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

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

Lower limits of detection for the real-time PCR tests using traditional and direct methods.^a

<p>Lower limits of detection for the real-time PCR tests using traditional and direct methods.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147765#t002fn001" target="_blank">^a</a></p

Diagnostic sensitivity and specificity for the real-time PCR tests by latent class analysis.

<p>Diagnostic sensitivity and specificity for the real-time PCR tests by latent class analysis.</p

Cerebrospinal fluid specimens that were positive by direct real-time PCR using PerfeCTa and negative by traditional real-time PCR using TaqMan or direct real-time PCR using 5x Omni.

<p>Cerebrospinal fluid specimens that were positive by direct real-time PCR using PerfeCTa and negative by traditional real-time PCR using TaqMan or direct real-time PCR using 5x Omni.</p

Final concentrations of primers and probes for the real-time PCR tests for detection of <i>Neisseria meningitidis</i> and its 6 serogroups, <i>Haemophilus influenzae</i>, and <i>Streptococcus pneumoniae</i> using traditional and direct methods^a.

<p>Final concentrations of primers and probes for the real-time PCR tests for detection of <i>Neisseria meningitidis</i> and its 6 serogroups, <i>Haemophilus influenzae</i>, and <i>Streptococcus pneumoniae</i> using traditional and direct methods<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147765#t001fn001" target="_blank">^a</a>.</p

Emergence of epidemic Neisseria meningitidis serogroup C in Niger, 2015: an analysis of national surveillance data.

International audienceTo combat Neisseria meningitidis serogroup A epidemics in the meningitis belt of sub-Saharan Africa, a meningococcal serogroup A conjugate vaccine (MACV) has been progressively rolled out since 2010. We report the first meningitis epidemic in Niger since the nationwide introduction of MACV. We compiled and analysed nationwide case-based meningitis surveillance data in Niger. Cases were confirmed by culture or direct real-time PCR, or both, of cerebrospinal fluid specimens, and whole-genome sequencing was used to characterise isolates. Information on vaccination campaigns was collected by the Niger Ministry of Health and WHO. From Jan 1 to June 30, 2015, 9367 suspected meningitis cases and 549 deaths were reported in Niger. Among 4301 cerebrospinal fluid specimens tested, 1603 (37·3%) were positive for a bacterial pathogen, including 1147 (71·5%) that were positive for N meningitidis serogroup C (NmC). Whole-genome sequencing of 77 NmC isolates revealed the strain to be ST-10217. Although vaccination campaigns were limited in scope because of a global vaccine shortage, 1·4 million people were vaccinated from March to June, 2015. This epidemic represents the largest global NmC outbreak so far and shows the continued threat of N meningitidis in sub-Saharan Africa. The risk of further regional expansion of this novel clone highlights the need for continued strengthening of case-based surveillance. The availability of an affordable, multivalent conjugate vaccine may be important in future epidemic response. MenAfriNet consortium, a partnership between the US Centers for Disease Control and Prevention, WHO, and Agence de Médecine Preventive, through a grant from the Bill & Melinda Gates Foundation