PCR-based identification of Burkholderia pseudomallei

Abstract

SUMMARYDNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99), but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.

The Aftermath of the Western Australian Melioidosis Outbreak

“…27 In time, a PCR assay was developed for B. pseudomallei identification based on a gene sequence associated with a bacterial fatty acid specific to B. pseudomallei . 28,29 This assay was more reliable than the earlier PCR assay used in WA during the first one-half of the decade. Another improvement was sequencing of a PCR-amplified RecA gene product.…”

Sri Lankan National Melioidosis Surveillance Program Uncovers a Nationwide Distribution of Invasive Melioidosis

“…Confirmed B. pseudomallei were then transferred to Perth, Western Australia for detailed characterization. Isolates were sent as suspensions of bacteria in sterile water and duplicated in nutrient agar-embedded bacteriology [13][14][15] Confirmed B. pseudomallei were also subjected to biogeographic attribution PCR assays for Yersinia-like fimbrial (YLF) and Burkholderia thailandensis-like flagellum and chemotaxis (BTFC) gene clusters. 16 Molecular epidemiology.…”

Pathological findings and diagnostic implications of a rhesus macaque (Macacca mulatta) model of aerosol exposure to Burkholderia mallei (glanders)

“…When specific assays are conducted in an effort to diagnose glanders, serology may be complicated by cross-reactivity between B. mallei and B. pseudomallei antisera. Although PCR assays for B. mallei have been available since the late 1990s, a search of the medical literature reveals no documentation of their use in a comparative study (Brook et al, 1997;Merritt et al, 2006). In the study reported here, PCR, culture and histopathology were compared for diagnostic applicability.…”