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00019606-200609000-00009ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2006 Lippincott Williams & Wilkins, Inc.15September 2006 p 174-179Seminested Polymerase Chain Reaction (PCR) for Detecting Helicobacter pylori DNA in Carotid AtheromasOriginal ArticlesArias, Eugenia MD, MSc*; Martinetto, Horacio PhD*; Schultz, Marcelo HT*; Ameriso, Sebastian MD*; Rivera, Santiago MD†; Lossetti, Oscar MD‡; Sevlever, Gustavo PhD, MD**FLENI, Montaneses 2325, Buenos Aires C1428AQK, Cuidad Autonoma de Buenos Aires†Facultad de Ciencias Biomedicas, Austral University, Pilar, Buenos Aires‡Department of Legal Medicine, Universidad Abierta Interamericana, Buenos Aires, ArgentinaReprints: Dr Gustavo Sevlever, PhD, MD, FLENI, Montaneses 2325, Buenos Aires C1428AQK, Cuidad Autonoma de Buenos Aires, Argentina (e-mail: [email protected]).AbstractA polymerase chain reaction (PCR) method for the detection of the glmM gene, selected as Helicobacter pylori target sequence, was improved. While performing pathogenicity island cagA gene detection to discriminate pathogenic strains in atherosclerotic carotid samples, several cagA-positive but glmM-negative samples were found. Polymorphisms present in the region amplified in the nested PCR reaction could explain this result; primers were therefore designed to perform a seminested reaction; this modification optimized sensitivity while maintaining specificity. A real-time PCR for Helicobacter DNA detection was also setup. The combination of all 4 PCR reactions detected 83% of H. pylori DNA-positive samples in atherosclerotic carotid tissue, 64% of which were cagA gene positive.Helicobacter pylori are gram-negative bacteria which produce a chronic infection in humans associated with peptic ulcer disease, gastritis, gastric mucosa-associated lymphoma, gastric adenocarcinoma, and other gastroduodenal diseases.1–3 These bacteria have been implicated in the pathogenesis of atherosclerosis, not only through serologic evidence,4,5 but also because of H. pylori DNA detection in atherosclerotic tissues,6–8 although this finding remains controversial.9–11 DNA detection by polymerase chain reaction (PCR) has varying levels of sensitivity and diagnostic accuracy. The glmM (UreC) gene encoding phosphoglucosamine mutase has been described as the most sensitive and specific target for H. pylori detection in gastric biopsy specimens in Taiwan.12 While testing for virulent strains (cagA gene), several cagA-positive and glmM-negative carotid atheroma samples were detected; modifications were therefore introduced to the method originally reported to solve this apparent paradox. The seminested PCR system consists of an initial PCR, primer combination glmMI(f)/glmMI(r) followed by a subsequent reaction, namely the primer glmMI(f)/glmMI(r) combination. The presence of H. pylori DNA was also detected using real-time PCR quantification and eventually by direct sequencing.MATERIALS AND METHODSSpecimen CollectionSpecimens were collected between March 1997 and August 2004. Patients with significant carotid artery stenosis, admitted for endarterectomy at the Vascular Surgery Department of the Institute of Neurological Research (FLENI), were enrolled. Atherosclerotic plaques were obtained from 90 endarterectomy specimens under aseptic conditions, cut into sections using a sterile blade and stored at −70°C. Tissue was also obtained from biopsies that had been previously fixed in 10% buffered formalin for 2 hours and embedded in paraffin wax; slices were cut and processed for DNA extraction. Thirteen control carotid samples from subjects who died of unrelated (nonvascular) causes were obtained from autopsy material, processed in a similar fashion, and used as nonatherosclerotic carotid controls. Absence of atherosclerosis in the carotid was assessed by histologic examination. Biopsy samples processed following the same diagnostic procedure included: 11 brain, 11 gastric, 1 breast, 1 small bowel, 1 large bowel, 1 pancreas, 1 aorta, 1 temporal artery, 1 mesenteric artery, 1 ovary, 1 myometrium, and 1 endometrium.DNA ExtractionFresh carotid tissue (20 mg) was homogenized and digested with proteinase K overnight at 55°C. Tissue slices (5 μm), cut from blocks subjected to paraffin extraction with xylene and washed with 100% ethanol, were prepared and digested. DNA was obtained using the DNA genomic Wizard purification kit (Promega, Madison, WI), following manufacturer's instructions. DNA was resuspended in a volume of 4 to 15 μL of DNA hydration solution. Several tissues from either frozen or paraffin-embedded specimens, as detailed above, were processed in parallel as negative controls for the DNA extraction procedure.Qualitative PCRInitially, bacterial DNA detection was performed using a nested procedure for the glmM gene (glmMI) and a single-step reaction for the cagA gene. The first step in the nested PCR required a set of glmM primers described elsewhere,12 and generated a 294-bp product. The specific primer pair glmMI (forward) and glmMI (reverse) generates a 129-bp amplicon. cagA and glmMI primers were designed with the Primer3 Input program (htpp://Frodo.wi.mit.edu/cgi-bin/primer3) after analysis of the published gene sequences [GenBank accession numbers: AF125214(ATCC43504), AJ269897, AJ269852, L11714, NC_000921(J99), AY353251(NCTC11637), NC_000915(26695), M60398]. Primer sequences are listed in Table 1. The first round of seminested PCR was performed using glmMI (forward) primer and glmMI (reverse) and rendered a 168-bp fragment; subsequently, the procedure was identical to the nested reaction. All carotid samples were analyzed using cagA-specific primers. Amplifications were performed in a final volume of 20 μL containing: 1 μL of template DNA in PCR buffer, 200 μM dNTP, 1.5 mM MgCl2, 1 μM of each primer, and 1 U Taq polymerase (for cagA reaction) or 2.5 U Taq polymerase (for glmM first reaction, nested or seminested) (Promega, Madison, WI). Samples were subjected to 35 cycles at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, followed by a final extension at 72°C for 7 minutes. Second-round reactions were carried out using 7 μL of the first-reaction product with 1 U of Taq polymerase, 2 mM MgCl,2 and 35 cycles at 60°C of annealing temperature. All these reactions were carried out using a PTC-100 thermal cycler (MJ Research, Inc, Watertown, MA). Amplified products were separated on a 12% polyacrylamide gel, stained with ethidium bromide, and visualized using ultraviolet light. DNA from ATCC 43504 D strain and gastric biopsies positive by histochemistry, were employed as positive controls. Human apex gene was amplified to verify DNA availability for the PCR reaction. Negative controls (water nuclease free instead of DNA, nonatherosclerotic carotids, and other tissues where H. pylori presence is not expected) were included in all reactions and every precaution was taken to avoid contamination.JOURNAL/dimp/04.03/00019606-200609000-00009/table1-9/v/2021-02-17T195928Z/r/image-tiff Primer Sets and Probes Used in Conventional Single Step, Nested, Seminested, and Real-time PCRQuantitative PCRQuantitative PCR was performed using the ABI Prism 7500 sequence detector system (Perkin/Elmer/Applied Biosystem, CA). Hpyl PCR primers and the TaqMan Hpyl probe were designed using the Primer Express 2.0 software program. Blast searches were performed to ensure specific target sequences for the glmM gene and to avoid differences between strains. Because these searches revealed some differences, a degenerate reverse primer was employed (Table 1). In this assay, the housekeeping gene GAPDH was amplified as a reference gene to control integrity and total amount of human DNA obtained from specimens. GAPDH primers and the TaqMan probe were described by Berggren et al.13 Duplicate real-time PCR assays were carried out in separate tubes for GAPDH and glmM (Hpyl). Standard H. pylori DNA and GAPDH human DNA curves were constructed using values obtained by plotting Ct of six 10-fold serial dilutions of strain ATCC43504 and standard human DNA. Concentration of human DNA was determined spectrophotometrically. H. pylori density in the specimen samples was expressed as number of bacterial genomes/μg of human DNA detected.14–16 Sample DNA (1 μL) solution was used for real-time PCR in a 25 μL volume consisting of 50 nM Hpyl (forward), 100 nM Hpyl (reverse) degenerated primer, 100 nM GAPDH (forward), and 300 nM GAPDH (reverse); and 150 nM Hpyl and GAPDH TaqMan probes. The initial thermal cycle conditions were 50°C for 2 minutes and 95°C for 10 minutes. Cycling conditions for both reactions in 65 cycles were 95°C for 15 seconds and 60°C for 1 minute.Sequencing of the glmM and cagA ProductsPCR product DNA sequences were determined (Macrogen, Inc, Seoul, Korea) after purification through Microspin S-400 HR Columns (Amersham Biosciences, Piscataway, NJ).RESULTSglmM and cagA Gene DetectionNinety atherosclerotic carotid samples were analyzed using nested PCR, seminested PCR, real-time PCR, and conventional PCR. All samples were first screened through nested PCR (63 fresh and 27 from archive), 59 of which were positive for glmMI (Table 2). When tested for cagA presence, 38 samples were positive; 10 samples were cagA positive but glmMI negative. When samples were amplified using the seminested procedure, 13 were glmMI* positive, including 7 of the 10 glmMI-negative and cagA-positive samples; 3 samples remained glmMI* negative and cagA positive. One of these was Hpyl positive when assayed using real-time PCR. From these results H. pylori DNA prevalence in atherosclerotic carotid samples was calculated to be 83% (75/90) and cagA positivity to be 64% (48/75) (Table 2).JOURNAL/dimp/04.03/00019606-200609000-00009/table2-9/v/2021-02-17T195928Z/r/image-tiff Results of Samples Analyzed and Methodology UsedWhen paraffin-embedded archive samples were compared with fresh ones, H. pylori positivity was 82% versus 84%, thus indicating similar efficiency in Helicobacter DNA detection combining all 4 methods. cagA presence in archive samples was 41% (9/22), contrasting with 74% (39/53) in fresh samples. This might be explained by the much lower DNA yield from archive samples, along with lower amounts of H. pylori DNA in atherosclerotic tissue compared with gastric tissue, which results in a lack of cagA assay sensitivity in this condition. To clarify this point, 11 formalin-fixed paraffin-embedded gastric biopsies, 10 of which were histochemistry positive were studied. Ninety percent (9/10) were glmMI positive and 67% (6/9) were cagA positive; all were glmMI* negative. This result is comparable to cagA gene prevalence in the stomach, confirming our assumption.Of the 13 cases of nonatherosclerotic carotid samples evaluated, 85% were glmMI, glmMI*, and cagA negative but 2 cases were positive for glmMI in nested reaction and one of them was cagA positive. None of these samples displayed signs of atherosclerosis, with the exception of one presenting a calcium deposit within the vascular wall. To rule out potential contamination during the procedure, other negative controls were also studied. Eleven fresh brain samples were chosen because this tissue has not been shown to be infected by H. pylori; additionally, other tissues such as ovary, small bowel, large bowel, breast, pancreas, aorta, temporal artery, mesenteric artery, brain, myometrium, and endometrium were also evaluated. All controls proved glmMI, glmMI*, and cagA negative.Analysis of cagA sequence: cagA primers were designed after careful analysis of the published gene sequences; amplification of the preserved right cag pathogenicity island region rendered a 101-bp fragment in a single-step PCR reaction; the cagA sequence was determined and a similarity search using the Blast Program showed that 101-bp amplicon actually corresponded to the H. pylori cagA gene. Sequences from the 101-bp amplicon showed a high degree of similarity to the NCTC 11637 and 26695 strains (Fig. 2). In some fresh samples, amplification fragments of 101-bp and 90-bp were observed (Fig. 1 Lane 5). The 90-bp amplicon did not match any sequence reported in the database. In general, the cagA reaction showed very high specificity (Fig. 2).JOURNAL/dimp/04.03/00019606-200609000-00009/figure1-9/v/2021-02-17T195928Z/r/image-jpeg PCR products of the glmM gene and cagA gene by electrophoresis in 12% polyacrylamide gel with ethidium bromide. Lanes 1, 2, and 3 glmMI positive atherosclerotic carotid samples. Lanes 4 and 5 cagA positive atherosclerotic carotid samples. Lane 6: PCR cagA-negative control. Lane 7: PCR glmMI-negative control. Lanes 8 and 9 glmMI and cagA PCR products in the ATCC 43504 D strain.JOURNAL/dimp/04.03/00019606-200609000-00009/figure2-9/v/2021-02-17T195928Z/r/image-tiff Nucleotide sequence alignment in some strains of Helicobacter pylori cagA gene. Sequence differences are underlined.Analysis of glmMI sequence: glmMI fragments were sequenced, allowing us to confirm the existence of different H. pylori strains in carotid tissue. Similarities in sequence were observed with strains from Sweden and to the NCTC11637 strain (Fig. 3). Interestingly, when the glmM(r) primer was compared with the glmM sequence from the NCTC11637 strain, several differences were identified.JOURNAL/dimp/04.03/00019606-200609000-00009/figure3-9/v/2021-02-17T195928Z/r/image-tiff Nucleotide sequence alignment in some strains of the Helicobacter pylori glmM gene. Sequence differences are underlined.H. pylori DNA Quantification in Human Carotid AtheromaEvaluation of H. pylori density in the atherosclerotic carotid tissue using real-time PCR was carried out. H. pylori DNA was detected by Hpyl real-time PCR in 43 (56.5%) out of 76 atherosclerotic carotid H. pylori DNA-positive samples; results obtained with the real-time PCR showed a range between 0 and 5000 H. pylori genomes/μg of human DNA. This corresponds to femto grams of H. pylori DNA detected in 1 μL of the DNA sample. Because the stomach is the natural niche for the bacteria, density, as expected, is much higher in stomach biopsies than in atherosclerotic carotid tissue. Gastric tissue has been described as containing 10 to 1000 H. pylori genomes/pg of human DNA.17 We obtained 7 H. pylori genomes/pg of human DNA in paraffin-embedded stomach biopsies.DISCUSSIONIn the present study the diagnostic potential of 4 PCR assays for the detection of H. pylori DNA in routinely processed atherosclerotic carotid biopsies was investigated. It was shown that PCR can in fact identify H. pylori DNA in atherosclerotic carotid tissue, but a combination of nested and seminested reactions is required to obtain accurate detection, especially in extragastrointestinal tissue where H. pylori is present in low numbers, or shows patchy distribution. Some groups have been unsuccessful in the detection of H. pylori DNA in carotid atheroma,9,10 whereas others have found the concept difficult to accept.18 In general, conventional PCR detection of H. pylori DNA requires the use of a set of primers that amplifies stable nonpolymorphic segments of the bacterial genome, to avoid potential false-negative results. Depending on the type of tissue tested, it is important to consider both target DNA concentration and quality of target DNA. Nested PCR is more sensitive than a single PCR when target DNA levels are limited; however, this also depends on the detection system being used. The presence of H. pylori DNA in human carotid atherosclerotic plaques has been previously described7 to be 53% in Argentinean samples tested from formalin-fixed paraffin-embedded tissue employing the PCR technique reported by Lu et al.12 The glmM gene may have a significant degree of sequence polymorphisms in clinical strains; when the literature was reviewed, discrepancies between strains were found on the 3′ extreme of the glmM sequence, where the glmM reverse primer should bind. These polymorphisms might have been responsible for the original failure to detect the glmM gene in some samples. Another advantage was the use of primers rendering fragments <200-bp in length, because evidence exists to suggest that DNA suffers disintegration after long periods in paraffin-embedded and stained tissues,19 as was the case in our archive samples (7 to 8 y in storage).Bacterial DNA was present in 2/13 (15%) of nonatherosclerotic carotid “control” cases; those samples belonged to 2 young male adults. This makes accurate lesion interpretation difficult. Detection of bacterial DNA in these 2 controls may represent a “bacteriemic phase” of the infection, occurring well before the presence of vascular wall lesions. In these cases, sample contamination cannot be entirely ruled out because sample-collecting conditions are rarely uniform from the operating theater to the autopsy table. In a central region of Argentina, the percentage of H. pylori seropositive subjects, with or without gastric symptoms was not significantly different between groups of adults (80.2% and 75.9%, respectively), suggesting high-degree exposure to H. pylori in this population.20 Among asymptomatic Japanese, who did not have cardiac disease or atherosclerosis, 45% were seropositive for the bacteria,21 in contrast to 70.9% in healthy Korean adults.22 From these observations we conclude: (1) nested PCR assay detected most of the H. pylori DNA in archived and fresh samples; (2) seminested PCR increased H. pylori DNA detection in both archived and fresh samples, with the greatest increase occurring in archived samples; this highlights the importance of fragment size, suggesting potential DNA degradation without excluding the possibility that this could have been secondary to sequence polymorphisms in atherosclerotic carotid samples, not seen in gastric biopsies. This in turn might reflect preference in some strains for atherosclerotic carotid tissue colonization; (3) the cagA gene detection system showed very high specificity and allowed positive H. pylori diagnosis in samples previously cataloged as false negatives; (4) limitation was observed for cagA detection in archived atherosclerotic carotid samples; single-step reactions may be insufficient to detect foreign DNA in nongastric samples. Supporting this, cagA DNA detection prevalence in paraffin-embedded gastric biopsies was comparable to data previously reported in our population23,24; we are currently trying to improve this reaction; (5) detection of H. pylori DNA by studying the glmM gene was accurate when a seminested assay was performed, either on archived or on fresh samples. Even though glmMI* seminested PCR represented a remarkable improvement in the results obtained, 3% of samples (2 archived and 1 fresh) remained cagA positive and glmM or glmMI* negative. Greater differences between strains in the samples being analyzed should have been present; real-time PCR was also conducted but the assay failed to detect H. pylori DNA in 33 of 76 samples where H. pylori DNA presence had already been demonstrated using nested/seminested/cagA conventional PCR. Despite the use of degenerated primers, the reason why we were unable to detect all the strains present in atherosclerotic carotid by applying this method may well have been the presence of sequence differences.Therefore, we believe that the presence of H. pylori DNA in carotid atheroma can no longer be considered controversial. This study illustrates the potential value of PCR for studying the glmM gene and the cagA system; however, target DNA concentration levels should be taken into consideration when selecting which bacterial DNA detection method to apply.REFERENCES1. Lee A, Fox JC, Hazell S. Pathogenicity of Helicobacter pylori: a perspective. 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Acta Gastroenterol Latinoam. 2004;34:16–20.[Context Link][Medline Link]cagA gene; atherosclerotic carotid samples; glmM gene; Helicobacter pylori; seminested PCR00019606-200609000-0000900000416_1999_83_1586_malnick_endarterectomy_|00019606-200609000-00009#xpointer(id(citation_FROM_JRF_ID_d3135e932_citationRF_FLOATING))|11065404||ovftdb|SL00000416199983158611065404citation_FROM_JRF_ID_d3135e932_citationRF_FLOATING[Full 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Lanes 1, 2, and 3 glmMI positive atherosclerotic carotid samples. Lanes 4 and 5 cagA positive atherosclerotic carotid samples. Lane 6: PCR cagA-negative control. Lane 7: PCR glmMI-negative control. Lanes 8 and 9 glmMI and cagA PCR products in the ATCC 43504 D strain. Nucleotide sequence alignment in some strains of Helicobacter pylori cagA gene. Sequence differences are underlined. Nucleotide sequence alignment in some strains of the Helicobacter pylori glmM gene. Sequence differences are underlined.Seminested Polymerase Chain Reaction (PCR) for Detecting <em xmlns:mrws="http://webservices.ovid.com/mrws/1.0">Helicobacter pylori</em> DNA in Carotid AtheromasArias Eugenia MD MSc; Martinetto, Horacio PhD; Schultz, Marcelo HT; Ameriso, Sebastian MD; Rivera, Santiago MD; Lossetti, Oscar MD; Sevlever, Gustavo PhD, MDOriginal ArticlesOriginal Articles315p 174-179