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      The medical diagnostic approaches with phylogenetic analysis for rare Brucella spp. diagnosis in Taiwan

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          Abstract

          Brucellosis is a bacterial zoonotic disease which can be easy to misdiagnose in clinical microbiology laboratories. In the present study, we have tried to improve the current clinical method for detecting Brucella spp. and its antibiotic characteristics. Our method begins with detecting the clinical isolate through traditional biochemical methods and automatic identification systems. Then, we move on to editing the sequence for BLAST allows us to compare 16s rRNA sequences with sequences from other species, allowing the gene level to be determined. Next, the phylogenetic analysis of multiple genetic loci is able to determine the evolutionary relationships between our bacteria strain and those from other locations. Finally, an anti-microbial susceptibility test hones in on the level of antibacterial activity that the bacteria displays. Employing these four steps in concert is extremely effective in identifying rare bacteria. Thus, when attempting to determine the identity of rare bacteria such as Brucella, utilizing these four steps from our research should be highly effective and ultimately prevent further identification errors and misdiagnoses. The standards we have suggested to identify rare bacteria strains is applicable not only to Brucella, but also to other rarely encountered bacteria.

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          16S ribosomal DNA amplification for phylogenetic study.

          A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.
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            Clinical manifestations and complications in 1028 cases of brucellosis: a retrospective evaluation and review of the literature.

            Brucellosis is the most prevalent bacterial zoonosis worldwide. In this study, we aimed to compare our 1028 brucellosis cases with other big series in the literature in view of epidemiological, clinical, and laboratory findings and therapeutic features. A total of 1028 brucellosis cases admitted to the Department of Infectious Diseases and Clinical Microbiology over a 10-year period were included in the study. A retrospective analysis was undertaken and patient files were reviewed for history, clinical and laboratory findings, and therapeutic features, as well as complications. Of the 1028 patients, 539 (52.4%) were female and 489 (47.6%) were male. The mean age of patients was 33.7+/-16.34 years and 69.6% of cases were aged 13-44 years. Four hundred and thirty-five cases (42.3%) had a history of raising livestock and 55.2% of the cases were found to have no occupational risk for brucellosis. Six hundred and fifty-four of the cases (63.6%) had a history of raw milk and dairy products consumption. The most frequently seen symptoms were arthralgia (73.7%) and fever (72.2%), while the most common clinical findings were fever (28.8%) and hepatomegaly (20.6%). The most frequent laboratory finding was a high C-reactive protein level (58.4%). The standard tube agglutination (STA) test+Coombs STA test was positive in 1016 cases (98.8%). Focal involvement was present in 371 (36.1%) cases. The most frequent involvement was osteoarticular involvement with 260 cases (25.3%). The overall relapse rate for patients with brucellosis was 4.7%. The highest relapse rate, 8.5%, was observed in the group of patients with osteoarticular involvement. Regimens including doxycycline and streptomycin with or without rifampin appeared more effective than other regimens in osteoarticular involvement. In humans, brucellosis may lead to serious morbidity, and it continues to be a major health problem in Turkey. There is no recommended treatment protocol for complicated brucellosis. Large multicenter studies are needed to determine the most appropriate treatment choices and durations in complicated brucellosis. Copyright 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
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              Characterisation of the genetic diversity of Brucella by multilocus sequencing

              Background Brucella species include economically important zoonotic pathogens that can infect a wide range of animals. There are currently six classically recognised species of Brucella although, as yet unnamed, isolates from various marine mammal species have been reported. In order to investigate genetic relationships within the group and identify potential diagnostic markers we have sequenced multiple genetic loci from a large sample of Brucella isolates representing the known diversity of the genus. Results Nine discrete genomic loci corresponding to 4,396 bp of sequence were examined from 160 Brucella isolates. By assigning each distinct allele at a locus an arbitrary numerical designation the population was found to represent 27 distinct sequence types (STs). Diversity at each locus ranged from 1.03–2.45% while overall genetic diversity equated to 1.5%. Most loci examined represent housekeeping gene loci and, in all but one case, the ratio of non-synonymous to synonymous change was substantially <1. Analysis of linkage equilibrium between loci indicated a strongly clonal overall population structure. Concatenated sequence data were used to construct an unrooted neighbour-joining tree representing the relationships between STs. This shows that four previously characterized classical Brucella species, B. abortus, B. melitensis, B. ovis and B. neotomae correspond to well-separated clusters. With the exception of biovar 5, B. suis isolates cluster together, although they form a more diverse group than other classical species with a number of distinct STs corresponding to the remaining four biovars. B. canis isolates are located on the same branch very closely related to, but distinguishable from, B. suis biovar 3 and 4 isolates. Marine mammal isolates represent a distinct, though rather weakly supported, cluster within which individual STs display one of three clear host preferences. Conclusion The sequence database provides a powerful dataset for addressing ongoing controversies in Brucella taxonomy and a tool for unambiguously placing atypical, phenotypically discordant or newly emerging Brucella isolates. Furthermore, by using the phylogenetic backbone described here, robust and rationally selected markers for use in diagnostic assay development can be identified.
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                Author and article information

                Contributors
                yclan@mail.cmu.edu.tw
                Journal
                Biomedicine (Taipei)
                Biomedicine (Taipei)
                BioMedicine
                China Medical University (Taichung )
                2211-8020
                2211-8039
                6 June 2015
                6 June 2015
                June 2015
                : 5
                : 2
                : 9
                Affiliations
                [ ]Department of Laboratory Medicine, China Medical University Hospital, 404, Taichung, Taiwan
                [ ]Department of Medical Laboratory Science and Biotechnology, China Medical University, 404 Taichung, Taiwan
                [ ]Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, 404 Taichung, Taiwan
                [ ]Department of Microbiology, UCLA, CA 90095 Los Angeles, USA
                [ ]Department of Pharmacy, College of Pharmacy, China Medical University, 404 Taichung, Taiwan
                [ ]Department of Laboratory Medicine, China Medical University Beigang Hospital, 651 Taichung, Taiwan
                [ ]Department of Internal Medicine, School of Medicine, China Medical University, 404 Taichung, Taiwan
                [ ]Graduate Institute of Microbiology and Public Health, National Chung Hsing University, 402 Taichung, Taiwan
                [ ]Department of Health Risk Management, China Medical University, No. 91, Hsueh-Shih Road, 404 Taichung, Taiwan
                Article
                9
                10.7603/s40681-015-0009-6
                4502044
                26048695
                9c69a227-8b27-40dd-b039-04814e038a61
                © China Medical University 2015
                History
                : 1 April 2015
                : 22 April 2015
                Categories
                Original Article
                Custom metadata
                © The Author(s) 2015

                brucella,traditional biochemical methods,6s rrna,phylogenetic analysis,anti-microbial,susceptibility test

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