Monoclonal antibody-based immunofluorescence microscopy for the rapid identification of Burkholderia pseudomallei in clinical specimens

Abstract

The diagnosis of melioidosis depends on the culture of Burkholderia pseudomallei, which takes at least 48 hours. We used a polyclonal-FITC-based immunofluorescence microscopic assay (Pab-IFA) on clinical samples to provide a rapid presumptive diagnosis. This has limitations including photobleaching and batch-to-batch variability. This study evaluated an IFA based on a monoclonal antibody specific to B. pseudomallei (Mab-IFA) and Alexa Fluor 488. A diagnostic evaluation was performed on a prospective cohort of 951 consecutive patients with suspected melioidosis. A total of 1,407 samples were tested. Test accuracy was defined against culture as the gold standard, and was also compared against Pab-IFA. A total of 88 samples from 64 patients were culture positive for B. pseudomallei. The diagnostic sensitivity and specificity of the Mab-IFA was comparable to the Pab-IFA (48.4% versus 45.3% for sensitivity, and 99.8% versus 98.8% for specificity). We have incorporated the Mab-IFA into our routine practice.

Figure 1.

Fluorescent microscopy of Burkholderia pseudomallei stained with Mab-IFA reagent. The bacteria shown were from laboratory cultures on Columbia agar (A) or LB broth (B), or from clinical samples (urine [C], pus [D], or sputum [E]) from patients with melioidosis. The atypical appearance of the bacterial morphology including bacterial elongation (D and E) and swollen cells (C) was not uncommon.