Optimization and prospective evaluation of sensitive real-time PCR assays with an internal control for the diagnosis of melioidosis in Thailand

Abstract

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. Effective treatment of melioidosis requires rapid detection of B. pseudomallei infection. Previous studies demonstrated that real-time PCR for the diagnosis of melioidosis in blood samples was insensitive. We optimized the DNA extraction method and evaluated real-time PCRs with an internal control for the detection of B. pseudomallei in plasma samples and compared the performance of real-time PCRs based on six target genes. The lower limit of detection (LOD) of TTS1-orf2, BPSS0745, BPSS1187, and BPSS1498 real-time PCRs was ≤10 genome equivalent (GE)/reaction, while the LOD of BPSS0087 and BPSS1492 was 100 GE/reaction. In the initial phase, we evaluated four targets (TTS1-orf2, BPSS0745, BPSS1187, BPSS1498) for the diagnosis of melioidosis using 87 plasma samples from 38 bacteremic-melioidosis patients and 49 other infection patients at a hospital in Northeast Thailand. Among these, BPSS1187 exhibited the highest sensitivity of 76.3% and specificity of 100%. A second evaluation phase was conducted at two hospitals in Northeast Thailand to compare two real-time PCRs between BPSS1187 and widely used TTS1-orf2 targets. A total of 705 clinical samples were collected from 421 suspected melioidosis cases; 77 of them were subsequently confirmed as melioidosis by culture using one or more specimens. The sensitivities of BPSS1187 and TTS1-orf2 were 89.6% and 79.2%, respectively (P = 0.004), and the specificities were 96% and 99%, respectively. The sensitivities of these two assays in plasma samples were 84.6% and 67.7%, respectively (P = 0.007). Therefore, the BPSS1187 real-time PCR was sensitive and specific for early diagnosis of melioidosis.