Design and comparative evaluation of oligonucleotide flow analysis versus microarray assays for non-culture confirmation of meningococcal and pneumococcal infections during trial and introduction of newly developed vaccines.
Dr Ed Kaczmarski, Dr Malcolm Guiver, Dr Ray Borrow
- Start Date:
01 January 2001
PHLS Meningococcal Reference Unit, Manchester, UK
Development of rapid, high throughput, sensitive and specific non-culture assays for the confirmation of meningococcal and pneumococcal disease is important for accurate estimation of the efficacy of novel vaccines against these conditions. PCR based confirmation on platforms such as the Applied Biosystems Taqman(tm) or the Roche Lightcycler(tm) is robust and rapid and suited for meningococci where disease is caused by a small number of serogroups. For meningococcal PorA outer membrane vesicle (OMV) and pneumococcal 7, 9 and 11-valent conjugate vaccines currently progressing to phase II studies in the UK, the numbers of assays required to detect all potentially infecting strains (200 meningococcal PorA variants and 96 pneumococcal serotypes) has increased beyond the scope of these platforms. Novel technologies need to be developed, ideally involving rapid, 'single tube' typing to provide timely data at a reasonable cost.
Current technologies capable of offering this are (i) oligonucleotide nucleotide flow analysis and (ii) DNA microarrays. For meningococcal confirmation and serogrouping, existing oligonucleotide primers and probes can be utilised in both assay formats. For PorA serosubtyping, probes will be designed. For pneumococci, primers and probes will be selected on capsular (cps) polymorphisms selected and serotypes whose cps sequence are not currently available will be characterised. Carboxylated microspheres for flow analysis are commercially available as are protocols for coupling of DNA probes to the microspheres. Microarray chips will be commercially synthesised with the required probe sequences. Once designed and developed, these assays will be evaluated on fully characterised isolates as well as PCR proven cases to determine performance characteristics. The final step will be a roll out to Meningococcal and Pneumococcal Reference Units in other European countries and elsewhere.