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Background: Brucellosis is a zoonosis that occurs worldwide. The disease has been completely eradicated in livestock in Sweden in 1994, and all cases of confirmed human brucellosis are imported into Sweden from other countries. However, due to an increase in the number of refugees and asylum seekers from the middle-east to Sweden, there is a need to improve the current diagnostic methodology for Brucella melitensis. Whilst culture of Brucella species can be used as a diagnostic tool, real-time PCR approaches provide a much faster result. The aim of this study was to set up a species-specific real-time PCR for the detection of all biovars of Brucella melitensis, which could be used routinely in diagnostic laboratories. Methods: A Brucella melitensis real-time PCR assay was designed using all available genomes in the public database of Brucella (N=96) including all complete genomes of Brucella melitensis (N=17). The assay was validated with a collection of 37 Brucella species reference strains, 120 Brucella melitensis human clinical isolates, and 45 clinically relevant non-Brucella melitensis strains. Results: In this study we developed a single real-time PCR for the specific detection of all biovars of Brucella melitensis. Conclusions: This new real-time PCR method shows a high specificity (100%) and a high sensitivity ( 1.25 GE/mu l) and has been implemented in the laboratories of four governmental authorities across Sweden.

In the case of a release of highly pathogenic bacteria (HPB), there is an urgent need for rapid, accurate, and reliable diagnostics. MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive technique that is becoming increasingly important in microbiological diagnostics to complement classical microbiology, PCR, and genotyping of HPB. In the present study, the results of a joint exercise with 11 partner institutions from nine European countries are presented. In this exercise, 10 distinct microbial samples, among them five HPB, Bacillus anthracis, Brucella canis, Burkholderia mallei, Burkholderia pseudomallei, and Yersinia pestis, were characterized under blinded conditions. Microbial strains were inactivated by high-dose gamma irradiation before shipment. Preparatory investigations ensured that this type of inactivation induced only subtle spectral changes with negligible influence on the quality of the diagnosis. Furthermore, pilot tests on nonpathogenic strains were systematically conducted to ensure the suitability of sample preparation and to optimize and standardize the workflow for microbial identification. The analysis of the microbial mass spectra was carried out by the individual laboratories on the basis of spectral libraries available on site. All mass spectra were also tested against an in-house HPB library at the Robert Koch Institute (RKI). The averaged identification accuracy was 77% in the first case and improved to >93% when the spectral diagnoses were obtained on the basis of the RKI library. The compilation of complete and comprehensive databases with spectra from a broad strain collection is therefore considered of paramount importance for accurate microbial identification.

An outbreak of canine brucellosis in Sweden was confirmed by the National Veterinary Institute (SVA) in August 2013. The whole genome of the causative agent was sequenced, assembled, and analyzed.

The potential use of bacteria and viruses as biological terror weapons makes certain highly pathogenic microorganisms a worldwide public health threat. In an outbreak investigation involving a possible deliberate spread of a biological warfare agent there is a need for a fast and reliable diagnostic method. Ideally, the same method should be usable for both bacteria and viruses. To distinguish between natural and deliberate spread, information on the prevalence/incidence of the organism/disease, ecology and natural mechanisms for spread is needed. In addition, knowledge of national and international subtypes reflecting both micro- and macro-evolution of the organisms is required for the analysis. The field of molecular epidemiology has evolved during the last decades with the introduction of several different methods to type and characterize bacteria. There is now a plethora of different molecular techniques available to identify and discriminate between different strains of bacteria. In this thesis, rapid protocols based on pyrosequencing technology were developed and used for discrimination of Bacillus anthracis from closely related bacillus species. The technique was also used for identification and subtyping of Francisella species, including the human pathogen Francisella tularensis and its subspecies. Also a rapid generic protocol for detection and genotyping of infectious agents including bacteria, parasites, and viruses was developed. Tularemia epidemics occur in limited geographical areas, and at variable intervals. The reasons for these irregularities are still unknown. There may be ecological niches in the affected areas, which harbor the bacteria between epidemics, or the bacterium may be reintroduced into an ecological system that permits its amplification. The establishment of a relevant typing system identifying individual strains is of great importance .One of the most promising methods, MLVA was evaluated as a tool for practical use in epidemiologica

In this study we describe a novel protocol for rapid molecular analysis of patient samples using a combination of real-time polymerase chain reaction (PCR) and Sanger sequencing. This would normally take 2 working days in the diagnostic laboratory, but using this protocol the process can be completed within 3 h using equipment normally found in the laboratory. The innovative steps in this protocol are the sequencing of the product generated in the diagnostic real-time PCR, addition of a sequencing tail to the PCR primer, which increases the quality of the sequence without loss of sensitivity or specificity, and optimization of the hands-on and instrument steps using modern reagents. The read length of the sequencing step is routinely 250 nucleotides, which is substantially longer than existing rapid sequencing methods, increasing the chances of covering several genetic markers within 1 analysis. As proof of the concept, we used the detection and genotyping of the intestinal parasite Giardia lamblia, but the protocol can be applied to any PCR and sequence-based analysis.