Melioidosis is a life-threatening disease in humans caused by the Gram- negative bacterium Burkholderia pseudomallei. As severe septicemic melioidosis can lead to death within 24 to 48 hours, a rapid diagnosis of melioidosis is critical for ensuring an optimal antibiotic course is prescribed to patients. Here, we report the development and evaluation of a bacteriophage tail fiber-based latex agglutination assay for rapid detection of B. pseudomallei infection. Burkholderia phage E094 was isolated from rice paddy fields in northeast Thailand, and whole genome sequenced to identify its tail fiber (94TF). The 94TF complex was structurally characterized, which involved identification of a tail assembly protein that forms an essential component of the mature fiber. Recombinant 94TF was conjugated to latex beads and developed into an agglutination-based assay (94TF-LAA). 94TF-LAA was initially tested against a large library of Burkholderia and other bacterial strains before a field evaluation was performed during routine clinical testing. The sensitivity and specificity of the 94TF-LAA were assessed alongside standard biochemical analyses on 300 patient specimens collected from an endemic area of melioidosis over 11 months. The 94TF-LAA took less than 5 minutes to produce positive agglutination, demonstrating 98% (95% CI; 94.2%−99.59%) sensitivity and 83% (95% CI; 75.64%−88.35%) specificity when compared to biochemical-based detection. Overall, we show how a Burkholderia-specific phage tail fiber can be exploited for rapid detection of B. pseudomallei. The 94TF-LAA has the potential for further development as a supplementary diagnostic to assist in clinical identification of this life-threatening pathogen.
IMPORTANCE
Rapid diagnosis of melioidosis is essential for ensuring optimal antibiotic courses are prescribed to patients, and thus warrants the development of cost-effective and easy-to-use tests for implementation in under-resourced areas such as Northeast Thailand and other tropical regions. Phage tail fibers are an interesting alternative to antibodies for use in various diagnostic assays for different pathogenic bacteria. As exposed appendages of phages, tail fibers are physically robust, easy to manufacture, and critically many tail fibers (such as 94TF investigated here) can target a given bacterial species with remarkable specificity. Here, we demonstrate the effectiveness of a latex agglutination assay using a Burkholderia-specific tail fiber 94TF against biochemical-based detection methods that are the standard diagnostic in many endemic areas of meilodosis.
Evaluation of a Latex Agglutination Assay for the Identification of Burkholderia pseudomallei and Burkholderia mallei
