Use of robotized DNA isolation and real-time PCR to quantify and identify close correlation between levels of Neisseria meningitidis DNA and lipopolysaccharides in plasma and cerebrospinal fluid from patients with systemic meningococcal disease

Abstract

The present study, using robotized DNA isolation and quantitative PCR based on the Neisseria meningitidis-specific capsular transport A gene, demonstrates the ease, rapidity, specificity, and sensitivity of quantifying neisserial DNA in plasma (n = 65) and cerebrospinal fluid (CSF) (n = 12) from patients with systemic meningococcal disease. We found a close correlation between the levels of neisserial DNA and lipopolysaccharides in plasma (r = 0.905) and in CSF (r = 0.964). The median concentration of neisserial DNA in plasma in 23 patients with persistent shock was 2 x 10(7) copies/ml, versus <10(3) copies/ml in 42 nonshock patients. Furthermore, quantitative PCR made possible estimates of the total number of meningococci in plasma, as opposed to conventional blood cultures, suggesting about 1,000 dead meningococci for every viable bacterium. Finally, with logistic regression analyses, neisserial DNA may predict a patient's disease severity and outcome at hospital admission. The number of meningococci in plasma and CSF appears to be the main determinant of the lipopolysaccharide levels, clinical presentation, and outcome.

FIG. 1.

Levels of neisserial DNA (a) and LPS (b) in plasma samples from patients with systemic meningococcal disease presenting with various clinical syndromes. The dotted lines indicate the detection limits, 10³ neisserial DNA copies/ml of plasma and 0.25 IU of LPS/ml of plasma. Horizontal bars denote median values.

FIG. 2.

Levels of neisserial DNA (a) and LPS (b) in CSF samples from patients with systemic meningococcal disease presenting with various clinical syndromes. The dotted lines indicate the detection limits, 10³ neisserial DNA copies/ml of CSF and 0.25 IU of LPS/ml of CSF.

FIG. 3.

Clearance of neisserial DNA (a) and LPS (b) in plasma samples collected after admission to the hospital from eight patients with fulminant meningococcal septicemia. The first sample (hour 0) was collected immediately before initiation of antibiotic therapy from seven of eight patients. The first sample from patient 7 was collected 1 h after the initiation of antibiotic therapy. Patients 2, 3, and 7 died. The half-life of neisserial DNA (a) was 3 to 4 h and that of LPS (b) was 1 to 3 h. The dotted lines indicate the detection limits, 10³ neisserial DNA copies/ml of plasma and 0.25 IU of LPS/ml of plasma.

FIG. 4.

Relationship between neisserial DNA and LPS in plasma samples from patients with systemic meningococcal disease at admission to hospital. The dotted lines separate samples as either positive or negative for neisserial DNA or LPS. ▪, fulminant septicemia, fatal cases (n = 9); ▪, fulminant septicemia, survivors (n = 12); ×, septicemia and meningitis (n = 2); ○, mild systemic meningococcal disease (n = 14); ▵, distinct meningitis (n = 28).