Multipoint identification of Enterobacteriaceae: report of the British Society for Microbial Technology collaborative study

AIMS: To evaluate the accuracy and reproducibility of multipoint identification schemes in a multicentre trial. METHODS: Forty two strains of Enterobacteriaceae were distributed to 22 laboratories for identification by routine multipoint methods. Analysis of results enabled inter- and intralaboratory reproducibility of a variety of tests, and the ability of laboratories to identify individual organisms to be determined. RESULTS: Interlaboratory reproducibility of most of the biochemical tests was acceptable. The least reproducible tests, both within and between laboratories, were citrate utilisation, production of urease and beta galactosidase, detection of motility, and decarboxylation of lysine and ornithine. Inconsistent results for these tests were often associated with misidentified strains. Most laboratories performed identifications satisfactorily. Most isolates (72.1%) were identified correctly to species level; 9.6% were incorrectly identified, and 6.4% could not be identified at all. The most difficult organisms to identify were Citrobacter freundii, Enterobacter cloacae, Hafnia alvei and Aeromonas hydrophila. Strains of Enterobacter, Serratia sp, and Providencia sp were difficult to speciate. Several laboratories could not identify organisms exhibiting at least one atypical biochemical reaction. CONCLUSION: This study emphasises the need for quality control of media and reagents for multipoint identification of Gram negative enteric bacilli.     

Identification of nasopharyngeal carriage of an outbreak strain of Neisseria meningitidis by pulsed-field gel electrophoresis versus phenotypic methods

BACKGROUND: Excessive stress can cause both physical and psychological health problems in GPs, and can lead to decreased quality of patient care. OBJECTIVE: This paper examines the range of coping strategies available to GPs and suggests several support mechanisms. DISCUSSION: Coping strategies include talking to family and peers, problem solving and specific stress management techniques. GPs might need to be taught how to be patients themselves. Divisions can provide support groups for stressed GPs. The profession and government must consider how extra demands on general practice, coupled with further financial pressure, can lead to excessive strain.     

Routine application of high-performance liquid chromatography for identification of mycobacteria

Mycolic acid analysis by high-performance liquid chromatography (HPLC) was introduced in our laboratory as the routine technique for identifying all clinical isolates of mycobacteria referred to us. HPLC identified 96.1% of the 1,103 strains analyzed, whereas the biochemical procedures and/or the commercial DNA probes identified 98.3% of strains, for an overall agreement of 94.4%. Compared with the probes, there was 100% specificity and 98.9% sensitivity for Mycobacterium tuberculosis identification. HPLC allowed early detection and identification of the rare mycobacterial species M. haemophilum, M. malmoense, M. shimoidei, and M. fallax as well as uncharacteristic strains of M. simiae. After 18 months of routine use, HPLC proved to be reliable, easy to perform, rapid, and less costly than other identification methods.     

p53 mutations in human bladder cancer: genotypic versus phenotypic patterns

Twelve sympathetic and 14 parasympathetic extra-adrenal paragangliomas were investigated immunohistochemically with antibodies against chromogranin A, chromogranin B, and secretogranin II. In sympathetic paragangliomas chromogranin A was found in 12/12 and chromogranin B in 11/12 tumors in almost all chief cells (the remaining tumor was focally chromogranin B positive), whereas secretogranin II was immunolocalized in the majority of chief cells in 5/12, in a focal distribution in 3/12, and only in a few scattered tumor cells in 3/12 cases. One case showed no secretogranin II immunoreactivity. In parasympathetic paragangliomas both chromogranin B and secretogranin II immunoreactivity was demonstrated in the majority of chief cells of all 14 tumors investigated. Chromogranin A showed a strong immunostaining in 2/14 cases; in 12 tumors chromogranin A was found in only a few chief cells or was completely absent. It is concluded that sympathetic and parasympathetic paragangliomas show a divergent expression of chromogranins/secretogranins that apparently reflects the different histogenetic origins of these tumors.     

Reproducibility of 1H-NMR integrals: a collaborative study

Respiratory distress syndrome (RDS) is characterized by intrapulmonary fibrin deposition, which can adversely affect surfactant function, and stimulate fibroblast proliferation, which may contribute to the development of bronchopulmonary dysplasia (BPD). We speculated that the premature lung may have impaired regulation of thrombin, thus making preterm infants susceptible to fibrin formation within the lung. Therefore, we studied the effect of stretch, which simulates fetal breathing movements (FBMs), on the generation and inhibition of a key hemostatic enzyme-thrombin-by rat fetal mixed lung cells (FMLCs). Our results showed that stretch induced glycosaminoglycan production with increased antithrombin activity due to an increase in the concentration of active chondroitin sulfate. Stretch downregulated secretion of tissue factor procoagulant activity, which may lead to decreased thrombin generation on the surface of FMLCs. Overall, stretch enhanced the local control of thrombin by FMLCs. These results suggest that premature infants, who will have experienced less FBM, may have impaired thrombin regulation. Impaired thrombin regulation likely contributes to increased fibrin deposition and, potentially, the development of BPD.     

Sensitivity of fluorochrome microscopy for detection of Mycobacterium tuberculosis versus nontuberculous mycobacteria

BACKGROUND: Sternal wound infection is a relatively rare but potentially devastating complication of open heart operations. The most common treatments after debridement are rewiring with antibiotic irrigation and muscle flaps. Here we present the results of a prospective trial to determine the appropriate roles of closed-chest catheter irrigation and muscle flap closure for sternotomy infection and to assess the effect of internal mammary artery bypass grafting on the outcome of each treatment modality. METHODS: Between 1990 and 1994, 5,658 sternotomies were performed at the University of Washington Medical Center. Sternal dehiscence occurred in 43 patients, 25 of whom had infection (overall incidence, 0.44%). Because of the infrequency of this complication, a prospective, randomized trial was developed in which the initial approach to sternal dehiscence was rewiring and catheter irrigation. Muscle flaps were used as the primary treatment if the sternum could not be restabilized or as secondary treatment if catheter irrigation failed. Wound resolution, length of hospital stay, and complications were evaluated. RESULTS: Sterile dehiscences were successfully closed with irrigation in 17 of 18 patients; the other patient required flap closure. Of the 25 patients with infection, 19 had irrigation and 6, closure with flaps primarily. In the group of infected patients, 17 of the 19 who received irrigation also had internal mammary artery bypass grafting. Irrigation failed in 15 (88.2%) of these 17 patients, and salvage was accomplished with muscle flap closure. All 6 patients with infection who were closed primarily with muscle flaps had a successful outcome. Hospitalization averaged 10.2 days when muscle flaps were used primarily and 14.3 additional days for unsuccessful irrigation. When irrigation was successful, the hospital stay averaged 11.2 days. CONCLUSIONS: Catheter irrigation should be reserved for patients without infection or patients with infection but without internal mammary artery bypass grafts in whom dehiscence occurs less than 1 month after sternotomy. All others should have closure with muscle flaps.     

Improved identification of mycobacteria by using the microbial identification system in combination with additional trimethylsulfonium hydroxide pyrolysis

The MIDI automated Microbial Identification System (MIS) uses gas chromatography (GC) analysis of whole-cell fatty acid methyl esters (FAMEs) between 9 and 20 carbons in length to characterize a wide range of bacterial genera and species, including mycobacteria. Mycolic acid cleavage products (MACPs) with chain lengths of C22 to C26 are not released by MIDI sample preparation of mycobacteria. Therefore, the MIS library search report often matches several mycobacterial species without any significant difference in the similarity indices. The problem is solved by adding trimethylsulfonium hydroxide (TMSH) instead of sodium sulfate in the last step of sample preparation, thus allowing the identification of MACPs in addition to FAMEs. Only one GC run parameter has to be changed: the temperature program must be extended from 260 to 310 degrees C. The MIS library search report for the identification of bacteria is not disturbed by TMSH. The combination of conventional library search report with the information of typical MACP patterns yields significantly better discrimination of mycobacterial species than the MIDI method allows.     

Differentiation of Yersinia enterocolitica serotype O:5,27 strains by phenotypic and molecular techniques

Restriction endonuclease analyses of virulence plasmid DNA (REAP) and chromosomal DNA and other phenotypic characteristics were used to study the differentiation of Yersinia enterocolitica serotype O:5,27 strains. There was a close correlation between REAP patterns and the geographical distribution of serotype O:5,27. Human isolates produced only one REAP pattern, which was also found with isolates from pigs and dogs.     

Measuring psychological uncertainty: Verbal versus numeric methods.

The authors argue that alternatives to the traditional numeric methods of measuring people's uncertainty may prove to hold important advantages under some conditions. In 3 experiments, the authors compared verbal measures involving responses such as very likely, and numeric measures involving responses such as 80% chance. The verbal measures were found to show more sensitivity to various manipulations affecting psychological uncertainty (Experiment 1), to be better predictors of individual preferences among options with unknown outcomes (Experiment 2), and to be better predictors of behavioral intentions (Experiment 3). Results suggest that numeric measures tend to elicit deliberate and rule-based reasoning from respondents, whereas verbal measures allow for more associative and intuitive thinking. Given that there may be many types of situations in which human decisions and behaviors are not based on deliberate and rule-based thinking, numeric measures may misrepresent how individuals think about uncertainty in those situations. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Evaluation of the home-based maternal record: a WHO collaborative study

Thirteen centres in eight countries (Egypt, India, Pakistan, Philippines, Senegal, Sri Lanka, Democratic Yemen and Zambia) participated in the WHO collaborative study to evaluate the home-based maternal record (HBMR). The evaluation showed that use of the HBMR had a favourable impact on utilization of health care services and continuity of the health care of women during their reproductive period. When adapted to local risk conditions, their cut-off points and the available resources, the HBMR succeeded in promoting self-care by mothers and their families and in enhancing the timely identification of at-risk cases that needed referral and special care. The introduction of the HBMR increased the diagnosis and referral of at-risk pregnant women and newborn infants, improved family planning and health education, led to an increase in tetanus toxoid immunization, and provided a means of collecting health information in the community. The HBMR was liked by mothers, community health workers and other health care personnel because, by using it, the mothers became more involved in looking after their own health and that of their babies. Apart from local adaptation of the HBMR, the training and involvement of health personnel (including those at the second and tertiary levels) from the start of the HBMR scheme influenced its success in promoting maternal and child health care. It also improved the collection of community-based data and the linking of referral networks.     

Mammalian Golgi apparatus UDP-N-acetylglucosamine transporter: molecular cloning by phenotypic correction of a yeast mutant

Transporters in the Golgi apparatus membrane translocate nucleotide sugars from the cytosol into the Golgi lumen before these can be substrates for the glycosylation of proteins, lipids, and proteoglycans. We have cloned the mammalian Golgi membrane transporter for uridine diphosphate-N-acetylglucosamine by phenotypic correction with cDNA from MDCK cells of a recently characterized Kluyveromyces lactis mutant deficient in Golgi transport of the above nucleotide sugar. Phenotypically corrected transformants were separated from mutants in a fluorescent-activated cell sorter after labeling of K. lactis cells with fluorescein isothiocyanate (FITC) conjugated to Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine. A 2-kb DNA fragment was found to restore the wild-type cell lectin binding phenotype, which reverted to the mutant one upon loss of the plasmid. The DNA fragment contained an ORF encoding a hydrophobic, multitransmembrane spanning protein of 326 aa that had only 22% amino acid sequence identity with the corresponding transporter from K. lactis but showed 53% amino acid sequence identity to the mammalian UDP-galactose transporters and 40% to the CMP-sialic acid transporter. Golgi vesicles from the transformant regained their ability to transport UDP-GlcNAc in an assay in vitro. The above results demonstrate that the mammalian Golgi UDP-GlcNAc transporter gene has all of the necessary information for the protein to be expressed and targeted functionally to the Golgi apparatus of yeast and that two proteins with very different amino acid sequences may transport the same solute within the same Golgi membrane.     

Variables affecting results of sodium chloride tolerance test for identification of rapidly growing mycobacteria

The sodium chloride tolerance test is often used in the identification of rapidly growing mycobacteria, particularly for distinguishing between Mycobacterium abscessus and Mycobacterium chelonae. This test, however, is frequently unreliable for the identification of some species. In this study we examined the following variables: medium manufacturer, inoculum concentration, and atmosphere and temperature of incubation. Results show that reliability is improved if the test and control slants are inoculated with an organism suspension spectrophotometrically equal to a 1 McFarland standard. Slants should be incubated at 35 degrees C in ambient air and checked weekly for 4 weeks. Growth on control slants should be critically evaluated to determine the adequacy of the inoculum; colonies should number greater than 50. Salt-containing media should be examined carefully to detect pinpoint or tiny colonies, and colonies should number greater than 50 for a positive reaction. Concurrent use of a citrate slant may be helpful for distinguishing between M. abscessus and M. chelonae. Molecular methodologies are probably the most reliable means for the identification of rapidly growing mycobacteria and should be used, if possible, when unequivocal species identification is of particular importance.     

Malaria: existing methods of vector control and molecular entomology

In general, the most effective means of malaria vector control is the killing of adult mosquitoes with a residual insecticide applied to bednets or sprayed on house walls and ceilings. Major reductions in all-cause child mortality have been achieved in Africa by these means. In some circumstances, personal protection and larval control may also make a contribution. We discuss the prospects of genetic control by release of sterile male mosquitoes or driving genes for refractoriness to malaria into wild populations. Many major malaria vectors belong to complexes of sibling species which differ in vectorial and biological characteristics. Distinguishing the species by cytogenetic or molecular methods is important for epidemiological studies and could improve the targeting of control.     

COAE thresholds: 1. Effects of equal-amplitude versus subtraction methods

Although research has demonstrated that click-evoked otoacoustic emissions (COAEs) elicited by high-level stimuli are useful for identifying hearing loss, the ability of COAEs to predict behavioral thresholds has not been adequately tested. Results of studies comparing COAE thresholds and behavioral thresholds have been equivocal, perhaps due to the need for a more rigorous approach to COAE threshold estimation. The present study was designed to address several methodological concerns in COAE threshold testing, particularly the effects of two methods of stimulus presentation on COAE testing and threshold calculation. In an attempt to make COAE threshold estimation consistent across participants, COAE threshold calculations were based on mean noise floor levels across participants. COAE and noise floor levels were measured in 15 participants using both equal-amplitude clicks and a subtraction method. Broadband COAEs were analyzed into 1/3 octave bands, so that input/output functions could be examined and COAE thresholds could be calculated for each 1/3 octave band. Comparison of the two stimulus methods indicated several differences. Mean noise floor levels for the equal-amplitude method were approximately 6 dB lower than those measured for the subtraction method across frequency. In many cases COAEs evoked using the equal-amplitude method were higher in amplitude than those evoked using the subtraction method. COAE thresholds measured using the equal-amplitude click stimuli were significantly lower than those measured using the subtraction method. The significantly higher thresholds obtained using the subtraction method may be attributed in part to the reduction of COAE amplitude by the subtraction procedure, and not merely to the higher noise level. Slopes of the input/output functions were not significantly different between the two stimulus methods. These results suggest that the equal-amplitude method is preferable for COAE threshold testing because lower noise floor and larger amplitude COAEs may be obtained in the same test time.     

Realpolitik versus fair process: Moderating effects of group identification on acceptance of political decisions.

Three studies examined the effects of perceived procedural justice and the favorability of a group-level outcome on the endorsement of a group-level decision and the evaluation of the authority responsible for the decision. Results showed that, contrary to findings usually seen with individual-level decisions, collective outcome favorability was more important than procedural justice in influencing the endorsement of the decision. Furthermore, increased identification with the group reduced the importance of procedural justice but accentuated the importance of collective outcome favorability. With regard to the evaluation of the authority, the results were similar to those obtained in individual-level decisions: Procedural fairness mattered more than collective outcome favorability. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Genus level identification of mycobacteria from clinical specimens by using an easy-to-handle Mycobacterium-specific PCR assay

An easy-to-handle Mycobacterium-specific PCR assay for detection of the presence of a wide range of mycobacterial species in clinical samples was evaluated. The performance of the genus probe was compared with the performance of probes specific for Mycobacterium tuberculosis and Mycobacterium avium and with that of standard culture. In addition, the utility of an internal control in monitoring amplification inhibitors was studied. Of 545 respiratory and 325 nonrespiratory specimens (a total of 870 specimens), 58 (6.7%) showed the presence of amplification inhibitors, as determined by a negative result for the internal control. Of these 58 specimens, 31 (53%) were stool specimens; other material, even citrate blood after lysis of erythrocytes, did not pose a problem with regard to inhibition of PCR amplification. Eighty-one of the remaining 812 specimens had a positive Mycobacterium culture result. Of these culture-positive specimens, 58 (71.6%) showed a positive result with the Mycobacterium genus-specific probe. Seventy-two samples had a positive result with the Mycobacterium-specific probe but a negative culture result. Of these 72 samples, 26 samples were regarded as true positive, either because the M. tuberculosis- or M. avium-specific probe was also positive at the same time or because other specimens from the same patient taken at the same time were culture positive. The sensitivity of the Mycobacterium-specific probe was 78.5% and the specificity was 93.5%. This study showed that pretesting of clinical specimens for mycobacteria to the genus level with a Mycobacterium-specific probe offers the routine clinical laboratory the possibility of detecting tuberculous and nontuberculous mycobacteria with one test. Furthermore, specimens testing positive with the genus-specific probe can be immediately identified with species-specific probes.