2, 10−4, and 10−6 dilutions of 45-μg/mL DNA from H pylori strain H37Rv, and the latter consisted of sterile water. Quality control was acceptable when the negative control had an undetectable cycle threshold (and the 10−2, 10−4, and 10−6 dilutions of H pylori DNA Mac Farland 1, had cycle threshold values between 17 and 19, and 27, and 33, respectively.

The specificity of the PCR method was evaluated in 20 biopsies samples without H pylori infection but positive with different microorganisms (Enterococcus faecalis, Pseudomonas aeruginosa, coagulase-negative Staphylococcus, Candida albicans, Escherichia coli, Klebsiella pneumoniae) for which the PCR remained negative. The PCR was also negative by testing Campylobacter sp.

H pylori Status

The results of the histological analysis, RUT, bacterial culture, and qPCR of the gastric biopsy specimens were carried out in a blind way, without knowing the results of any other examination.

H pylori infection was considered by “the reference method” when culture was positive alone or both histology and RUT. Results were compared with those obtained by the qPCR. A negative H pylori status was considered when all 4 tests, such as culture, histology, RUT, and qPCR gave concordant negative results.

Statistical Analysis

This is a descriptive study; calculation of median and range (min-max) of all quantitative parameters were done using the Stat-View System (Abacus, CA). The difference between the qualitative parameters was calculated by using the χ2 test. Analysis of variance was also used for the crossing of qualitative and quantitative parameters. P values ≤0.05 were considered significant.

The results of the gastric biopsy–based qPCR, with calculation of sensitivity, specificity, positive and negative predictive values, and test accuracy with their 95% confidence interval (95% CI), were compared with those of the reference biopsy–based methods.

RESULTS

Patient Characteristics

Among the 403 enrolled children (median age 7.5 years [6 months-18 years]), 62 (15.3%) exhibited H pylori infection according to the reference method or to the qPCR method (Fig. 1). H pylori–positive children were significantly older than H pylori–negative children, median (range) 108.5 months (7–204) versus 86.4 (6–216), P = 0.003. Female-to-male ratio in the 62 H pylori–positive children was 40/22 versus 177/168 in the 341 H pylori–negative children.

F1-11
FIGURE 1:
Discordant and concordant results of Helicobacter pylori antimicrobial susceptibility testing according to interventional culture versus the quantitative real-time polymerase chain reaction (qPCR).

According to the qPCR results, in 2010 H pylori–positive children versus H pylori–negative ones showed 13 versus 84; in 2011, 33 versus 139; and in 2012, 16 versus 118.

Results of the Different Tests

Antimicrobial susceptibility testing was obtained for 25/62 children (Fig. 1). The positivity of the different diagnostic tests is summarized in Table 1. Concordant results (H pylori–positive and H pylori–negative children), among the different diagnostic tests, were obtained in 372/403 children (92.3%).

T1-11
TABLE 1:
Concordant results between different diagnostic tools in Helicobacter pylori–positive children, n = 62

The positivity of the different diagnostic tests is summarized in Table 1. Thirty-seven children had a positive histology according to the Sydney classification; inflammation grade was as follows: grade 1 in 7, grade 2 in 13, and grade 3 in 17. The RUT was positive for 37 children, and the qPCR was positive for 62. Concordance results between culture, histology, RUT, and qPCR in 62 H pylori–positive children are summarized in Table 2.

T2-11
TABLE 2:
Concordant results between culture, histology, RUT, and qPCR bacterial load in Helicobacter pylori–positive children, n = 62

Among the 20 patients with a 101 copies of qPCR bacterial load, 18 showed negative histology, 1 showed histologic inflammation of grade 1, and 1 of grade 3. Whereas, among the 11 patients with a 103 copies of qPCR bacterial load; 2 showed negative histology; and 4, 3, and 2 a histologic inflammation of grades 1, 2, and 3, respectively. Finally, 2 patients with a 105 copies of qPCR bacterial load showed histologic inflammation of grade 3 (Table 2). Moreover, among the 20 patients with a 101 copies of qPCR bacterial load, 8 showed absence of histologic activity, and 4, 6, and 2 showed a histologic activity of grades 1, 2, and 3, respectively. Whereas, among the 11 patients with a 103 copies of qPCR bacterial load 1 showed absence of histologic activity, and 1, 6, and 3 a histologic activity of grades 1, 2, and 3, respectively. Finally, 2 patients with a 105 copies of qPCR bacterial load showed a histologic activity of grade 3 (Table 2).

The qPCR showed a bacterial load ≥103 copies when culture, histology, and RUT were all positive (29/31 children) versus <103 when culture, histology, and RUT were all negative (25/31 children). Grades 2 and 3 histological gastritis was associated with a bacterial load ≥103 copies per milliliter for (28/35 children) versus (27/27 children) of grades 0 to 1 <103 copies per milliliter (Table 2).

Thirty-one children had negative culture and positive qPCR (22 susceptible clarithromycin strains and 9 resistant), and 25 children had both positive qPCR and culture without any discordant antimicrobial susceptibility testing results (17 sensitive clarithromycin strains and 8 resistant). Finally, 6 children had both positive qPCR and culture, but unfortunately antimicrobial susceptibility could not be obtained (3 were susceptible to clarithromycin and 3 resistant based on qPCR only) (Fig. 1, Table 3).

T3-11
TABLE 3:
Concordant results between antimicrobial susceptibility testing by interventional culture versus clarithromycin point mutation by qPCR in Helicobacter pylori–positive children, n = 62

Finally, clarithromycin resistance was the result of the mutation A2143G in 8 cases and A2142G in 1 case. The mutation A2142C was not detected as mixed population of H pylori in the biopsies.

Performances of the Different Diagnostic Tests

The qPCR method detected H pylori in all reference method “criterion standard” or routine histology, RUT, and culture-positive samples. This method also detected H pylori infection in 25 negative biopsies for culture, histology, RUT, and the reference method, as well as in 6 negative biopsies for culture and positive for histology and RUT (Table 4).

T4-11
TABLE 4:
Test performance of the different diagnostic tests, n = 403

The reference method versus H pylori qPCR positivity showed, respectively (95% CI) a sensitivity of 100% versus 100%, a specificity of 93.2% (86.9–99.4) versus 100%, a positive predictive value of 59.7% (47.4–71.9) versus 100%, a negative predictive value of 100% versus 100%, and finally a test accuracy of 59.6 % (47.3–71.8) versus 100% (Table 4).

DISCUSSION

In this study, we found that gastric mucosa qPCR is more sensitive than routine histology, RUT, culture, or the commonly used reference method “criterion standard” for detecting H pylori infection in children with gastritis. Furthermore, there was no discordant in the detection of antimicrobial susceptibility between the cultures compared with the qPCR.

In adults, PCR-based techniques have been shown to be more sensitive than conventional methods for H pylori detection. Lage et al (15) used an ureC gene amplification PCR assay in gastric biopsy specimens and found it more sensitive than histology staining and 13C-UBT for diagnosing H pylori infection. Ramírez-Lázaro et al (16) showed that qPCR was positive for 16 sRNA+urea+23sRNA in 35 (67%) of the 52 histology-negative biopsy specimens, being more sensitive than conventional histology or immunohistochemistry. Belda et al (9) showed that a new qPCR system for detecting H pylori in gastric biopsies was more sensitive than traditional diagnostic methods. In 154 symptomatic patients, the traditional criterion standard detected 85 patients (55.19%) with H pylori infection, and the qPCR was positive for H pylori in antral biopsies of 101 patients (65.6%). Owing to significantly high sensitivity, qPCR detected additional H pylori infection in 8% of criterion standard negative or 10.7% of routine histology-negative gastric mucosa specimens (10).

In children, Agudo et al (17) evaluated a commercially available kit, MutaREAL H pylori (Inmundiagnostik, Bensheim, Germany) qPCR for the detection of H pylori infection and clarithromycin resistance in gastric biopsies. This qPCR assay showed sensitivity of 93.33% (negative predictive value 90.90%) and specificity of 86.95% (positive predictive value 90.32%) for H pylori detection. Clarithromycin resistance was detected in 26 cases by qPCR with a sensitivity and specificity of 90.62% and 95.83%, respectively. MutaREAL kit was able to detect H pylori and its clarithromycin susceptibility with high efficacy (17). In another pediatric study, Mitui et al (18), examining clarithromycin resistance rate using a DNA PCR/sequencing assay of “the 23S rRNA gene” of H pylori on histology archived gastric biopsy specimens, found that 19/38 (50%) of the specimens contained H pylori with mutations significant for clarithromycin resistance. The overall concordance between clarithromycin resistance by E-test and the presence of 23S rRNA gene mutations was 81% to 91%. Considering phenotype resistance testing as the standard criterion, it was shown that the sensitivity and specificity of point mutations at position 2142 and 2143 were 75% and 98.3%, respectively (18). These results are in agreement with ours because qPCR was positive in 25 of both routine histology and reference method–negative patients. Furthermore, H pylori clarithromycin-resistant strains were detected in 8 cases by both culture and qPCR methods without discordant results. Despite the low cases of detected resistance (n = 8), we confirm the predominance of the mutation A2143G responsible for the clarithromycin resistance as previously described in adults and in children in France (19,20). MET resistance in H pylori is complex and is primarily associated with mutational inactivation of the redox-related genes (frxA, rdxA). FrxA may act indirectly by affecting cellular reductive potential in low-level MET resistant isolates. RdxA gene inactivation confers resistance by saturation transpose on mutagenesis of the H pylori genome. Thus, factors that lead to the loss of or inactivation of the 2 genes may lead to contribute to MET resistance per se. Meanwhile, there are reports that the MET-resistant phenotype may arise in H pylori without mutations in rdxA or frxA, suggesting the presence of additional MET resistance mechanisms (20). Resistance of H pylori to MET is therefore difficult to estimate in vitro, particularly using molecular tools because several genes are involved and the mechanism of action of MET is not yet fully elucidated.

Thus, qPCR method is quicker than culture and will lead to the reduction in the total time (17). Based on the infecting H pylori strains antibiotic resistance profiles using the qPCR, H pylori treatment regimens could be implemented to optimize therapy against the H pylori strain infecting a symptomatic child with upper digestive disease (21).

Zsikla et al (7) have shown that qualitative nested and quantitative PCR can detect H pylori in approximately 20% of histologic-negative gastric biopsies. Weiss et al (22) demonstrated that in gastric biopsy specimens, H pylori was detected in 52% of cases by PCR, and only 20% by CLO test or immunohistochemical analysis. Ramírez-Lázaro et al (16) showed that qPCR detected H pylori infection in 67% of histology-negative formalin-fixed paraffin-embedded biopsy samples obtained during peptic ulcer bleeding episodes, whereas immunohistochemical analysis detected the infection in only 6% of the patients. Another limitation for gastric mucosa qPCR is its invasiveness for endoscopic procedure and tissue biopsies. In clinical practice, esophagogastroduodenoscopy examination with biopsy is usually performed to diagnose H pylori infection in symptomatic children. Hematoxylin and eosin is routinely used by a pathologist to identify histologic gastritis, and in order to improve the detection of H pylori, a special stain, that is, Giemsa staining is currently used, in conjunction with RUT. Owing to low numbers of H pylori organisms and minimal gastric inflammation, either histology stain or RUT may not identify H pylori infection. In this situation, PCR method would accurately diagnose H pylori infections that would otherwise be missed. According to our European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition Consensus (3), where once H pylori is detected treatment should be offered. Thus, the clinical implications of identifying low H pylori density in children with recurrent abdominal pain remains to be investigated. Belda et al (9) realized a prospective study to determine the main diagnostic validity parameters of a qPCR system for detecting H pylori in gastric biopsies. This system has a good diagnostic capacity compared with the criterion standard and was superior in antral mucosa. The optimum cutoff point was 3.56 microorganisms per cell for antrum (sensitivity 83.5% [95% CI 74.2–89.9], specificity 91.3% [95% CI 82.3–96.0], positive predictive value 92.2%, negative predictive value 81.8%). The positive likelihood ratios were 9.61 and 8.52 for antrum and corpus, respectively. With the cutoff point that maximizes the Youden index, 8.7% false-positives were obtained. They conclude that the qPCR is useful for diagnosing infection caused by H pylori and the false-positives detected probably correspond to patients who were actually infected but the infection was not detected using traditional techniques. (9). In our study, we showed that the positivity of culture and histology was correlated with a high bacterial load ≥103 copies. Similarly, the grade of histologic gastritis and activity was also correlated with the bacterial load.

In adults with upper GI bleeding, biopsy-based methods, such as RUT, histology, and bacterial culture, have a low sensitivity at 45% to 70% and a high false-negative rate at 30% to 55% (4). Saez et al (23) suggest that qPCR has a good diagnostic capacity compared with the classical criterion standard in bleeding adults’ patients and is slightly greater in antrum than in corpus. A global analysis of all the techniques used showed that the test with the greatest odds of giving a positive result in bleeding patients is the 13C-UBT, followed by real-time PCR, which confirms previously published findings (23). In our study there was, however, no patient presented with upper GI bleeding seeking problems for diagnostic test accuracy of H pylori infection.

CONCLUSIONS

The biopsy-based qPCR test is a sensitive, reliable, and specific test for the detection of H pylori infection and the identification of clarithromycin-resistant strains. This method is quicker and more sensitive than culture and allows adapting rapidly antimicrobial treatment. Furthermore, its overall high specificity and negative predictive values allow ruling out H pylori infection in case of negativity. We also demonstrated that the grade of gastritis was correlated to the bacterial load in children.

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Keywords:

children; diagnosis; Helicobacter pylori; quantitative real-time polymerase chain reaction

© 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,