Detection and genotyping of meningococci using a nested PCR approach

Abstract

An effective vaccine against Neisseria meningitidis serogroup B is required. Outer-membrane protein vaccines have been developed, which may provide protection against common circulating serotypes and serosubtypes in some countries. However, limited genosubtyping data are available because most laboratories use mAbs directed against a limited number of specific serotypes and serosubtypes and laboratories do not genosubtype directly from body fluids due to the lack of a sensitive PCR method. A nested PCR was therefore developed that enables the amplification of the porA gene directly from clinical samples and has the required sensitivity for nucleotide sequencing of the three main variable regions, VR1, VR2 and VR3. Data were compared with those from culturebased nucleotide sequencing, and the use of this method increased the availability of genosubtype information by 45 %, thereby indicating the impact that this methodology has on the data provided and the implications for vaccine design. INTRODUCTIONMeningococcal disease remains a significant public health problem, and a vaccine against Neisseria meningitidis serogroup B is urgently needed (Rosenstein et al., 2001). However, the use of a capsule-based vaccine is unlikely because the meningococcal polysaccharide is identical to a human carbohydrate [AE (2 ! 8) N-acetyl neuraminic acid or polysialic acid] (Finne et al., 1987;Hayrinen et al., 1995). Vaccines based on other antigens therefore require development and the outer-membrane proteins (OMPs) of the meningococcus have been investigated (Rosenstein et al., 2001;Al'Aldeen & Cartwright, 1996). Although OMP-based vaccines are not the solution for combating serogroup B meningococcal infection, they may be useful in some circumstances (Al'Aldeen & Cartwright, 1996;Rappuoli, 2001). However, such proteins are highly antigenically variable, and an OMP vaccine cannot protect against all serotypes and serosubtypes of N. meningitidis. Therefore, selected OMPs must be used, based on the prevalent serotypes and serosubtypes in a given population. However, to develop such vaccines, high-quality data must be gained to determine such prevalence. To date, the characterization of meningococci has relied on serogrouping, serotyping and serosubtyping using serological methods, thereby limiting the designation of serotypes and serosubtypes because a panel of mAbs is used. Newer nucleotide sequence methods, based on the detection and sequencing of the porB and porA genes for serotype (class 2/3 OMP) and serosubtype (class 1 OMP), respectively, have led to a better understanding of the variability of these proteins (Urwin et al., 1998a, b, c;Clarke et al., 2001a;Jelfs et al., 2000;Molling et al., 2000;Feavers et al., 1996;Saunders et al., 1993;Smith et al., 1995). However, most of these methods are only useful when N. meningitidis has been isolated from blood or cerebrospinal fluid (CSF) and they therefore rely on the availability of a culture (Jelfs et al., 2000;Feavers et al., 1996). Other methods rely on sufficient numbers of me...

Prospects for Vaccine Prevention of Meningococcal Infection

“…Permanent sequelae are common among survivors of meningococcal infection (93,95). Definitive diagnosis requires the isolation of N. meningitidis from a normally sterile body fluid or the detection of meningococcal DNA by PCR (36,71,87,259,321).…”

PorA Variable Antigenic Regions VR1, VR2, and VR3 of Neisseria meningitidis Serogroups B and C Isolated in Brazil from 1999 to 2004

“…A third variable region, designated VR3, is present on the top of loop V (24). The genetic variability of this region has also been studied previously (1,6,7,10,14,19), and research has revealed a lower level of genetic variability than that found within VR1 and VR2. One potential explanation is that loop V, where VR3 is located, is slightly shorter than loops I and IV, where VR1 and VR2 are located.…”

Impact of MenBvac, an outer membrane vesicle (OMV) vaccine, on the meningococcal carriage

“…Volunteers underwent a swabbing from the posterior pharynx (i.e., soft palate and pillars) performed by a trained physician. Swabs were immediately plated on chocolate agar supplemented with vancomycin, colimycin and amphotericin B (VCAT agar, Biomérieux, France) and incubated at 35-37 • C under 7.5% CO 2 for 48 h. Meningococci were detected and identified by standard methods (colony morphology with one to three colonies from each morphotype, Gram stain, oxidase, biochemical profile using API-NH strip [Biomerieux, France]) and by PCR targeting ctrA and porA genes [12,13]. Isolates were characterized by genogrouping, serogrouping, serotyping, serosubtyping, and sequence typing by multilocus sequence typing (MLST) [14].…”