Comparison of DNA concentration and bacterial pathogen PCR detection when using two DNA extraction kits for nasopharyngeal/oropharyngeal samples.

INTRODUCTION: Several important human pathogens that cause life-threatening infections are asymptomatically carried in the Nasopharynx/Oropharynx (NP/OP). DNA extraction is a prerequisite for most culture-independent techniques used to identify pathogens in the NP/OP. However, components of DNA extraction kits differ thereby giving rise to differences in performance. We compared the DNA concentration and the detection of three pathogens in the NP/OP using the discontinued DNeasy PowerSoil Kit (Kit DP) and the DNeasy PowerLyzer PowerSoil Kit (Kit DPP). METHODS: DNA was extracted from the same set of 103 NP/OP samples using the two kits. DNA concentration was measured using the Qubit 2.0 Fluorometer. Real-time Polymerase Chain reaction (RT-PCR) was done using the QuantStudio 7-flex system to detect three pathogens: S. pneumoniae, H. influenzae, and N. meningitidis. Bland-Altman statistics and plots were used to determine the threshold cycle (Ct) value agreement for the two kits. RESULTS: The average DNA concentration from kit DPP was higher than Kit DP; 1235.6 ng/ml (SD = 1368.3) vs 884.9 ng/ml (SD = 1095.3), p = 0.002. Using a Ct value cutoff of 40 for positivity, the concordance for the presence of S. pneumoniae was 82% (84/102); 94%(96/103) for N. meningitidis and 92%(95/103) for H. influenzae. Kit DP proportionately resulted in higher Ct values than Kit DPP for all pathogens. The Ct value bias of measurement for S. pneumoniae was +2.4 (95% CI, 1.9-3.0), +1.4 (95% CI, 0.9-1.9) for N. meningitidis and +1.4 (95% CI, 0.2-2.5) for H. influenzae. CONCLUSION: The higher DNA concentration obtained using kit DPP could increase the chances of recovering low abundant bacteria. The PCR results were reproducible for more than 90% of the samples for the gram-negative H. influenzae and N. meningitidis. Ct value variations of the kits must be taken into consideration when comparing studies that have used the two kits.

Pediatric Bacterial Meningitis Surveillance in Niger: Increased Importance of Neisseria meningitidis Serogroup C, and a Decrease in Streptococcus pneumoniae Following 13-Valent Pneumococcal Conjugate Vaccine Introduction.

BACKGROUND: Meningitis is endemic in Niger. Haemophilus influenzae type b (Hib) vaccine and the 13-valent pneumococcal conjugate vaccine (PCV13) were introduced in 2008 and 2014, respectively. Vaccination campaign against Neisseria meningitidis serogroup A was carried out in 2010-2011. We evaluated changes in pathogen distribution using data from hospital-based surveillance in Niger from 2010 through 2016. METHODS: Cerebrospinal fluid (CSF) specimens from children <5 years old with suspected meningitis were tested to detect vaccine-preventable bacterial pathogens. Confirmatory identification and serotyping/grouping of Streptococcus pneumoniae, N. meningitidis, and H. influenzae were done. Antimicrobial susceptibility testing and whole genome sequencing were performed on S. pneumoniae isolates. RESULTS: The surveillance included 2580 patients with suspected meningitis, of whom 80.8% (2085/2580) had CSF collected. Bacterial meningitis was confirmed in 273 patients: 48% (131/273) was N. meningitidis, 45% (123/273) S. pneumoniae, and 7% (19/273) H. influenzae. Streptococcus pneumoniae meningitis decreased from 34 in 2014, to 16 in 2016. PCV13 serotypes made up 88% (7/8) of S. pneumoniae meningitis prevaccination and 20% (5/20) postvaccination. Neisseria meningitidis serogroup C (NmC) was responsible for 59% (10/17) of serogrouped N. meningitidis meningitis. Hib caused 67% (2/3) of the H. influenzae meningitis isolates serotyped. Penicillin resistance was found in 16% (4/25) of S. pneumoniae isolates. Sequence type 217 was the most common lineage among S. pneumoniae isolates. CONCLUSIONS: Neisseria meningitidis and S. pneumoniae remain important causes of meningitis in children in Niger. The decline in the numbers of S. pneumoniae meningitis post-PCV13 is encouraging and should continue to be monitored. NmC is the predominant serogroup causing N. meningitidis meningitis.