Loop-mediated isothermal amplification method targeting the TTS1 gene cluster for detection of Burkholderia pseudomallei and diagnosis of melioidosis

Melioidosis is a severe infection caused by <em>Burkholderia pseudomallei</em>. The timely implementation of effective antimicrobial treatment requires rapid diagnosis. Loop-mediated isothermal amplification (LAMP) targeting the TTS1 gene cluster was developed for the detection of <em>B. pseudomallei</em>. LAMP was sensitive and specific for the laboratory detection of this organism. The lower limit of detection was 38 genomic copies per reaction, and LAMP was positive for 10 clinical <em>B. pseudomallei</em> isolates but negative for 5 <em>B. thailandensis</em> and 5 <em>B. mallei</em> isolates. A clinical evaluation was conducted in northeast Thailand to compare LAMP to an established real-time PCR assay targeting the same TTS1 gene cluster. A total of 846 samples were obtained from 383 patients with suspected melioidosis, 77 of whom were subsequently diagnosed with culture-confirmed melioidosis. Of these 77 patients, a positive result was obtained from one or more specimens by PCR in 26 cases (sensitivity, 34%; 95% confidence interval [CI], 23.4 to 45.4%) and by LAMP in 34 cases (sensitivity, 44%; 95% CI, 32.8 to 55.9%) (<em>P</em> = 0.02). All samples from 306 patients that were culture negative for <em>B. pseudomallei</em> were negative by PCR (specificity, 100%; 95% CI, 98.8 to 100%), but 5 of 306 patients (1.6%) were positive by LAMP (specificity, 98.4%; 95% CI, 96.2 to 99.5%) (<em>P</em> = 0.03). The diagnostic accuracies of PCR and LAMP were 86.7% (95% CI, 82.9 to 89.9%) and 87.5% (95% CI, 83.7 to 90.6%), respectively (<em>P</em> = 0.47). Both assays were very insensitive when applied to blood samples; PCR and LAMP were positive for 0 and 1 of 44 positive blood cultures, respectively. The PCR and LAMP assays evaluated here are not sufficiently sensitive to replace culture in our clinical setting.