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Comparison of PCR and clinical laboratory tests for diagnosing H. pylori infection in pediatric patients.

Vinette KM, Gibney KM, Proujansky R, Fawcett PT - BMC Microbiol. (2004)

Bottom Line: Especially in children, these tests may result in a false negative outcome because of patchy distribution of the organism in the stomach mucosa.Of the seropositive patients, 26 were ELISA positive, 13 were positive by Western blot, and 11 by both serologic methods.In addition, we found the current commercially available approved ELISA method appears unable to accurately detect H. pylori in this population.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunology Laboratory, Department of Research, Alfred I, duPont Hospital for Children, Wilmington, Delaware, USA.kvinette@nemours.org

ABSTRACT

Background: Histology and/or culture are generally considered the gold standard for the detection of H. pylori infection. Especially in children, these tests may result in a false negative outcome because of patchy distribution of the organism in the stomach mucosa. We have developed a PCR assay utilizing nested primer pairs directed against a subunit of the H. pylori urease gene (ureA). As part of a prospective evaluation of diagnostic tests to aid in detecting H. pylori infection in children, the aim of this study was to compare our PCR and Western blot assays with results obtained from histologic examination of biopsy specimens, rapid urease tests, and an FDA approved serologic assay and published PCR results to determine if we could validate the assays for diagnostic use on our patient population.

Results: Gastric biopsy specimens obtained from 101 pediatric patients were evaluated for the presence of H. pylori using histologic techniques, rapid urease (CLOtest) test and the PCR assay. Serum samples from each patient were assayed using both ELISA and Western Blot for antibodies to H. pylori. A total of 32 patients tested were positive by at least one of the methods evaluated. Thirteen patients had positive histology, 13 had a positive CLOtest, and 17 patients had positive H. pylori PCR. Out of the 13 CLO positive patients, 12 were positive by histologic analysis and all 13 were positive by PCR. Results of serologic tests on the same population did not correlate well with other assays. Twenty-eight patients showed serologic evidence of H. pylori infection, of which 9 were both CLO and histology positive and 12 were positive by PCR. Of the seropositive patients, 26 were ELISA positive, 13 were positive by Western blot, and 11 by both serologic methods.

Conclusions: The results obtained suggest that our nested PCR assay has the specificity and sensitivity necessary for clinical application when compared to standard histologic examination and rapid urease test. In addition, we found the current commercially available approved ELISA method appears unable to accurately detect H. pylori in this population. The Western blot assay yielded better concordance with CLOtest and histology, but not as good as the nested PCR assay.

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Results of patient testing for H. pylori. Results of all testing (including biopsy-based methods and serology) are shown for the thirty-four samples included in the study which gave at least one positive result. Samples 20 and 21 were biopsies collected during endoscopic examinations performed 4 months apart on the same patient. Matched serum samples for this patient were obtained at each visit. Sample 31 was a biopsy collected from the antrum of a patient undergoing endoscopy, while sample 32 was from the body of the stomach of the same patient. Only one serum sample from this patient was received for analysis. The remaining sixty-seven patients (71 samples) were negative for all assays performed (data not shown). The BIOPSY column includes all specimens for which at least one test result was positive (CLOtest, PCR, or histology). The SEROLOGY column includes all serum samples considered reactive by either H. pylori ELISA (IgG and/or IgA), Western Blot (IgG and/or IgA), or both.
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Figure 1: Results of patient testing for H. pylori. Results of all testing (including biopsy-based methods and serology) are shown for the thirty-four samples included in the study which gave at least one positive result. Samples 20 and 21 were biopsies collected during endoscopic examinations performed 4 months apart on the same patient. Matched serum samples for this patient were obtained at each visit. Sample 31 was a biopsy collected from the antrum of a patient undergoing endoscopy, while sample 32 was from the body of the stomach of the same patient. Only one serum sample from this patient was received for analysis. The remaining sixty-seven patients (71 samples) were negative for all assays performed (data not shown). The BIOPSY column includes all specimens for which at least one test result was positive (CLOtest, PCR, or histology). The SEROLOGY column includes all serum samples considered reactive by either H. pylori ELISA (IgG and/or IgA), Western Blot (IgG and/or IgA), or both.

Mentions: Complete data for this study was obtained on 101 pediatric patients (49 males, 52 females). Three patients had samples taken from two separate locations at the time of biopsy while one patient underwent endoscopic examination twice in a four month time period, yielding 105 biopsies and 102 serum samples for testing. Of these 105 samples, a total of 34 (32.4%) gave positive results for at least one of the methods examined (Figure 1). Fourteen samples were both CLOtest positive (13.3%) and positive by our nested PCR assay. Fourteen biopsies were positive by histologic staining (13.3%), of which 2 gave a negative result by PCR. A total of 18 samples were positive by the nested PCR assay (17.1%). Patient samples were considered to be positive for H. pylori by PCR amplification if the 279 bp band was seen in the initial reaction and/or the 120 bp was seen in the internal nested reaction. Of the 18 PCR positive samples, 12 showed both the 279 and the 120 bp bands, while the remaining 6 gave a positive reaction only after the internal nested amplification. When invasive methods were considered together, 20 samples were positive by one or more of these tests (19.0%). Twenty-eight (27.4%) of the matching serum specimens on this patient population showed reactivity to H. pylori. Of these, 26 were positive by ELISA, 13 were positive by Western blot, and 11 were positive by both serologic methods. For 14 (13.3%) of the serum samples, serologic testing yielded the only H. pylori positive results. Interestingly, one patient had clearly negative CLOtest and PCR results, while the histologic examination of both of this child's biopsies (obtained from endoscopies performed on two separate dates) showed the presence of organisms consistent with H. pylori. At both examination dates, both ELISA and Western blot on this patient were also positive. Conversely, our PCR assay yielded the only positive result for another patient shown included in the study. Using our nested assay, we were able to detect H. pylori-specific sequences at an estimated concentration of 1 picogram (pg) after just the initial round of amplification. This corresponds to approximately 540 H. pylori bacteria, based on a size of 1.67 Mbp for the complete H. pylori genome [11]. Amplification of the internal gene segment using the nested primer pair further increased the reliable detection limit sensitivity to approximately 30 femtograms (fg), corresponding to about 16 bacterial genomes.


Comparison of PCR and clinical laboratory tests for diagnosing H. pylori infection in pediatric patients.

Vinette KM, Gibney KM, Proujansky R, Fawcett PT - BMC Microbiol. (2004)

Results of patient testing for H. pylori. Results of all testing (including biopsy-based methods and serology) are shown for the thirty-four samples included in the study which gave at least one positive result. Samples 20 and 21 were biopsies collected during endoscopic examinations performed 4 months apart on the same patient. Matched serum samples for this patient were obtained at each visit. Sample 31 was a biopsy collected from the antrum of a patient undergoing endoscopy, while sample 32 was from the body of the stomach of the same patient. Only one serum sample from this patient was received for analysis. The remaining sixty-seven patients (71 samples) were negative for all assays performed (data not shown). The BIOPSY column includes all specimens for which at least one test result was positive (CLOtest, PCR, or histology). The SEROLOGY column includes all serum samples considered reactive by either H. pylori ELISA (IgG and/or IgA), Western Blot (IgG and/or IgA), or both.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC338288&req=5

Figure 1: Results of patient testing for H. pylori. Results of all testing (including biopsy-based methods and serology) are shown for the thirty-four samples included in the study which gave at least one positive result. Samples 20 and 21 were biopsies collected during endoscopic examinations performed 4 months apart on the same patient. Matched serum samples for this patient were obtained at each visit. Sample 31 was a biopsy collected from the antrum of a patient undergoing endoscopy, while sample 32 was from the body of the stomach of the same patient. Only one serum sample from this patient was received for analysis. The remaining sixty-seven patients (71 samples) were negative for all assays performed (data not shown). The BIOPSY column includes all specimens for which at least one test result was positive (CLOtest, PCR, or histology). The SEROLOGY column includes all serum samples considered reactive by either H. pylori ELISA (IgG and/or IgA), Western Blot (IgG and/or IgA), or both.
Mentions: Complete data for this study was obtained on 101 pediatric patients (49 males, 52 females). Three patients had samples taken from two separate locations at the time of biopsy while one patient underwent endoscopic examination twice in a four month time period, yielding 105 biopsies and 102 serum samples for testing. Of these 105 samples, a total of 34 (32.4%) gave positive results for at least one of the methods examined (Figure 1). Fourteen samples were both CLOtest positive (13.3%) and positive by our nested PCR assay. Fourteen biopsies were positive by histologic staining (13.3%), of which 2 gave a negative result by PCR. A total of 18 samples were positive by the nested PCR assay (17.1%). Patient samples were considered to be positive for H. pylori by PCR amplification if the 279 bp band was seen in the initial reaction and/or the 120 bp was seen in the internal nested reaction. Of the 18 PCR positive samples, 12 showed both the 279 and the 120 bp bands, while the remaining 6 gave a positive reaction only after the internal nested amplification. When invasive methods were considered together, 20 samples were positive by one or more of these tests (19.0%). Twenty-eight (27.4%) of the matching serum specimens on this patient population showed reactivity to H. pylori. Of these, 26 were positive by ELISA, 13 were positive by Western blot, and 11 were positive by both serologic methods. For 14 (13.3%) of the serum samples, serologic testing yielded the only H. pylori positive results. Interestingly, one patient had clearly negative CLOtest and PCR results, while the histologic examination of both of this child's biopsies (obtained from endoscopies performed on two separate dates) showed the presence of organisms consistent with H. pylori. At both examination dates, both ELISA and Western blot on this patient were also positive. Conversely, our PCR assay yielded the only positive result for another patient shown included in the study. Using our nested assay, we were able to detect H. pylori-specific sequences at an estimated concentration of 1 picogram (pg) after just the initial round of amplification. This corresponds to approximately 540 H. pylori bacteria, based on a size of 1.67 Mbp for the complete H. pylori genome [11]. Amplification of the internal gene segment using the nested primer pair further increased the reliable detection limit sensitivity to approximately 30 femtograms (fg), corresponding to about 16 bacterial genomes.

Bottom Line: Especially in children, these tests may result in a false negative outcome because of patchy distribution of the organism in the stomach mucosa.Of the seropositive patients, 26 were ELISA positive, 13 were positive by Western blot, and 11 by both serologic methods.In addition, we found the current commercially available approved ELISA method appears unable to accurately detect H. pylori in this population.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunology Laboratory, Department of Research, Alfred I, duPont Hospital for Children, Wilmington, Delaware, USA.kvinette@nemours.org

ABSTRACT

Background: Histology and/or culture are generally considered the gold standard for the detection of H. pylori infection. Especially in children, these tests may result in a false negative outcome because of patchy distribution of the organism in the stomach mucosa. We have developed a PCR assay utilizing nested primer pairs directed against a subunit of the H. pylori urease gene (ureA). As part of a prospective evaluation of diagnostic tests to aid in detecting H. pylori infection in children, the aim of this study was to compare our PCR and Western blot assays with results obtained from histologic examination of biopsy specimens, rapid urease tests, and an FDA approved serologic assay and published PCR results to determine if we could validate the assays for diagnostic use on our patient population.

Results: Gastric biopsy specimens obtained from 101 pediatric patients were evaluated for the presence of H. pylori using histologic techniques, rapid urease (CLOtest) test and the PCR assay. Serum samples from each patient were assayed using both ELISA and Western Blot for antibodies to H. pylori. A total of 32 patients tested were positive by at least one of the methods evaluated. Thirteen patients had positive histology, 13 had a positive CLOtest, and 17 patients had positive H. pylori PCR. Out of the 13 CLO positive patients, 12 were positive by histologic analysis and all 13 were positive by PCR. Results of serologic tests on the same population did not correlate well with other assays. Twenty-eight patients showed serologic evidence of H. pylori infection, of which 9 were both CLO and histology positive and 12 were positive by PCR. Of the seropositive patients, 26 were ELISA positive, 13 were positive by Western blot, and 11 by both serologic methods.

Conclusions: The results obtained suggest that our nested PCR assay has the specificity and sensitivity necessary for clinical application when compared to standard histologic examination and rapid urease test. In addition, we found the current commercially available approved ELISA method appears unable to accurately detect H. pylori in this population. The Western blot assay yielded better concordance with CLOtest and histology, but not as good as the nested PCR assay.

Show MeSH
Related in: MedlinePlus