Assessment of Resolution and Intercenter Reproducibility of Results of Genotyping Staphylococcus aureus by Pulsed-Field Gel Electrophoresis of SmaI Macrorestriction Fragments: a Multicenter Study

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.The use of electric field pulsing techniques in conjunction with agarose gel electrophoresis for discrimination of large DNA molecules was introduced by Schwarz and Cantor in 1984 (9). During the past decade the methodology has been adapted and improved by various research groups to the point that pulsed-field gel electrophoresis (PFGE) for bacterial strain typing is now utilized with relative ease in a variety of laboratories (1). The combination of contour-clamped homogeneous field electrophoresis and PFGE for the molecular analysis of Staphylococcus aureus has been reported since the late 1980s (7, 19). At present, PFGE is considered to have both the reproducibility and resolving power of a standard technique for the epidemiological typing of bacterial isolates (10, 15).Molecular typing systems can identify different strains within a species, generating data useful for taxonomic or epidemiologic purposes (10, 14). A frequently observed shortcoming of typing systems in general is their lack of reproducibility: most typing systems do not provide a definitive strain identification, which is usually due to the variability of the technique and the lack of large databases containing fragment patterns from a wide variety of organisms to which unknowns can be compared. These problems were recently described in detail for two molecular typing systems. A multicenter study on random amplification of polymorphic DNA for discrimination of S. aureus strains revealed a lack of interlaboratory reproducibility among the banding patterns generated by the participating centers, although the epidemiological interpretation of the data was similar for all the centers involved (16). For PFGE, a similar lack of interlaboratory reproducibility of patterns was observed, although the interpretation of the experimental data also differed per participating center (2). The latter study analyzed 12 different methicillin-resistant S. aureus (MRSA) strains with different techniques optimized in each center and different sources and types of equipment. Since interlaboratory discrepancies with respect to classification of the strains were observed, the study concluded that there is a clear need for standardization of the technique, including the construction of a panel of reference strains to assist the individual researcher in the optimization of the PFGE protocol.The aim of the present study was to compare the fragment patterns of a well-defined collection of MRSA isolates in 12 laboratories using in-house and a standard set of PFGE parameters to determine whether standardization of experimental parameters (DNA preparation and switching protocols) would improve intercenter reproducibility of PFGE analysis.     

Menstrual cycle effects on the action of buspirone on food intake in healthy female volunteers

We studied the effect of single doses of the 5-HT(1A) agonist buspirone (10 mg and 20 mg) on food intake during the menstrual cycle of nine healthy women, none of whom were on a contraceptive pill. In a double-blind placebo-controlled trial, subjects were tested over three cycles, receiving one treatment per cycle. Within each cycle, they were tested at two time points; mid-follicular and late luteal. Food intake following 20 mg buspirone was significantly higher in the luteal phase, suggesting the possibility of pre-menstrually enhanced 5-HT receptor sensitivity.     

CAMPATH-1H IN RHEUMATOID ARTHRITIS--AN INTRAVENOUS DOSE-RANGING STUDY

Forty-one patients with active and refractory rheumatoid arthritis(RA) received a total of 100, 250 or 400 mg of CAMPATH-1H (CAMPATHis a trademark of Glaxo-Wellcome group companies, registeredin the US Patent and Trademark Office) over 5 or 10 days inan open, uncontrolled study. Following therapy, patients weremonitored for adverse effects and disease activity for 6 months.Therapy was associated with prolonged peripheral blood lymphopeniain all dosing cohorts. During the month immediately followingtherapy, lymphopenia was most profound in the 400 mg cohorts.The first dose of monoclonal antibody (Mab) was associated witha ‘flu’-like syndrome, more pronounced at higherinitial doses. One patient developed haemolytic-uraemic syndrome.There were a number of dose-related infections during the earlypost-treatment period and one fatal opportunistic infectionwhich followed additional immunosuppressive therapy. Antiglobulinresponses developed in 9 of 31 patients tested. The majorityof patients showed symptomatic improvement following therapyand 20% of patients maintained a 50% Paulus response at 6 months,all of whom were in the 250 or 400 mg cohorts. CAMPATH- 1H appearsto be an effective treatment for RA. Allowing for the smallnumber of patients treated, infections were more common withhigher doses, although this was not true for adverse eventsoverall, and therapeutic responses were more sustained at higherdosing levels. The broad specificity of CAMPATH- 1H may be appropriatefor the immunotherapy of RA and future studies should aim todefine a dose with an optimal therapeutic ratio. KEY WORDS: CAMPATH-1H, Rheumatoid arthritis, Immunotherapy, Monoclonal antibody, Antiglobulin response     

Adult familial cryptogenic fibrosing alveolitis in the United Kingdom

BACKGROUND: Familial cases of cryptogenic fibrosing alveolitis (CFA) have previously been reported; however, the prevalence and genetic background of this disorder are not known. The clinical and epidemiological findings of 25 families identified within the UK are reported. METHODS: Adult pulmonary physicians in the UK were asked to identify all families under their care in which two or more individuals had been diagnosed with fibrosing alveolitis of unknown cause. A detailed structured questionnaire was sent to each proband to delineate possible environmental/occupational exposures and to obtain complete pedigree data. Physicians were also asked to provide clinical and diagnostic information. RESULTS: Twenty five families were identified comprising 67 cases. Suitable data for analysis were available for 21 families (57 cases). The male:female ratio was 1. 75:1 (p<0.05). A high resolution computed tomographic (HRCT) scan was performed in 93% and a diagnosis of CFA confirmed on biopsy specimens in 32%. The mean age at diagnosis was 55.5 (2.5) years. Fifty percent of cases were ever smokers and 18% had been diagnosed as asthmatic. Exposure to known fibrogenic agents was recorded by 36% of patients. Clinical signs/symptoms and histological findings were indistinguishable from non-familial cases. CONCLUSIONS: This study represents the largest cohort of familial CFA cases reported to date and confirms a prevalence of 1.34 cases per 10(6) in the UK population. Although rare, such cases represent an important subgroup in which a genetic susceptibility to pulmonary fibrosis is particularly evident. Familial patients are younger at diagnosis but otherwise indistinguishable from non-familial cases. The mode of inheritance is as yet unclear but a number of genetic loci are likely to be involved and are the subject of ongoing studies.     

Outcome of children with respiratory symptoms without objective evidence of asthma: a two-year, prospective, follow-up study

This study evaluated the outcome of 33 children with asthma-like symptoms without objective evidence of asthma, and the role of certain factors in predicting the development of clinical asthma in these children. Data on symptom histories, lung functions (flow-volume spirometry, free running test and methacholine inhalation challenge test) and atopic sensitization (skin prick tests and markers of eosinophilic inflammation) were collected twice with an interval of 2 y, and the diagnoses were re-evaluated after the follow-up period. Based on the results, it was concluded that one-third of the children with prolonged or recurrent lower airway symptoms, such as cough or wheeze, either have mild asthma or will develop asthma in the near future. Children who had a significant response [≥ 10% fall in forced expiratory volume in 1 s (FEV1)] in the free running test formed a risk group for active asthma, whereas other baseline characteristics seemed not to predict the outcome.     

Randomized trial examining cerebral haemodynamics following artificial or animal surfactant

To determine the effects of animal and artificial surfactants on cerebral haemodynamics, 20 premature babies receiving mechanical ventilation were randomized to receive Curosurf or Exosurf surfactant. Anterior cerebral artery blood flow velocity (CABFV) was measured using Doppler ultrasound before and up to 2 h after treatment. Following animal surfactant there was a rapid reduction in CABFV (median -36%, range -43% to +8%, p < 0:01), whereas artificial surfactant resulted in a slower rise which was less marked (median +20%, range -7% to +62%, p < 0:05). There were no significant changes in blood pressure. Two hours after administration, the oxygenation index (OI) improved significantly only in babies receiving animal surfactant. In this group there was a significant association between the change in CABFV at 1 min and the change in OI at 2 h ( r = 0:66, p < 0:05). Animal surfactant produces rapid improvements in ventilation which are associated with marked alterations in cerebral haemodynamics.