Optimization and prospective evaluation of sensitive real-time PCR assays with an internal control for the diagnosis of melioidosis in Thailand
1
Issued Date
2023-12-01
Resource Type
eISSN
21650497
Scopus ID
2-s2.0-85180011869
Pubmed ID
37819125
Journal Title
Microbiology Spectrum
Volume
11
Issue
6
Rights Holder(s)
SCOPUS
Bibliographic Citation
Microbiology Spectrum Vol.11 No.6 (2023)
Suggested Citation
Noparatvarakorn C., Jakkul W., Seng R., Tandhavanant S., Ottiwet O., Janon R., Saikong W., Chantratita N. Optimization and prospective evaluation of sensitive real-time PCR assays with an internal control for the diagnosis of melioidosis in Thailand. Microbiology Spectrum Vol.11 No.6 (2023). doi:10.1128/spectrum.01039-23 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/95984
Title
Optimization and prospective evaluation of sensitive real-time PCR assays with an internal control for the diagnosis of melioidosis in Thailand
Corresponding Author(s)
Other Contributor(s)
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.