Artitaya Yatsomboon1,2, Rasana W Sermswan3,2, Surasakdi Wongratanacheewin1,2. 1. Department of Microbiology. 2. Melioidosis Research Center, Khon Kaen University, Khon Kaen 40002, Thailand. 3. Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Abstract
BACKGROUND: Septicemic melioidosis caused by Burkholderia pseudomallei is a serious cause of morbidity and mortality. An effective, rapid and simple diagnostic method is required for detection of B. pseudomallei infection. OBJECTIVE: To develop immunomagnetic beads (IMB) coupled with ELISA (IMB-ELISA) for detection of B. pseudomallei in blood samples of patients with suspected melioidosis. METHODS: For separation of B. pseudomallei from buffer, blood samples and hemoculture, 200 nm immunomagnetic beads (IMBs) coated with 4B11 monoclonal antibody (4B11-IMBs) against exopolysaccharide antigens were used. The detection was done by an ELISA based biotin-streptavidin system. The sensitivity and specificity were evaluated. RESULTS: 4B11-IMBs (100 μg) were successfully developed and used for detection of B. pseudomallei in 1 ml samples. Transmission electron microscopy (TEM) imaging demonstrated B. pseudomallei was captured by 4B11-IMBs. The IMBs showed high capture efficiency (98%) with B. pseudomallei in buffer. The IMB-ELISA assay was highly specific for B. pseudomallei. It showed no cross-reactions with other bacteria, except B. mallei. The limits of the B. pseudomallei assay detection for detecting B. pseudomallei in either buffer solution or blood was 102 CFU/ml. The IMB-ELISA detection sensitivity in blood samples was 44.5%. Although it did not give the highest sensitivity, it was useful for detection with hemoculture that was faster than conventional methods. CONCLUSION: This study suggests the IMB-ELISA assay offers a simple and highly specific method with a turnaround time of 6 h for detection of B. pseudomallei. The developed assay can be applied in hospitals for surveillance of B. pseudomallei.
BACKGROUND:Septicemic melioidosis caused by Burkholderia pseudomallei is a serious cause of morbidity and mortality. An effective, rapid and simple diagnostic method is required for detection of B. pseudomallei infection. OBJECTIVE: To develop immunomagnetic beads (IMB) coupled with ELISA (IMB-ELISA) for detection of B. pseudomallei in blood samples of patients with suspected melioidosis. METHODS: For separation of B. pseudomallei from buffer, blood samples and hemoculture, 200 nm immunomagnetic beads (IMBs) coated with 4B11 monoclonal antibody (4B11-IMBs) against exopolysaccharide antigens were used. The detection was done by an ELISA based biotin-streptavidin system. The sensitivity and specificity were evaluated. RESULTS: 4B11-IMBs (100 μg) were successfully developed and used for detection of B. pseudomallei in 1 ml samples. Transmission electron microscopy (TEM) imaging demonstrated B. pseudomallei was captured by 4B11-IMBs. The IMBs showed high capture efficiency (98%) with B. pseudomallei in buffer. The IMB-ELISA assay was highly specific for B. pseudomallei. It showed no cross-reactions with other bacteria, except B. mallei. The limits of the B. pseudomallei assay detection for detecting B. pseudomallei in either buffer solution or blood was 102 CFU/ml. The IMB-ELISA detection sensitivity in blood samples was 44.5%. Although it did not give the highest sensitivity, it was useful for detection with hemoculture that was faster than conventional methods. CONCLUSION: This study suggests the IMB-ELISA assay offers a simple and highly specific method with a turnaround time of 6 h for detection of B. pseudomallei. The developed assay can be applied in hospitals for surveillance of B. pseudomallei.