Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction

Details

• Alternative Title:
  PLoS One
• Personal Author:
  Vuong, Jeni ; Collard, Jean-Marc ; Whaley, Melissa J. ; Bassira, Issaka ; Seidou, Issaka ; Diarra, Seydou ; Ouédraogo, Rasmata T. ; Kambiré, Dinanibè ; Taylor, Thomas H. ; Sacchi, Claudio ; Mayer, Leonard W. ; Wang, Xin
• Description:
  Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR) requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA) and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y) targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824-135,982 for 5x Omni, and 168-6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0-99.9%, 97.5-99.9%, and 92.9-99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume.
• Subjects:
  DNA, Bacterial Humans Limit Of Detection Meningitis, Bacterial Real-Time Polymerase Chain Reaction Sensitivity And Specificity
• Source:
  PLoS One. 11(2).
• Pubmed ID:
  26829233
• Pubmed Central ID:
  PMC4735509
• Document Type:
  Journal Article
• Volume:
  11
• Issue:
  2
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:8fd175c37c7703754764d60eaf2c4fc66081ba8b806bd481b39610b36b9ffc0b
• Download URL:
  https://stacks.cdc.gov/view/cdc/38137/cdc_38137_DS1.pdf
• File Type:
  [PDF - 193.48 KB ]