Alternative molecular methods for improved detection of meningococcal carriage and measurement of bacterial density

Abstract

BACKGROUND: Conventional methods for detecting pharyngeal carriage of Neisseria meningitidis (Nm) are complex. There is a need for simpler methods with improved performance. We have investigated two alternative approaches. METHODS: Three pharyngeal swabs were collected from 999 pupils aged 10 to 18 years in The Gambia. Carriage of Nm was investigated using three different methods: (i) plating on Thayer-Martin selective medium and testing by conventional microbiological methods followed by PCR testing, (ii) seeding in Todd Hewitt broth (THB) and, after overnight culture, testing by PCR, (iii) compression of the swab on filter paper and, after DNA concentration, testing by PCR. RESULTS: PCR after culture in THB was more than twice as sensitive as conventional methods in detecting Nm (13.2% versus 5.7%; p<0.0001). PCR after DNA extraction from filter paper had a similar sensitivity to that of conventional methods (4.9% vs 5.7%, p=0.33). Capsular genogroups detected by broth culture were W (21), B (12), Y (8), E (3), and X (2), and 68 meningococci had the capsule null intergenic region. The distribution of genogroups and of capsule null organisms was similar with each of the three methods. Carriage density in samples extracted from filter paper ranged from 1 to 25,000 DNA copies. CONCLUSIONS: PCR of overnight broth culture doubled the yield of Nm carriage isolates compared with conventional methods. This approach could improve the efficiency of carriage studies. Collection on filter paper followed by quantitative PCR could be useful for density measurement and for carriage studies in areas with limited resources.