A single-tube 5 nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport (ctrA), capsulation (bexA), and pneumolysin (ply) gene targets specific for N. meningitidis, H. influenzae, and S. pneumoniae, respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for N. meningitidis, H. influenzae, and S. pneumoniae were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrA primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the ply primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the bexA primers amplified H. influenzae types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number (n ‫؍‬ 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal, H. influenzae, and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The ctrA primer set used in the multiplex PCR was found to be more sensitive (P < 0.0001) than the ctrA primers that had been used for meningococcal PCR testing at that time.

The immune response to SARS-CoV-2 is critical in controlling disease but there is concern that waning immunity may predispose to re-infection. We analysed the magnitude and phenotype of the SARS-CoV-2-specific T cell response in 100 donors at six months following infection. T-cell responses were present by ELISPOT and/or ICS analysis in all donors and characterised by predominant CD4+ T cell responses with strong IL-2 cytokine expression. Median T-cell responses were 50% higher in donors who had experienced a symptomatic infection indicating that the severity of primary infection establishes a ‘setpoint’ for cellular immunity. T-cell responses to spike and nucleoprotein/membrane proteins were correlated with peak antibody levels. Furthermore, higher levels of nucleoprotein-specific T cells were associated with preservation of NP-specific antibody level although no such correlation was observed in relation to spike-specific responses. In conclusion, our data are reassuring that functional SARS-CoV-2-specific T-cell responses are retained at six months following infection.

SummaryObjectivesNeisseria meningitidis is a leading cause of meningitis and septicaemia. The hyperinvasive ST-11 clonal complex (cc11) caused serogroup C (MenC) outbreaks in the US military in the 1960s and UK universities in the 1990s, a global Hajj-associated serogroup W (MenW) outbreak in 2000–2001, and subsequent MenW epidemics in sub-Saharan Africa. More recently, endemic MenW disease has expanded in South Africa, South America and the UK, and MenC cases have been reported among European and North American men who have sex with men (MSM). Routine typing schemes poorly resolve cc11 so we established the population structure at genomic resolution.MethodsRepresentatives of these episodes and other geo-temporally diverse cc11 meningococci (n = 750) were compared across 1546 core genes and visualised on phylogenetic networks.ResultsMenW isolates were confined to a distal portion of one of two main lineages with MenB and MenC isolates interspersed elsewhere. An expanding South American/UK MenW strain was distinct from the ‘Hajj outbreak’ strain and a closely related endemic South African strain. Recent MenC isolates from MSM in France and the UK were closely related but distinct.ConclusionsHigh resolution ‘genomic’ multilocus sequence typing is necessary to resolve and monitor the spread of diverse cc11 lineages globally.