Development of rapid detectionfor burkholderia pseudomallei by loop-mediated isothermal amplification

Burkholderia pseudomallei, a gram-negative bacteria, is the causative agent of melioidosis, a tropical infectious disease, which is endemic to Southeast Asia and Northeast Australia. Because of its clinical manifestation as septicemic and acute melioidosis which can progress up to the severe infection, a rapid laboratory diagnosis is needed in order to give the effective treatment to the patients confirmed as having infected by B.pseudomallei in timely manner. Culture and PCR assays are commonly used to detect B.pseudomallei, but both tests require experienced microbiologists and are only available in a few research laboratories worldwide. Despite the higher sensitivity than culture, PCR assays also require experienced personnel and positive controls, and are relatively costly for resource-limited settings. The aim of this study was to develop a rapid detection for B.pseudomallei by loop-mediated isothermal amplification (LAMP) which could be an affordable alternative molecular assay which can be used as point-of-care diagnostic test applicable in poor resource health system therefore achieving an effective treatment of patients in case. The LAMP developed in this study was sensitive and specific for the laboratory detection of B.pseudomallei. The lower limit of detection was 10 pg/μl and LAMP developed in this study was positive for 73 clinical samples collected from patients diagnosed with cultured-confirmed melioidosis (analytical sensitivity 100%), but it was negative for others bacteria used in this study including Salmonella spp., Shigella spp., Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, B.thailandesis, and B.mallei. All 18 negative samples for melioidosis by bacterial culture were negative by LAMP (analytical specificity, 100%). The diagnostic accuracy of LAMP was 100%. Considering the specificity, sensitivity and diagnostic accuracy, the LAMP assay developed in this study can be used in the health care system as a point-of-care laboratory assay for rapid detection of B.pseudomallei.