Modified rapid urease test for Helicobacter pylori detection in relation to an immunohistochemical stain.

BACKGROUND: The rapid urease test and touch cytology have been used for the rapid detection of Helicobacter pylori infection. Recently, a modified rapid urease (MRU) test, which provides results in 20 min has been available on a commercial basis. To date, few reports have evaluated the accuracy of this test. This study evaluated the sensitivity, specificity, and accuracy of the MRU test and touch cytology to detect H. pylori in relation to the density of H. pylori infection determined semi-quantitatively by using immunohistochemical stains. METHODS: Biopsy specimens obtained from a total of 60 patients who underwent endoscopy for evaluation of gastroduodenal diseases were studied by using the MRU test, Giemsa stain for touch smear tissue and histological methods. An immunohistochemical stain was used as a standard, and the density of H. pylori infection was graded according to the number of individual bacteria seen as follows: grade 0 = 0; grade 1+ = 1-9; grade 2+ = 10-29; grade 3+ = 30-99; grade 4+ > or = 100. The severity of gastritis was evaluated histologically in each specimen and compared with the density of H. pylori infection. RESULTS: The MRU test had an overall sensitivity of 73%, specificity of 100% and accuracy of 85%. The Giemsa stain had a sensitivity of 91%, specificity of 100% and accuracy of 95%.The sensitivities of the MRU test and Giemsa stain decreased in mild H. pylori infection. In the MRU test, the sensitivity was 47% when the density of H. pylori infection was 1+, while 80-100% sensitivities were obtained when the densities of infection were > or = 2+. With the Giemsa stain, the sensitivity was 80% when the density was 1+, while the sensitivity increased to 100% when the densities were > or = 2+. The severity of gastritis determined by the Rauws scores showed a positive correlation with the density of H. pylori infection as evaluated by immunohistochemical staining. CONCLUSIONS: The MRU test had high sensitivity and specificity for moderate to severe H. pylori infection, but it may result in false-negative results in tests for mild infection. As the MRU test has the advantages of shorter incubation times and low cost, a combination of the MRU test and the Giemsa stain for touch cytology may be the most time- and cost-efficient tests in a clinical setting for the diagnosis of H. pylori infection.

Semiquantitative evaluation for diagnosis of Helicobacter pylori infection in relation to histological changes.

OBJECTIVES: Several methods are used to detect Helicobacter pylori (HP) infection. However, few reports have evaluated the accuracy of each method and compared the grade of HP infection with the severity of histological changes. HP infection was evaluated semiquantitatively in relation to the severity of gastritis, and the sensitivity, specificity, and accuracy of several methods to detect HP infection were compared. METHODS: Biopsy specimens, obtained from a total of 64 patients who underwent endoscopy for evaluation of gastroduodenal diseases, were studied using a rapid urease test, culture, and histological assessment. An immunohistochemical method was used as the gold standard and graded according to the number of individual bacteria seen, as follows: 0 = 0; 1+ = <10; 2+ = 10-29; 3+ = 30-99; 4+ = >100. The severity of gastritis was evaluated histologically in each specimen and compared with the grade of HP infection. RESULTS: The rapid urease test had a sensitivity of 53%, specificity of 100%, and accuracy of 73%. The culture method had a sensitivity of 75%, specificity of 100%, and accuracy of 86%. Sensitivities of the rapid urease test and the culture method decreased in a positive correlation with the decrease in total number of HP bacteria counted. Using the rapid urease test, sensitivity was <30% when the grade of HP infection was < or =2+, whereas 100% sensitivity was obtained when the grade of HP infection was 4+. On the other hand, sensitivity of the culture method remained between 80% and 90% when HP infection was > or =2+. The severity of gastritis determined with Rauws scores increased in a positive correlation with the grade of HP infection as evaluated by immunohistochemical stain. CONCLUSIONS: The rapid urease test and culture of HP may result in false-negative tests for a mild infection, although they had high sensitivity and specificity for moderate to severe infection. Immunohistochemical stain provides a reliable semiquantitative diagnosis of HP infection and a positive correlation with histological changes. Clinicians should be aware of the characteristics of each method to detect HP infection and select the appropriate one(s) for their purposes.

[Semi-quantitative detection of Helicobacter pylori using immunohistochemical staining].

Several methods have been used for the detection of Helicobacter pylori (HP) infection. However, few reports have evaluated the accuracy of each method and compared the level of HP infection. HP infection was evaluated semi-quantitatively using immunohistochemical staining and accuracy of several methods to detect HP infection were compared. Biopsy specimens, obtained from a total 64 patients who underwent endoscopy for evaluation of gastroduodenal diseases, were studied using a rapid urease test, culture method, and immunohistochemical method. The infection was graded according to the number of the individual bacteria seen in a highly magnified visual field, and defined as follows: (0) = 0; (1+) < 10; (2+) = 10-29; (3+) = 30-99, (4+) > 100. The rapid urease test had a sensitivity of 53%, specificity of 100%, and accuracy of 73%. The culture method had a sensitivity of 75%, specificity of 100%, and accuracy of 86%. Sensitivity of rapid urease test and the culture method decreased in a positive correlation to the decrease in total number of HP bacteria counted. Using the rapid urease test, sensitivity was < 30% when the grade of HP infection was (2+) or less, while 100% sensitivity was obtained only when the grade of HP infection was (4+). On the other hand, sensitivity of culture method, remained between 80 and 90% when HP infection was (2+) or more. The rapid urease test and culture of HP may result in false negative tests for mild infection although those have high sensitivity and specificity for moderate to severe infection. Immunohistochemical stain provides a reliable semi-quantitative diagnosis of HP infection. Clinicians should be aware of the characteristics of each method to detect HP infection, and select the appropriate ones for their purpose.

Effects of dilazep on arachidonic acid metabolism by neutrophils and platelet/neutrophil interactions.

The effect of dilazep (tetrahydro-1H-1,4-diazepine-1,4(5H)-dipropanolbis(3,4, 5-trimethoxy-benzoate)dihydrochloride monohydrate, Comelian), a coronary and cerebral vasodilator and an anti-platelet agent, on endogenous and exogenous arachidonic acid (AA) metabolism by human neutrophils and platelet/neutrophil interactions was studied in vitro. Neutrophils preincubated with dilazep (up to 300 mumol/l) were incubated with the Ca ionophore A23187 in the absence or presence of AA. Platelet/neutrophil mixtures preincubated with dilazep (up to 100 mumol/l) were incubated with thrombin plus N-formylmethionylleucylphenylalanine (FMLP), FMLP plus AA, and platelet-activating factor (PAF) plus AA. AA metabolites including leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (5-HETE) and 5S,12S-dihydroxyeicosatetraenoic acid (5S,12S-DiHETE) were analyzed and quantitated by reversed-phase high-performance liquid chromatography. The formation of LTB4 and 5-HETE from endogenous AA by neutrophils was inhibited by dilazep, whereas their production from exogenous AA was enhanced. LTB4 synthesis from endogenous AA by platelet/neutrophil interactions was inhibited by dilazep, while 5S,12S-DiHETE production was increased. The production of 5-lipoxygenase metabolites from exogenous AA by these interactions was increased by this drug. Thus, dilazep inhibits endogenous AA metabolism by neutrophils and by platelet/neutrophil interactions, whereas it stimulates exogenous AA metabolism by these blood cells and their interactions.

Effects of dilazep on cytoplasmic calcium ion concentrations and arachidonic acid metabolism in activated human platelets.

The effects of dilazep (tetrahydro-1H-1,4-diazepine-1,4(5H)-dipropanol bis(3,4,5-trimethoxybenzoate)-di-hydrochloride monohydrate, Comelian), a coronary and cerebral vasodilator and an antiplatelet drug, on the cytoplasmic Ca2+ concentration ([Ca2+]i) and arachidonic acid (AA) metabolism in activated human platelets were investigated. [Ca2+]i (free calcium ion concentration) of aequorin-loaded platelets was estimated by using the platelet ionized calcium aggregometer. AA metabolism was studied by the determination of AA metabolites, hydroxyheptadecatrienoic acid and 12-hydroxyeicosatetraenoic acid, using reversed-phase high performance liquid chromatography. When platelets were preincubated with dilazep (0-0.5 mmol/l), the drug inhibited both platelet aggregation and [Ca2+]i elevation induced by thrombin, AA and collagen in a concentration dependent manner, while only aggregation was inhibited after stimulation with the Ca ionophore A23187 (calcimycin). Both influx and release of Ca2+ into platelet cytoplasm induced by thrombin or AA were inhibited by dilazep, while neither of them was affected when induced by A23187. Oral ingestion of dilazep as a 100-mg capsule significantly depressed the [Ca2+]i elevation induced by thrombin, AA and collagen after 3 h. Dilazep inhibited endogenous AA metabolism by platelets induced by thrombin, although it enhanced exogenous one. Thus, dilazep inhibited platelet aggregation induced by any agonists including A23187, while [Ca2+]i elevation was inhibited by the drug only when the receptor-mediated agonist was used. Furthermore, it is suggested that dilazep inhibited AA liberation from platelet membrane phospholipids, leading to reduced production of all endogenous AA metabolites after platelet activation although metabolites of exogenous AA could be increased.

Lipoxygenase activities of human platelets and their subcellular fractions: comparison between lipoxygenase-deficient platelets and normal platelets.

Lipoxygenase activities were estimated in washed platelets (intact platelets) and their subcellular fractions obtained from 7 patients with deficient platelet lipoxygenase activities and 9 normal subjects. From sonicated platelet preparations, 12,000 g supernatant (F-I), cytosol (F-II) and microsomal fractions (F-III) were prepared by differential centrifugation. When 12-hydroxyeicosatetraenoic acid (12-HETE) produced by the incubation of arachidonic acid with intact platelets or each of their subcellular fractions from normal subjects was measured by reversed-phase high-performance liquid chromatography analysis, the lipoxygenase activities of F-I, F-II and F-III were 87%, 31% and 17%, respectively, of the enzyme activity of intact platelets. One of the patients showed no detectable lipoxygenase activity in any preparation, while the other patients showed reduced enzyme activities in all preparations. The addition of CaCl2 significantly increased 12-HETE synthesis solely by F-I from these patients. In most of these patients, contrary to normal subjects, it appeared that the lipoxygenase activity was not fully expressed in intact platelets, since the F-I produced more 12-HETE than the intact platelets.

A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia.

We found a novel platelet aggregating factor in a patient with steroid-responsive immune thrombocytopenic purpura that is associated with defective collagen-induced platelet functions. The aggregating factor and platelet functions were analyzed. The patient, a 58-year-old female, had purpura and prolonged bleeding time despite adequate platelet counts (greater than 140,000/microL) after steroid therapy. The patient's platelets responded normally to all agonists except collagen. Platelet adhesion to collagen fibrils was decreased. The patient's plasma induced irreversible aggregation and ATP release in normal platelet-rich plasma (PRP). This platelet aggregating factor was found in F(ab')2 fragments of the patient's IgG, which caused thromboxane B2 synthesis, elevation of cytoplasmic Ca2+ levels, and phosphorylation of 40 kDa protein in normal platelets. Platelet aggregation by the patient's IgG was inhibited by prostacyclin, dibutyryl cAMP, diltiazem, disodium ethylenediaminetetraacetate, and antimycin A plus iodoacetate, but ADP scavengers, cyclo-oxygenase inhibitors, and heparin had little or no effect. The aggregating activity of the patient's IgG absorbed to and eluted from normal platelets. The patient's Fab fragments did not induce platelet aggregation in eight of ten normal PRP but specifically inhibited aggregation induced by collagen and by the patient's IgG. The major component of an immunoprecipitate made with the patient's IgG from radiolabeled membrane proteins of normal platelet extract had a 62 kDa mol wt, while no such precipitate appeared in extracts of the patient's platelets. These results indicated that platelet aggregation by the patient's IgG was induced by the reaction of an antibody with a specific antigen on the normal platelet membrane through stimulus-response coupling. This antigen may be a collagen receptor on the platelet, most likely a polypeptide of 62 kDa under reducing condition. The defect of collagen-induced aggregation of the patient's platelets seemed to be due to alteration of the membrane protein related to this putative collagen receptor.

Hemorrhagic thrombocytopathy with platelet thromboxane A2 receptor abnormality: defective signal transduction with normal binding activity.

Subnormal platelet responses to thromboxane A2 (TXA2) were found in a patient with polycythemia vera, and the mechanism of this dysfunction was analyzed. The patient's platelets showed defective aggregation and release reaction to arachidonic acid, enzymatically generated TXA2 and synthetic TXA2 mimetics (STA2, U-46619). In contrast, they showed normal responses to thrombin. When the platelet TXA2 receptor was examined with both a 125I-labelled derivative of a TXA2 receptor antagonist ([125I]-PTA-OH) and a 3H-labelled TXA2 agonist ([3H]U-46619), the equilibrium dissociation rate constants (Kd) and the maximal concentrations of binding sites (Bmax) of the patient's platelets to both ligands were within normal ranges, suggesting that the binding capacity of their TXA2 receptor was normal. STA2 failed to induce normal elevation in the cytoplasmic free calcium ion concentration, phosphatidic acid formation and 40 kD protein phosphorylation in the patient's platelets, whereas these responses to thrombin were within normal ranges. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) also evoked normal response in the 40 kD protein phosphorylation in the patient's platelets. These results suggested that the patient's platelets had TXA2 receptor abnormalities which were characterized by defective transduction of the binding signal to postreceptor reactions after normal TXA2 binding.

Requirement of free arachidonic acid for leukotriene B4 biosynthesis by 12-hydroperoxyeicosatetraenoic acid-stimulated neutrophils.

Stimulation of human neutrophils with 12-hydroperoxyeicosatetraenoic acid (12-HPETE) led to formation of 5S, 12S-dihydroxyeicosatetraenoic acid (DiHETE), but leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE) was not detectable by reversed-phase high-performance liquid chromatography analysis. N-formylmethionylleucylphenylalanine (FMLP) induced the additional synthesis of small amounts of LTB4 in 12-HPETE-stimulated neutrophils. The addition of arachidonic acid greatly increased the synthesis of LTB4 and 5-HETE by neutrophils incubated with 12-HPETE. In experiments using [1-14C]arachidonate-labeled neutrophils, little radioactivity was released by 12-HPETE alone or by 12-HPETE plus FMLP, while several radiolabeled compounds, including LTB4 and 5-HETE, were released by A23187. These findings demonstrate that LTB4 biosynthesis by 12-HPETE-stimulated neutrophils requires free arachidonic acid which may be endogenous or exogenous.

Deficient induction of leukotriene synthesis in human neutrophils by lipoxygenase-deficient platelets.

The effect of human platelets with deficient lipoxygenase activities on leukotriene B4 (LTB4) synthesis by neutrophils was studied. When arachidonic acid (AA) metabolites obtained from the incubation of washed normal neutrophils and platelets with N-formylmethionylleucylphenylalanine (FMLP), cytochalasin B, and AA were analyzed by reversed-phase high-performance liquid chromatography, the synthesis of 5-lipoxygenase products, including LTB4, was remarkably stimulated by platelets, with their maximal effect at a ratio of platelets to neutrophils of 15:1. However, the use of lipoxygenase-deficient platelets obtained from four patients with myeloproliferative disorders instead of normal platelets showed the deficient production of 5-lipoxygenase-derived products, whereas platelets with normal lipoxygenase activities obtained from MPD patients stimulated the 5-lipoxygenase pathway similarly to the way in which normal platelets did. The addition of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), a labile AA metabolite via the platelet lipoxygenase pathway, could activate the 5-lipoxygenase pathway in neutrophils incubated with FMLP, cytochalasin B and AA, but its stable end product, 12-hydroxyeicosatetraenoic acid, could not. Thus, it is suggested that lipoxygenase-deficient platelets did not sufficiently stimulate LTB4 synthesis during platelet-neutrophil interactions because of defective formation of 12-HPETE. This altered interaction between platelets and neutrophils through the lipoxygenase pathway might result in deficient responses at sites of thrombosis or inflammation in patients with deficient platelet lipoxygenase activities.