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Detection of Helicobacter pylori in contaminated samples using polymerase chain reaction based on the isocitrate dehydrogenase gene
Esophageal, Gastric, and Duodenal Disorders A155
April 1998 Drug (arms)
N. pts
% Healed(PP) % Healedat 4 wk 2wk 3-4wk G8wk 12wk Gradel II m-IV
S. relief n/n %
H2RAvs H2RA 351 513 665 447 219 S-dose(8) 1091 412 614 726 52~ 261o H-dose(14) 2099 PPIs vs H2RA 83e 95g 89i 74k 60m PPIs (17) 1472 67a 70c 52f 62h 52) 40 10~ H2RA(17) 1192 38b 39~ Refi~y 75° 84q 81s 50u PPIs (3) 217 55P 31r 50t 9v H2RAO) 133 PPI vs PPI 77 87 79 85 67 542//87 69* Lan (6) 1219 62 74 87 67 82 70 371/588 63 Ome (5) 1001 61 76 92 197/265 74# Pan (2) 311 79 93 147/187 79 Ome (2) 216 N. pts=number of patients, PP=per-protocol analysis, wk=weeks, S-dose=standard dose H-dose=high d o s e , -=not available. Lan=lansoprazole, Ome=omeprazole, Pan=pantoprazole. S. relief=symptom relief at 1-2 wk. Data on intention-to-treat and healing by grades at 8 and 12 wk are not shown due to space limit, p < 0.0l 1 vs 2, 3 vs 4, 5 vs 6, a vs b, c vs d, e vs f, g vs h, i vs j, k vs 1, m vs n, q vs r, s vs t; p=0.05 7 vs 8; p=0.012 o vs p; p=O.021 u vs v; p---ns 9 vs 10, Lan vs Ome, Pan vs Ome. *p=0.025 Lan vs Ome, #p=ns Pan vs Ome by Z2 test. Conclusions: H2RAs are not effective agents for treating patients with EE even with higher doses, especially in those with severe EE. PPIs heal significantly more EE than H2RAs including patients resistant to H2RAs. There are no significant differences in the healing of EE between the PPIs. However, Lan relieved significantly more symptoms than Ome at the first 2 weeks of treatment. • G0634 D E T E C T I O N O F HELICOBACTER PYLORI IN CONTAMINATED SAMPLES USING P O L Y M E R A S E C H A I N R E A C T I O N BASED ON T H E I S O C I T R A T E D E H Y D R O G E N A S E GENE. L.L. Huang, P. GriJbel, N. Masubuchi, P.J. Goddard, J.S. Hoffman and D.R. Cave. Division of Gastroenterology, St. Elizabeth's Medical Center of Boston, Boston, MA Polymerase chain reaction (PCR) has been extensively used for the detection of H. pylori in clinical and environmental samples, however, technical problems with DNA polymerase inhibitors and non-specific and insensitive primers make interpretation difficult. In this study we report a newly developed PCR assay, based on the H. pylori isocitrate dehydrogenase (ICD) gene sequence, which can rapidly and specifically detect H. pylori in contaminated specimens. Methods: We recently identified a functional icd gene from H. pylori that was present in all strains of H. pylori tested. One pair of oligonucleotide primers, which is unique for the icd gene of H. pylori based on genebank data analysis, was selected to amplify a 1200-bp fragment. One-step DNA template extraction from the samples was performed using the XTRAX T M DNA extraction kit. The specificity of the PCR assay was tested against DNA derived from various bacteria, including several Helicobacter spp. and a variety of Gram negative bacteria, as well as from laboratory raised houseflies, and stool from H. pylori-negative patients. Results: None of the control specimens were positive for the icd gene of H. pylori. The sensitivity of the PCR was determined by spiking control samples with a known number of H. pylori cells. The lower limit of detection was 50 11. pylori cells in flies and 500 H. pylori cells in stool samples, respectively. 152 batches of flies, with each batch containing five flies, were examined by this PCR assay. The identity of the PCR products was confirmed by DNA sequencing. Results showed that as with clinical isolates of H. pylori, the sequences of PCR products from wild flies were 95-96% identical to that present in the H. pylori reference strain 26695. Conclusions: The isocitrate dehydrogenase gene PCR is a specific and sensitive assay for H. pylori. It can be used to determine the presence of H. pylori in contaminated samples when combined with the XTRAX T M DNA extraction kit.
• G0635 I S O L A T I O N AND E X P R E S S I O N O F AN I S O C I T R A T E DEHYDROG E N A S E G E N E F R O M HELICOBACTER PYLORI. L.L. Huang, D.R. Cave and A, Wright*. Division of Gastroenterology, St. Elizabeth's Medical Center of Boston, and Department of Molecular Biology and Microbiology*, Tufts University School of Medicine, Boston, M A
Helicobacter pylori is implicated as the cause of gastritis and peptic ulcers, and is associated with gastric cancer. There is little known about the physiology of this organism. In this study we isolated and characterized a functional gene encoding the isocitrate dehydrogenase (ICD), a key enzyme in Kreb's cycle, from H. pylori. Methods and Results: Identification and cloning of the icd gene from H. pylori was performed by selecting clones for their ability to complement the glutamate auxotrophy of E. coli KKL1 (icd::Kan recA56 his-4 rpsL31). We used a partial San3A H. pylori library, carded by the plasmid pSK, to transform KKL1 cells. Colonies which grew on the minimal plate without glutamate supplement were selected. Plasmid DNAs were isolated from two independent colonies and found to contain 2.5 and 4.1 kb H. pylori DNA fragments. These plastids, pKHI1 and pKHI9 respectively, were confirmed to complement the icd defect in strain KKL1. The presence of ICD activity was verified by enzyme assay using extracts from strains KKL1 (pKHI1) or KKL1 (pKHI9). Sequencing of both strands of plasmid DNA was carried out by the dideoxy chain termination method using insert-specific oligonucleotide primers. A single open reading frame of 1278 base pairs corresponding to a 426 amino acid protein with a calculated molecular weight of 47,479 Da was found. The DNA sequence determined in this work is 96% identical to that present in the total genome sequence of H. pylori strain 26695. The deduced amino acid sequence of the H. pylori gene is 68% identical to E. coli isocitrate dehydrogenase. Expression of the gene in an E. coli icd- mutant allowed the mutant strain to grow in the absence of glutamate, indicating that it complements the enzyme defect. The results of PCR analysis of eight other clinical isolates of H. pylori suggest that they also contain the gene. Conclusions: A gene (icd) responsible for activity of isocitrate dehydrogenase in H. pylori has been identified, isolated, sequenced and expressed. This gene exists in all of H. pylori strains we have tested. G0636 A POPULATION-BASED I N V E N T A R I S A T I O N O F L O N G T E R M ACID SUPPRESSANT USE IN 24 G E N E R A L P R A C T I C E S IN T H E NETHERLANDS. GJB Hurenkamp l, HGLM Grundmeyer 1, PJE Bindels 1, GNJ Tytgat2, RWM van der Hulst 2. Departments of General Practice 1 and Gastroenterology2, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands. A considerable portion of the medication budget of the Dutch General Practitioners(GP) is spent on acid suppressant drugs. These high expenses relate to the high cost of the drugs, the high frequency of prescription and particularly the long duration of prescription. The aim of this study was to investigate the magnitude of longterm prescription of acid suppressant therapy in general practice, the clinical - grounds for the first prescription and the frequency and way of objectivation of the primary working diagnosis. Patients on long term acid suppressant therapy were identified in a population of 46,813 patients in 24 general practices in the Amsterdam region by extraction from computerized medication databases of the pharmacologists. The indications for the prescription and the investigations performed to confirm the working diagnosis, were extracted from the patients files in the GP-practices. Long term acid suppressant therapy was defined as a prescription for at least 12 weeks in one year. 922 patients (2% of the population) were on long-term acid suppressant therapy (0.9%-4.2% per practice). Mean duration of prescription was 33 weeks (range 12 (in 8%) to > 52 (in 23%) weeks). In more than 50% of the patients acid suppressant therapy was prescribed in a continuous fashion. In 25% of the patients no objective investigations were performed. In 75% of the patients endoscopy or radiology was done; the predominant diagnoses were ulcer disease (39.2%), GERD (38.3%) and other (gastritis, H.D., normal aspect) (29.5%). The H. pylori status had been established in 29% of the patients with ulcer disease. Eradication therapy was reported in 40% of these patients. Long term acid suppressant therapy is often administered without a firm clinical indication and without a confirmed working diagnosis.
April 1998 Drug (arms)
N. pts
% Healed(PP) % Healedat 4 wk 2wk 3-4wk G8wk 12wk Gradel II m-IV
S. relief n/n %
H2RAvs H2RA 351 513 665 447 219 S-dose(8) 1091 412 614 726 52~ 261o H-dose(14) 2099 PPIs vs H2RA 83e 95g 89i 74k 60m PPIs (17) 1472 67a 70c 52f 62h 52) 40 10~ H2RA(17) 1192 38b 39~ Refi~y 75° 84q 81s 50u PPIs (3) 217 55P 31r 50t 9v H2RAO) 133 PPI vs PPI 77 87 79 85 67 542//87 69* Lan (6) 1219 62 74 87 67 82 70 371/588 63 Ome (5) 1001 61 76 92 197/265 74# Pan (2) 311 79 93 147/187 79 Ome (2) 216 N. pts=number of patients, PP=per-protocol analysis, wk=weeks, S-dose=standard dose H-dose=high d o s e , -=not available. Lan=lansoprazole, Ome=omeprazole, Pan=pantoprazole. S. relief=symptom relief at 1-2 wk. Data on intention-to-treat and healing by grades at 8 and 12 wk are not shown due to space limit, p < 0.0l 1 vs 2, 3 vs 4, 5 vs 6, a vs b, c vs d, e vs f, g vs h, i vs j, k vs 1, m vs n, q vs r, s vs t; p=0.05 7 vs 8; p=0.012 o vs p; p=O.021 u vs v; p---ns 9 vs 10, Lan vs Ome, Pan vs Ome. *p=0.025 Lan vs Ome, #p=ns Pan vs Ome by Z2 test. Conclusions: H2RAs are not effective agents for treating patients with EE even with higher doses, especially in those with severe EE. PPIs heal significantly more EE than H2RAs including patients resistant to H2RAs. There are no significant differences in the healing of EE between the PPIs. However, Lan relieved significantly more symptoms than Ome at the first 2 weeks of treatment. • G0634 D E T E C T I O N O F HELICOBACTER PYLORI IN CONTAMINATED SAMPLES USING P O L Y M E R A S E C H A I N R E A C T I O N BASED ON T H E I S O C I T R A T E D E H Y D R O G E N A S E GENE. L.L. Huang, P. GriJbel, N. Masubuchi, P.J. Goddard, J.S. Hoffman and D.R. Cave. Division of Gastroenterology, St. Elizabeth's Medical Center of Boston, Boston, MA Polymerase chain reaction (PCR) has been extensively used for the detection of H. pylori in clinical and environmental samples, however, technical problems with DNA polymerase inhibitors and non-specific and insensitive primers make interpretation difficult. In this study we report a newly developed PCR assay, based on the H. pylori isocitrate dehydrogenase (ICD) gene sequence, which can rapidly and specifically detect H. pylori in contaminated specimens. Methods: We recently identified a functional icd gene from H. pylori that was present in all strains of H. pylori tested. One pair of oligonucleotide primers, which is unique for the icd gene of H. pylori based on genebank data analysis, was selected to amplify a 1200-bp fragment. One-step DNA template extraction from the samples was performed using the XTRAX T M DNA extraction kit. The specificity of the PCR assay was tested against DNA derived from various bacteria, including several Helicobacter spp. and a variety of Gram negative bacteria, as well as from laboratory raised houseflies, and stool from H. pylori-negative patients. Results: None of the control specimens were positive for the icd gene of H. pylori. The sensitivity of the PCR was determined by spiking control samples with a known number of H. pylori cells. The lower limit of detection was 50 11. pylori cells in flies and 500 H. pylori cells in stool samples, respectively. 152 batches of flies, with each batch containing five flies, were examined by this PCR assay. The identity of the PCR products was confirmed by DNA sequencing. Results showed that as with clinical isolates of H. pylori, the sequences of PCR products from wild flies were 95-96% identical to that present in the H. pylori reference strain 26695. Conclusions: The isocitrate dehydrogenase gene PCR is a specific and sensitive assay for H. pylori. It can be used to determine the presence of H. pylori in contaminated samples when combined with the XTRAX T M DNA extraction kit.
• G0635 I S O L A T I O N AND E X P R E S S I O N O F AN I S O C I T R A T E DEHYDROG E N A S E G E N E F R O M HELICOBACTER PYLORI. L.L. Huang, D.R. Cave and A, Wright*. Division of Gastroenterology, St. Elizabeth's Medical Center of Boston, and Department of Molecular Biology and Microbiology*, Tufts University School of Medicine, Boston, M A
Helicobacter pylori is implicated as the cause of gastritis and peptic ulcers, and is associated with gastric cancer. There is little known about the physiology of this organism. In this study we isolated and characterized a functional gene encoding the isocitrate dehydrogenase (ICD), a key enzyme in Kreb's cycle, from H. pylori. Methods and Results: Identification and cloning of the icd gene from H. pylori was performed by selecting clones for their ability to complement the glutamate auxotrophy of E. coli KKL1 (icd::Kan recA56 his-4 rpsL31). We used a partial San3A H. pylori library, carded by the plasmid pSK, to transform KKL1 cells. Colonies which grew on the minimal plate without glutamate supplement were selected. Plasmid DNAs were isolated from two independent colonies and found to contain 2.5 and 4.1 kb H. pylori DNA fragments. These plastids, pKHI1 and pKHI9 respectively, were confirmed to complement the icd defect in strain KKL1. The presence of ICD activity was verified by enzyme assay using extracts from strains KKL1 (pKHI1) or KKL1 (pKHI9). Sequencing of both strands of plasmid DNA was carried out by the dideoxy chain termination method using insert-specific oligonucleotide primers. A single open reading frame of 1278 base pairs corresponding to a 426 amino acid protein with a calculated molecular weight of 47,479 Da was found. The DNA sequence determined in this work is 96% identical to that present in the total genome sequence of H. pylori strain 26695. The deduced amino acid sequence of the H. pylori gene is 68% identical to E. coli isocitrate dehydrogenase. Expression of the gene in an E. coli icd- mutant allowed the mutant strain to grow in the absence of glutamate, indicating that it complements the enzyme defect. The results of PCR analysis of eight other clinical isolates of H. pylori suggest that they also contain the gene. Conclusions: A gene (icd) responsible for activity of isocitrate dehydrogenase in H. pylori has been identified, isolated, sequenced and expressed. This gene exists in all of H. pylori strains we have tested. G0636 A POPULATION-BASED I N V E N T A R I S A T I O N O F L O N G T E R M ACID SUPPRESSANT USE IN 24 G E N E R A L P R A C T I C E S IN T H E NETHERLANDS. GJB Hurenkamp l, HGLM Grundmeyer 1, PJE Bindels 1, GNJ Tytgat2, RWM van der Hulst 2. Departments of General Practice 1 and Gastroenterology2, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands. A considerable portion of the medication budget of the Dutch General Practitioners(GP) is spent on acid suppressant drugs. These high expenses relate to the high cost of the drugs, the high frequency of prescription and particularly the long duration of prescription. The aim of this study was to investigate the magnitude of longterm prescription of acid suppressant therapy in general practice, the clinical - grounds for the first prescription and the frequency and way of objectivation of the primary working diagnosis. Patients on long term acid suppressant therapy were identified in a population of 46,813 patients in 24 general practices in the Amsterdam region by extraction from computerized medication databases of the pharmacologists. The indications for the prescription and the investigations performed to confirm the working diagnosis, were extracted from the patients files in the GP-practices. Long term acid suppressant therapy was defined as a prescription for at least 12 weeks in one year. 922 patients (2% of the population) were on long-term acid suppressant therapy (0.9%-4.2% per practice). Mean duration of prescription was 33 weeks (range 12 (in 8%) to > 52 (in 23%) weeks). In more than 50% of the patients acid suppressant therapy was prescribed in a continuous fashion. In 25% of the patients no objective investigations were performed. In 75% of the patients endoscopy or radiology was done; the predominant diagnoses were ulcer disease (39.2%), GERD (38.3%) and other (gastritis, H.D., normal aspect) (29.5%). The H. pylori status had been established in 29% of the patients with ulcer disease. Eradication therapy was reported in 40% of these patients. Long term acid suppressant therapy is often administered without a firm clinical indication and without a confirmed working diagnosis.