Direct Real-Time PCR for the Detection and Serotyping of Haemophilus influenzae without DNA Extraction.

To monitor the burden and changes in Haemophilus influenzae (Hi) disease, direct real-time PCR (drt-PCR) assays have been developed for Hi detection in monoplex form and its six serotypes in triplex form, directly from cerebrospinal fluid (CSF) specimens. These assays target the phoB gene for the species detection (Hi-phoB) and serotype-specific genes in region II of the capsule biosynthesis locus (Hi-abf and Hi-cde), identified through comparative analysis of Hi and non-Hi whole-genome sequences. The lower limit of detection (LLD) is 293 CFU/mL for the Hi-phoB assay and ranged from 11 to 130 CFU/mL for the triplex serotyping assays. Using culture as a reference method, the sensitivity and specificity of Hi-phoB, Hi-abf, and Hi-cde were 100%. Triplex serotyping assays also showed 100% agreement for each serotype compared to their corresponding monoplex serotyping assay. These highly sensitive and specific drt-PCR assays do not require DNA extraction and thereby reduce the time, cost, and handling required to process CSF specimens. Furthermore, triplex drt-PCR assays combine the detection of three serotypes in a single reaction, further improving testing efficiency, which is critical for laboratories that process high volumes of Hi specimens for surveillance and diagnostic purposes.

Genomic basis of a polyagglutinating isolate of Neisseria meningitidis.

Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate (M16917) from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene (synD) of M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the M16917 genome sequence, which led to the introduction of an ∼2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis, this kind capsule-switching event could have public health relevance as a vaccine escape mutant.

Carriage of Neisseria lactamica in 1- to 29-year-old people in Burkina Faso: epidemiology and molecular characterization.

Neisseria lactamica is a true commensal bacterium occupying the same ecological niche as the pathogenic Neisseria meningitidis, which is responsible for outbreaks and large epidemics, especially in sub-Saharan Africa. To better understand the epidemiology of N. lactamica in Africa and its relationship to N. meningitidis, we studied N. lactamica carriage in 1- to 29-year-old people living in three districts of Burkina Faso from 2009 to 2011. N. lactamica was detected in 18.2% of 45,847 oropharyngeal samples. Carriage prevalence was highest among the 2-year-olds (40.1%) and decreased with age. Overall prevalence was higher for males (19.1%) than females (17.5%) (odds ratio [OR], 1.11; 95% confidence interval [CI], 1.04 to 1.18), while among the 18- to 29-year-olds, carriage prevalence was significantly higher in women (9.1%) than in men (3.9%) (OR, 2.49; 95% CI, 1.94 to 3.19). Carriage prevalence of N. lactamica was remarkably homogeneous in the three districts of Burkina Faso and stable over time, in comparison with carriage of N. meningitidis (P. A. Kristiansen et al., Clin. Vaccine Immunol. 18:435-443, 2011). There was no significant seasonal variation of N. lactamica carriage and no significant change in carriage prevalence after introduction of the serogroup A meningococcal conjugate vaccine, MenAfriVac. Multilocus sequence typing was performed on a selection of 142 isolates. The genetic diversity was high, as we identified 62 different genotypes, of which 56 were new. The epidemiology of N. lactamica carriage and the molecular characteristics of carried isolates were similar to those reported from industrialized countries, in contrast to the particularities of N. meningitidis carriage and disease epidemiology in Burkina Faso.

sodC-based real-time PCR for detection of Neisseria meningitidis.

Real-time PCR (rt-PCR) is a widely used molecular method for detection of Neisseria meningitidis (Nm). Several rt-PCR assays for Nm target the capsule transport gene, ctrA. However, over 16% of meningococcal carriage isolates lack ctrA, rendering this target gene ineffective at identification of this sub-population of meningococcal isolates. The Cu-Zn superoxide dismutase gene, sodC, is found in Nm but not in other Neisseria species. To better identify Nm, regardless of capsule genotype or expression status, a sodC-based TaqMan rt-PCR assay was developed and validated. Standard curves revealed an average lower limit of detection of 73 genomes per reaction at cycle threshold (C(t)) value of 35, with 100% average reaction efficiency and an average R(2) of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC-positive, with a range of average C(t) values from 13.0 to 29.5. The mean sodC C(t) value of these Nm isolates was 17.6±2.2 (±SD). Of the 626 Nm tested, 178 were nongroupable (NG) ctrA-negative Nm isolates, and 98.9% (176/178) of these were detected by sodC rt-PCR. The assay was 100% specific, with all 244 non-Nm isolates testing negative. Of 157 clinical specimens tested, sodC detected 25/157 Nm or 4 additional specimens compared to ctrA and 24 more than culture. Among 582 carriage specimens, sodC detected Nm in 1 more than ctrA and in 4 more than culture. This sodC rt-PCR assay is a highly sensitive and specific method for detection of Nm, especially in carriage studies where many meningococcal isolates lack capsule genes.