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Mikrobiyol Bul. 2013 Jul;47(3):514-22.

[Development of a novel Francisella tularensis antigen stained with tetrazolium-blue for tularemia microagglutination test].

[Article in Turkish]

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  • 1Public Health Institution of Turkey, National Tularemia Reference Laboratory, Ankara, Turkey. mdskilic2003@yahoo.com.


The isolation of Francisella tularensis in cultures is the reference method for the laboratory diagnosis of tularemia. However, due to the limitations such as the low sensitivity and need for high safety level and equipped laboratories, serologic methods are frequently used as diagnostic tools. F.tularensis-specific antibodies may be demonstrated by several methods, however microagglutination test (MA) remains the most common method with its high sensitivity and specificity. The aim of this study was to develop a novel MA test antigen prepared from whole F.tularensis cells and stained with tetrazolium-blue for more clear and easier evaluation. F.tularensis NCTC 10857 strain was cultured on the cysteine heart agar supplemented with 9% sheep blood and bacterial cells were harvested by scraping, collected in physiological saline (PS) and centrifuged at 1500 rpm for 10 minutes. For preparing stock antigen suspension cell concentration was adjusted to OD600=1.5, spectrophotometrically. Tetrazolium-blue solution (BTC [3,3'-(3,3'-Dimethoxy[1,1'-biphenyl]-4,4'-diyl) bis [2,5-diphenyl-2H-tetrazolium dichloride], T4375 Sigma-Aldrich) at the final concentration of 1% was added to cell suspension and incubated at 37°C for 5 hours for absorption. Then, the living cells were chemically inactivated by formaldehyde. Repeating centrifugations were performed to discard excess dye and formaline, then 0.4% formaline saline was added on the sediment. Optimum concentration of this novel antigen (BTC-Ag) for MA test was determined by plate titration method by using standard serum sample with a known MA titer (1/128). The performance of BTC-Ag in MA test was evaluated by using 100 patient sera positive for F.tularensis antibodies, and 100 tularemia negative patient sera (of them 50 were seropositive for brucellosis). All of the results were compared with standard MA test in which safranin-O stained antigen (SO-Ag) was used. There was 100% agreement between the two tests performed with BTC-Ag and SO-Ag in tularemia seropositive (in ≥ 1/20 titers and when ±1 dilution variation was accepted as normal) and seronegative sera. No significant cross reactivity with Brucella spp. was observed. Accuracy, sensitivity and specificity of BTC-Ag were found to be 100%. In conclusion, newly developed BTC-Ag for MA test provides better agglutination patterns resulting in a clear supernatant in wells, thus provides easy evaluation for the agglutination reaction, and is expected to facilitate tularemia serodiagnosis.

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