Priyapa Najomtien,1 Wisitsak Phoksawat,1 Sukanya Khammanthoon,2 Wadcharin Klasuk,3 Nuttiya Srisurat,3 Supaksorn Chattagul,5 Chotima Photisap,5 Chonlatip Pipattanaboon,1 Rasana W. Sermswan,4,5 Surasakdi Wongratanacheewin1,5

Affiliations:
1 Department of Microbiology, Faculty of Medicine, Khon Kaen University, Thailand
2 Srinagarind Hospital, Khon Kaen, Thailand
3 Khon Kaen Hospital, Khon Kaen, Thailand
4 Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Thailand
5 Melioidosis Research Center, Khon Kaen University, Thailand

Abstract

Background: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. Septicemic melioidosis patients have a high mortality rate within 48 hours.
Objectives: To develop a polymerase chain reaction (PCR) combined with a lateral flow dipstick (LFD) assay for detection of B. pseudomallei in blood samples.
Methods: The PCR with wcbG gene primers and a PCR-LFD test were developed. The specificity and sensitivity were determined using the B. pseudomallei and other bacterial DNAs. They were evaluated using 43 B. pseudomallei positive blood samples and another 43 blood samples positive for other microbial infections.
Results: The detection limit of the PCR-LFD test was 50 fg of bacterial gDNA or 1.0 CFU per 200 µl of blood. All B. pseudomallei were positive while B. thailandensis and selected gram-negative bacterial strains were negative. The PCR-LFD gave all positives with all 43 B. pseudomallei culture positive patient blood samples and all negative with 43 blood samples that were culture positive for K. pneumoniae, E. gallinarum, E. faecium, E. coli, S. aureus, A. baumannii, A. hydrophila, S. haemolyticus, S. pneumoniae, P. aeruginosa, E. cloacae, S. hominis, E. aerogenes, P. mirabilis, C. neoformans, C. albicans, A. caviae, E. faecalis and K. variicola.
Conclusions: The developed PCR-LFD assay provided 100% sensitivity and 100% specificity compared to the conventional blood culture. The technique took only 1.5 hours that is easy and quick to perform compared to the 3-7 days of culture method. The new method of PCR with LFD could facilitate the detection to be a semi-point-of-care testing (POCT).
Key words: Burkholderia pseudomallei, polymerase chain reaction, lateral flow dipstick assay, melioidosis, blood samples