Development of Real-Time PCR Assays and Evaluation of Their Potential Use for Rapid Detection of Burkholderia pseudomallei in Clinical Blood Specimens

Abstract

The early initiation of appropriate antimicrobial therapy is critical for improving the prognosis of patients with septicemic melioidosis. Thus, the use of a rapid molecular diagnosis may affect the outcome of this disease, which has a high mortality rate. We report the development of two TaqMan real-time PCR assays (designated 8653 and 9438) that detect the presence of two novel genes unique to Burkolderia pseudomallei. The analytical sensitivity and specificity of the assays were assessed with 91 different B. pseudomallei isolates, along with 96 isolates and strains representing 28 other bacterial species, including the closely related Burkholderia/Ralstonia. The two assays performed equally well with both purified DNA and crude cell lysates, with 100% analytical specificity for the detection of B. pseudomallei. The limit of detection was 50 fg of DNA (equivalent to six bacterial genomes) per PCR for both assay 8563 and 9438. We also evaluated these assays with DNA extracted from blood specimens taken from 45 patients with culture-confirmed septicemic melioidosis or other septicemias. Of the 28 melioidosis blood specimens, assays 8653 and 9438 gave sensitivities of 71% (20/28) and 54% (15/28), respectively. Effectively, all fatal cases of septicemic melioidosis were detected by 8653. For the 17 non-melioidosis blood specimens, specificities of 82% (14/17) and 88% (15/17) were obtained for assays 8653 and 9438, respectively. The real-time PCR assays developed in this study provide alternative, rapid molecular tools for the specific detection of B. pseudomallei, and this may be of particular use in the early diagnosis and treatment of septicemic melioidosis.

PCR-based Methodologies Used to Detect and Differentiate theBurkholderia pseudomalleicomplex:B. pseudomallei,B. mallei, andB. thailandensis

“…A total of 10 false positive soil samples were observed by the two environmental follow-up studies (Kaestli et al, 2007;Trung et al, 2011). These false positives agreed with other qPCR tests (Kaestli et al, 2007;Supaprom et al, 2007), indicating Novak et al's qPCR sensitivity beyond culture for soil samples. Overall, the test approached an environmental accuracy of 100%.…”

“…The single false negative soil sample was detected by another method (Supaprom et al, 2007;Trung et al, 2011). Therefore, both Novak et al and Trung et al's procedures could be used on environmental samples (Trung et al, 2011).…”

“…One false negative decreased the assay's sensitivity on soil samples (Trung et al, 2011). This false negative was detected by a different qPCR method (Supaprom et al, 2007;Trung et al, 2011). A total of 10 false positive soil samples were observed by the two environmental follow-up studies (Kaestli et al, 2007;Trung et al, 2011).…”

“…Two novel qPCR methods targeting separate B. pseudomallei loci were developed, and both tests (9438 and 8653) had 100% accuracy (Supaprom et al, 2007). These procedures were then evaluated for their ability to detect B. pseudomallei in clinical samples from septicemic patients.…”

Forensic Microbiology

“…Real-time PCR assays have been developed as rapid identification tools, but several assays were evaluated with strain collections and spiked samples only (30; 40; 41; 43). In fatal septicaemia the amount of bacterial DNA that can be extracted from blood is high enough to be detectable using real-time PCR (38), but a study in Thailand has shown that this diagnostic tool may be of little clinical value when compared with conventional diagnostic approaches (5). MLST analyses have shown that strains from Thailand and Australia can be discriminated and that some sequence types found in environmental samples are underrepresented among clinical isolates thus indicating that they may be less pathogenic for humans (12; 48).…”