Abstract
The aim of this study was the evaluation of application two (2) molecular methods in diagnosis of acute brucellosis, polymerase chain reaction-enzyme immunoassay (PCR-EIA) and real time PCR (RT-PCR). RT-PCR was also used for monitoring bacterial DNA load before, during and after treatment in order to find a possible correlation with the outcome and course of the disease. Whole blood and serum specimens from 243 patients with acute brucellosis were examined by PCR-EIA. Following the amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenic 31 kDa membrane protein specific for the Brucella genus (BCSP31), the amplified product was detected in a microtiter plate by hybridization with specific biotinylated probe. For the evaluation of a quantitative RT PCR assay, whole blood and serum specimen from 126 patients with acute brucellosis were examined. The evolution of bacterial DNA load was then monitored in 78 from these patients with at least 3 whole blood ...
The aim of this study was the evaluation of application two (2) molecular methods in diagnosis of acute brucellosis, polymerase chain reaction-enzyme immunoassay (PCR-EIA) and real time PCR (RT-PCR). RT-PCR was also used for monitoring bacterial DNA load before, during and after treatment in order to find a possible correlation with the outcome and course of the disease. Whole blood and serum specimens from 243 patients with acute brucellosis were examined by PCR-EIA. Following the amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenic 31 kDa membrane protein specific for the Brucella genus (BCSP31), the amplified product was detected in a microtiter plate by hybridization with specific biotinylated probe. For the evaluation of a quantitative RT PCR assay, whole blood and serum specimen from 126 patients with acute brucellosis were examined. The evolution of bacterial DNA load was then monitored in 78 from these patients with at least 3 whole blood specimens. The RT PCR assay was based on direct amplification of a 207-base pair DNA sequence of the same membranic protein (BCSP31). The amplification product was detected by using the hybridization probes fluorescence technique. The amplification reaction and the detection of amplified product were performed at the same glass capillary of thermal cycler (LightCycler). After amplification, melting curve analysis was performed to verify the specificity of PCR products. Two hundred forty-one of the 243 patients, who were studied with PCR-EIA, had detectable Brucella DNA in either whole blood or serum specimens: 149 (61.3%) patients were positive in both blood and serum specimens, 43 (17.7%) were positive in serum specimens only, and 49 (20.2) were positive in whole blood specimens only. The diagnostic specificity of the PCR-EIA assay for both specimen categories was 100%, while the sensitivity was 81.5% for whole blood specimens, 79% for serum specimens, and 99.2% for whole blood and serum specimens combined. One hundred twenty-six of the 126 patients, who were studied with PCR-EIA, had detectable Brucella DNA in either whole blood or serum specimens: 108 (85.7%) patients were positive in both blood and serum specimens, 6(4.8%) were positive in serum specimens only, and 9 (7.1%) were positive in whole blood specimens only. The diagnostic specificity of the RT PCR assay for both specimen categories was 100%, while the sensitivity was 92.8% for whole blood specimens, 90.5% for serum specimens, and 97.6% for whole blood and serum specimens combined. The majority of 78 patients (87% at the end of treatment, 77% at 6 months after treatment completion, and 70% at >2 years after treatment) exhibited persistent detectable microbiological load despite being asymptomatic. The 6 patients who experienced relapse did not exhibit any statistically significant difference in their bacterial load at any stage of disease or during follow-up. In conclusion, the application of two assays for the detection of Brucella DNA showed that they are sensitive and specific methods which can assist the acurate rapid diagnosis of acute brucellosis efficiently. Moreover, RT PCR assay can be used for monitoring bacterial DNA load and, subsequently, the course of the disease in order to improve the clinical management of patients with brucellosis.
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