Detection of Burkholria pseudomallei DNA in whole blood samples of bacterial septicemia patients using conventional PCR and real-time PCR

Abstract

Burkholderia pseudomallei is a bacterium that causes a disease known as melioidosis. Infections of B. pseudomallei appear in several organs including acute septicemia which showed high mortality rate. The rapid and high efficient diagnostic test may reduce the mortality rate of melioidosis patients. We performed conventional PCR using LPS primers and real-time PCR using 16S rDNA primers for detection of B. pseudomallei in clinical blood specimens. Bacterial culture was used as a gold standard. Blood specimens from 32 suspected bacterial septicemia patients were obtained from admitted patients in Khon Kaen Hospital and other hospitals in the region. These consist of the patients with 19 B. pseudomallei and 13 other bacterial infections including 1 Burkholderia cepacia, 1 Escherichia coli, 4 Klebsiella pneumoniae, 1 Enterobacter species, 3 Staphylococcus aureus, 1 Streptococcus pneumoniae and 2 group A Streptococcus. The sensitivity, specificity, positive and negative predictive values were determined. Conventional PCR showed low sensitivity of 37 % (95% CI:15-59) and high specificity of 92 % (95% CI:78-100) with 88 % (95% CI: 65-100) and 50 % (95% CI:30-70) of positive predictive value (PPV) and negative predictive value (NPV), respectively. In contrast, a real-time PCR and using of combination test showed higher sensitivity (63 %, 95% CI: 41-85) and lower specificity (69 %, 95% CI: 44-94) with 75 % (95% CI:54-96)  and 56 % (95% CI:32-81) of PPV and NPV, respectively. In 5 melioidosis patients who have died, both PCR methods showed rising of sensitivity [80 % (95 % CI: 45-100) and 100 % (95% CI: 100-100)], respectively. The lower detection limit of B. pseudomallei by conventional PCR was 10³ cfu/ml. The conventional PCR and real-time PCR could detect B. pseudomallei DNA of 10 pg and 50 fg per PCR reaction, respectively. The higher sensitivity of real-time PCR may be useful for early screening test, whereas the conventional PCR with higher specificity and PPV may be used as a confirmatory test for diagnosis of B. pseudomallei. Therefore, the two assays may be used in combination as rapid molecular diagnostic for melioidosis which might lead to a reduction in the mortality rate of melioidosis patients.