Comparison of loop-mediated isothermal amplification and conventional methods for rapid diagnosis of Brucella species

Brucellosis poses a significant threat to people who are in direct contact with livestock. Traditional diagnostic methods rely on isolation and cultivation, which can be time-consuming. The development of LAMP (loop-mediated isothermal amplification) and other molecular techniques offers the potential for faster diagnosis with high sensitivity and specificity compared to conventional culture-based methods. The aim of this study was to evaluate the utility of LAMP for diagnosing Brucella spp. in patients’ blood samples. A total of 60 patients presenting with clinical signs suggestive of brucellosis and with positive initial serological test results were enrolled in the study. Ten milliliters of blood were collected from each patient under sterile conditions: 5 mL was designated for blood culture, and 5 mL was collected in EDTA for molecular analyses. Culture samples were inoculated into BACTEC aerobic bottles and sub cultured onto Brucella Agar (Merck, Germany), upon instrument alert, with incubation at 37°C for up to one weeks.LAMP assay was performed on part of the sample using Brucella-specific primers to enable rapid detection at a constant temperature, with results interpreted within 60 minutes. Additionally, conventional PCR targeting Brucella-specific genes was conducted on DNA extracted from peripheral blood samples. A commercial kit (Bioneer Co., South Korea) was used to extract the DNA and confirm the presence of Brucella DNA. Amplification products were analyzed by gel electrophoresis or real-time detection. The results from each patient's culture, LAMP, and PCR were recorded and compared to assess diagnostic concordance, sensitivity, and specificity using SPSS version 17. Of the 60 blood samples examined using the culture method, 9 (15%) were positive and 51 (85%) were negative. The PCR and LAMP assays showed 38 (63.33%) and 47 (78.33%) of the samples were positive for Brucella spp., respectively. Assuming the culture and PCR methods are the gold standard and using Stata/MP v17.0 software with a 95% confidence interval, the predicted clinical sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the LAMP method compared to the PCR method are 86%., 36.36% (95% CI: 14.49%-58.23%), 70.21% (95% CI: 51.67%-88.75%), 61.54% (95% CI: 33.92%-89.16%), and 68.33% (95% CI: 56.49%-80.17%), respectively. The LAMP assay is a highly specific and sensitive method for detecting Brucella strains in clinical samples. Due to the importance of an early diagnosis and appropriate treatment of brucellosis, the LAMP technique offers a faster, more sensitive, and more cost-effective way to identify Brucella species in blood samples. Brucellosis is a well-known animal disease that is found all over the world. The LAMP assay help with faster diagnosis and save time while providing highly remarkable and precise results. LAMP assay is specific and sensitive for the detection of Brucella strains in clinical samples. LAMP requires only a temperature-controlled water- or dry-bath, and the results can be analyzed directly by the naked eye.