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Effective treatment of melioidosis requires rapid detection of\n                    <jats:italic>B. pseudomallei<\/jats:italic>\n                    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\n                    <jats:italic>B. pseudomallei<\/jats:italic>\n                    in plasma samples and compared the performance of real-time PCRs based on six target genes. The lower limit of detection (LOD) of TTS1-\n                    <jats:italic>orf2<\/jats:italic>\n                    , BPSS0745, BPSS1187, and BPSS1498 real-time PCRs was \u226410 genome equivalent (GE)\/reaction, while the LOD of BPSS0087 and BPSS1492 was 100 GE\/reaction. In the initial phase, we evaluated four targets (TTS1-\n                    <jats:italic>orf2<\/jats:italic>\n                    , 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-\n                    <jats:italic>orf2<\/jats:italic>\n                    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-\n                    <jats:italic>orf2<\/jats:italic>\n                    were 89.6% and 79.2%, respectively (\n                    <jats:italic>P<\/jats:italic>\n                    = 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 (\n                    <jats:italic>P<\/jats:italic>\n                    = 0.007). Therefore, the BPSS1187 real-time PCR was sensitive and specific for early diagnosis of melioidosis.\n                  <\/jats:p>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Melioidosis is a serious infectious disease caused by\n                      <jats:italic>Burkholderia pseudomallei<\/jats:italic>\n                      , an environmental Gram-negative bacterium. Early detection of\n                      <jats:italic>B. pseudomallei<\/jats:italic>\n                      infection is crucial for successful antibiotic treatment and reducing mortality rates associated with melioidosis. Bacteria culture is currently used to identify\n                      <jats:italic>B. pseudomallei<\/jats:italic>\n                      in clinical samples, but the method is slow. Therefore, there is a need for more accurate and sensitive molecular-based diagnostic methods that can detect\n                      <jats:italic>B. pseudomallei<\/jats:italic>\n                      in all sample types, including samples from blood. We developed an optimal DNA extraction method for\n                      <jats:italic>B. pseudomallei<\/jats:italic>\n                      from plasma samples and used an internal control for real-time PCR. We evaluated six PCR target genes and identified the most effective target for the early detection of\n                      <jats:italic>B. pseudomallei<\/jats:italic>\n                      infection in patients. To prevent delays in the treatment of melioidosis that can lead to fatal outcomes, we recommend implementing this new approach for routine early detection of\n                      <jats:italic>B. pseudomallei<\/jats:italic>\n                      in clinical settings.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.01039-23","type":"journal-article","created":{"date-parts":[[2023,10,11]],"date-time":"2023-10-11T09:12:51Z","timestamp":1697015571000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":11,"title":["Optimization and prospective evaluation of sensitive real-time PCR assays with an internal control for the diagnosis of melioidosis in Thailand"],"prefix":"10.1128","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6888-3431","authenticated-orcid":false,"given":"Chawitar","family":"Noparatvarakorn","sequence":"first","affiliation":[{"name":"Department of 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