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

• Published in: PloS one Vol. 11; no. 2; p. e0147765
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.02.2016; Public Library of Science (PLoS)
• Subjects: Analysis; Bacteria; Bacterial meningitis; Bacteriology; Biology and Life Sciences; Cerebrospinal fluid; Contamination; Deoxyribonucleic acid; Diagnosis; Diagnostic systems; Disease control; Disease prevention; DNA; DNA polymerase; DNA, Bacterial; DNA, Bacterial - isolation & purification; Epidemiology; Extraction; Genetic aspects; Haemophilus influenzae; Health risks; Health surveillance; Human health and pathology; Humans; Infectious diseases; Laboratories; Lead; Life Sciences; Limit of Detection; Medicine and Health Sciences; Meningitis; Meningitis, Bacterial; Meningitis, Bacterial - cerebrospinal fluid; Meningitis, Bacterial - diagnosis; Methods; Microbiology and Parasitology; Neisseria meningitidis; Pathogens; Polymerase chain reaction; Public health; Real time; Real-Time Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction - methods; Research and Analysis Methods; Risk reduction; Santé publique et épidémiologie; Sensitivity; Sensitivity and Specificity; Streptococcus infections; Streptococcus pneumoniae; Vaccines; Brazil; Atlanta Georgia; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0147765

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.