Pulsed-field gel electrophoresis (PFGE) is the most common genotypic method used in reference and clinical laboratories for typing methicillin-resistant Staphylococcus aureus (MRSA). Many different protocols have been developed in laboratories that have extensive experience with the technique and have established national databases. However, the comparabilities of the different European PFGE protocols for MRSA and of the various national MRSA clones themselves had not been addressed until now. This multinational European Union (EU) project has established for the first time a European database of representative epidemic MRSA (EMRSA) strains and has compared them by using a new "harmonized" PFGE protocol developed by a consensus approach that has demonstrated sufficient reproducibility to allow the successful comparison of pulsed-field gels between laboratories and the tracking of strains around the EU. In-house protocols from 10 laboratories in eight European countries were compared by each center with a "gold standard" or initial harmonized protocol in which many of the parameters had been standardized. The group found that it was not important to standardize some elements of the protocol, such as the type of agarose, DNA block preparation, and plug digestion. Other elements were shown to be critical, namely, a standard gel volume and concentration of agarose, the DNA concentration in the plug, the ionic strength and volume of running buffer used, the running temperature, the voltage, and the switching times of electrophoresis. A new harmonized protocol was agreed on, further modified in a pilot study in two laboratories, and finally tested by all others. Seven laboratories' gels were found to be of sufficiently good quality to allow comparison of the strains by using a computer software program, while two gels could not be analyzed because of inadequate destaining and DNA overloading. Good-quality gels and inclusion of an internal quality control strain are essential before attempting intercenter PFGE comparisons. A number of clonally related strains have been shown to be present in multiple countries throughout Europe. The well-known Iberian clone has been demonstrated in Belgium,

Human brucellosis poses a significant public health problem in many developing countries and requires fast and accurate diagnosis. A PCR assay amplifying part of the 31-kDa Brucella abortus antigenic protein gene sequence was developed and applied to whole-blood and serum samples from 31 brucellosis patients and 45 healthy individuals. All patients except one had detectable Brucella DNA in either whole blood or serum (combined sensitivity, 97%), but the assay sensitivity was higher with serum samples (94%) than with wholeblood samples (61%). The assay specificity was excellent (100%). A confirmatory PCR assay targeting another Brucella gene region (omp-2) was also developed but lacked sensitivity. Serum is the optimal specimen for the diagnosis of brucellosis by PCR, a choice that leads to assay simplification and shortens turnaround time.Brucellosis is still an important zoonosis of both public health and economic significance in many developing countries. Half a million new cases are reported worldwide each year, but according to the World Health Organization, these numbers greatly underestimate the true incidence of human disease (22). As the clinical picture of human brucellosis is extremely variable, diagnosis can be established only by laboratory methods. Since the disease constitutes a serious infection necessitating treatment with a prolonged course of antibiotics, accuracy and short turnaround time are required for these tests (21).Blood cultures represent the "gold standard" of laboratory diagnosis. Automated systems have been reported to detect more than 95% of Brucella melitensis-positive cultures within 7 days of incubation (23). Unless this technology is not available, prolonged incubation, blind subcultures, and special growth media are no longer required (23). Ironically, however, the technology indeed is lacking in developing countries or rural areas where the disease is prevalent. In addition, due to their comparatively long doubling time, Brucella species grow slowly on primary cultures and subcultures, while their inert biochemical profiles hamper fast identification of isolates (9). Distinct disease conditions like focal, relapsing, or chronic disease and disease caused by species other than B. melitensis are characterized by low blood culture yields and pose special diagnostic problems (1, 2). Consequently, detection and identification of Brucella spp. in clinical specimens by cultures may still be a difficult task with significant delays.Several agglutination tests (Rose Bengal, Wright's tube, Wright's card, and Wright-Coombs) and indirect immunofluorescence, complement fixation, and enzyme-linked immunosorbent assays are also available for diagnosis of brucellosis (3, 14, 24). The standard, with which all other methods should be compared, is Wright's tube agglutination test (1,14). A broad range of test sensitivity, low specificity in areas of endemicity, lack of usefulness in diagnosing chronic disease and relapse, presence of cross-reacting antibodies, and lack of timeliness constitute ...

These findings indicate wide dissemination of OXA-58 carbapenemase, which contributes, at least partially, to the imipenem resistance of unrelated A. baumannii isolates in our ICU.

In our hospital, where MBL-producing P. aeruginosa were previously prevalent, meropenem resistance due to the overexpression of efflux pumps has also now emerged. Early recognition of this resistance mechanism should allow the use of alternative beta-lactams, such as ceftazidime, which would be inactive even against phenotypically susceptible MBL producers.

No abstract