Laboratory handling of Helicobacter pylori critically influences the results of in-vitro metronidazole resistance determination.

In-vitro metronidazole resistance rates of Helicobacter pylori determined by Etest are high, and the predictive value of metronidazole resistance is low. It was hypothesised that altered laboratory methods could reduce the overestimation of resistance and improve the predictive value of the Etest. Pre-treatment isolates (n = 150) of H. pylori from 150 patients were investigated by Etest with incubation for 72 h. Treatment with metronidazole, tetracycline and bismuth for 10 days failed to eradicate H. pylori in 23 patients. After isolate storage for 3 years, resistance determination results by agar dilution and Etest, with incubation for 72 and 31 h, were compared. The rate of metronidazole resistance was reduced significantly during storage, and instability of resistance was associated significantly with treatment outcome. Isolates that retained in-vitro resistance had significantly (p 0.008) higher treatment failure rates (n = 13; 42%) than isolates that lost resistance (n = 3; 9%). The reproducibility achieved by dual testing with agar dilution and Etest was 41% and 70% for +/- 1 and +/- 2 log2 dilutions, respectively, after incubation for 72 h, and 85% and 92%, respectively, after incubation for 31 h. Thus, the predictive value was improved from 25% to 50% by the altered laboratory conditions (p 0.04). MIC values of 2-8 mg/L signified an intermediate risk of treatment failure.

Assessment of optimal atmospheric conditions for growth of Helicobacter pylori.

The number and diameter of colonies of Helicobacter pylori isolates growing on agar plates were determined to compare five methods that produce a culture atmosphere. No catalyst was applied. No significant difference was found between two hydrogen-based kits that have a different capacity for production of H2. These hydrogen-based methods were significantly better than all others evaluated, including one kit that produces ascorbic acid that binds with oxygen. Growth was significantly improved when the atmosphere outside the plastic incubation jars was enriched with 10% CO2, but carbon dioxide enrichment alone (i.e., no reduction of the oxygen concentration) gave a very poor yield. The colony diameter was a sensitive and reliable measure of atmospheric conditions, as the mean intra- and interobserver difference between repeated readings was < or = 0.1 mm for 82% and < or = 0.2 mm for 95% of the isolates.

Pressure response of protein backbone structure. Pressure-induced amide 15N chemical shifts in BPTI.

The effect of pressure on amide 15N chemical shifts was studied in uniformly 15N-labeled basic pancreatic trypsin inhibitor (BPTI) in 90%1H2O/10%2H2O, pH 4.6, by 1H-15N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15N signals were low field shifted linearly and reversibly with pressure (0.468 +/- 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0-1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three-dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 A/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15N shifts is considered to reflect site-specific changes in phi, psi angles. For beta-sheet residues, a decrease in psi angles by 1-2 degrees/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 +/- 0.343 and 0.519 +/- 0.261 ppm/2 kbar, respectively) than for beta-sheet (0.295 +/- 0.195 ppm/2 kbar), suggesting that the pressure-induced structural changes (local compressibilities) are larger in helical and loop regions than in beta-sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in beta-sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place.

A simple method for determining metronidazole resistance of Helicobacter pylori.

The reliability of methods for determination of Helicobacter pylori resistance to metronidazole has been found to depend upon the incubation time. Because the disk diffusion method is more vulnerable than other methods to prolonged incubation, this method has not been recommended for H. pylori. However, because media designed for rapid growth of H. pylori have been introduced, the time has come to look at the clinical usefulness of this inexpensive and simple method again. The correlation of readings obtained with the E test (AB Biodisk, Solna, Sweden) and Rosco's (Taastrup, Denmark) disk diffusion method for in vitro metronidazole resistance determination for H. pylori with a short incubation time (24 to 31 h) was studied. Plates which could not be read after 24 to 31 h were reincubated for another night. Fifty-seven consecutive clinical strains were tested. Because the rate of regrowth of H. pylori depends upon the age of the colonies inoculated, the reproducibility of resistance test results for young colonies versus old colonies was also studied. Resistance plates could be read after 24 to 31 h of incubation for 28 of 29 strains when the inoculum consisted of young colonies (3 to 4 days old). For these 29 strains, a high correlation (r = -0.937) was found between results obtained with the E test and those obtained with the disk diffusion test. A poorer correlation was found for old colonies (> or = 5 days old) (r = -0.742), which required a prolonged incubation for 8 of 23 strains. In conclusion, short incubation was successfully applied with young colonies. Results obtained with the simple and inexpensive disk diffusion method correlated well with those obtained with the E test.

Risks related to lack of standardization of tests to detect in vitro metronidazole resistance in Helicobacter pylori.

The correlation between metronidazole MIC readings obtained in vitro on two different media for two different inoculum dilutions was studied after two, three, and five days of incubation using 20 clinical isolates of Helicobacter pylori. The PDM epsilometer (E test) was used. After two days of incubation, log MIC values on chocolate PDM and Vestfold charcoal medium, a new charcoal medium, showed good correlation. Charcoal media can thus be used for metronidazole sensitivity tests. In terms of double-dilution gradients, prolongation of incubation time from two to three days and change of inoculum dilution from 0.5 to 4 McFarland had little impact when studied separately. However, the combined effect of the two variables was marked. The matching of a sparse inoculum incubated for two days against a heavy inoculum incubated for three days resulted in readings which, for 30% of the pairs, differed by two double-dilution gradients or more. On the other hand, the separate influence of both variables was found to affect the correlation coefficients significantly. Five days of incubation was associated with a poor correlation when the variables on MIC readings was not exposed in a scale of double-dilution gradients because the intervals were too large. For this reason, scales without thresholds are recommended.

Rapid growth of Helicobacter pylori.

The ability of six variants of a new charcoal medium and Skirrow's medium to grow Helicobacter pylori in 3 and 5 days was studied using 20 different strains of Helicobacter pylori. The main admixtures for the charcoal media were serum, whole blood, and egg yolk emulsion. For this purpose, serum was significantly better and egg yolk emulsion significantly worse than whole blood. The addition of Iso Vitalex resulted in significantly improved growth on the charcoal media. Skirrow's medium showed very poor performance after three days of incubation and needed a long incubation time.

Rapid diagnosis of respiratory syncytial virus and influenza A virus infections by immunofluorescence: experience with a simplified procedure for the preparation of cell smears from nasopharyngeal secretions.

A simplified procedure for preparing direct smears of exfoliated cells from nasopharyngeal secretions is presented. The specimens were examined by the indirect immunofluorescence (IF) technique for the presence of respiratory syncytial (RS) virus and influenza A virus antigens. Specimens from 457 children hospitalised at twelve different hospital departments in Norway were examined during the winter season 1980-81. RS virus was identified in samples from 139 children and influenza A virus from 28. Results of IF examinations for RS virus were copositive with serology in 19 of 23 cases (83%) and with probably no false positive results. Samples from 29 children were also prepared by the conventional washing technique. RS virus was identified in 18 of these specimens. Seventeen of these were also found positive by the simplified method. This comparison was done blind. Compared to results of virus isolation and serology the IF examinations for influenza A virus were somewhat less sensitive with copositivity in 12 of 17 cases (71%) and in one instance the IF finding was probably false positive. However, also for influenza A virus our IF findings were fully comparable with figures obtained by others who performed the conventional washing technique for preparation of exfoliated cells from nasopharyngeal secretions. General practitioners submitted samples from 65 patients, and RS virus and influenza A virus were identified in eleven and four cases respectively. The simplicity of preparation of cell smears from nasopharyngeal secretions with our simplified method opens up the possibility of the application of rapid respiratory virus diagnostic service to an increasing number of hospital departments and also for general practitioners. We feel that this method is a suitable tool for surveillance of certain respiratory virus infections.

Radioimmmunoassay for melatonin in human serum.

Goat antisera raised against N-acetyl-5-methoxytryptophan conjugated to bovine thyroglobulin by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is utilized in a radioimmunoassay for melatonin. The raised antibodies are coupled to Sepharose 4B and melatonin in human serum is isolated by affinity chromatography, thereby avoiding the time-consuming extractions by organic solvents. A detection limit of 1.9 pg (8.2 X 10(-15) mol) melatonin is achieved. The antibody specificity has been analysed and none of the common melatonin analogues influence this method of melatonin measurement.

Art, evolution, and history: a case study of paradigm change in anthropology.

An ethnological controversy over the origin and evolution of decorative art is documented for the period 1896-1904 and is used to test the relevance in anthropology of Thomas Kuhn's outline of the structure of scientific revolutions. Using a combination of archival materials and content analysis of professional periodicals, both the appropriateness and the limitations of Kuhn's scheme are explored. The conclusion is that paradigms and scientific revolutions are valid and useful concepts for use in the history of anthropology, but that for the particular period under study they are insufficient. Nonparadigmatic aspects of anthropology's supporting communities must also be considered, especially anthropology's "permeable boundaries".