Genotypic Analysis of Meningococcal Factor H-Binding Protein from Non-Culture Clinical Specimens

Abstract

Factor H-Binding Protein (fHbp) is an outer membrane protein antigen included in two novel meningococcal group B vaccines and, as such, is an important typing target. Approximately 50% of meningococcal disease cases in England and Wales are confirmed using real-time PCR on non-culture clinical specimens only. Protocols for typing fHbp from this subset of cases have not yet been established. Here we present a nested PCR-based assay designed to amplify and sequence fHbp from non-culture clinical specimens. From analytical sensitivity experiments carried out using diluted DNA extracts, an estimated analytical sensitivity limit of 6 fg/µL of DNA (<3 genome copies/µL) was calculated. The sensitivity of the assay was shown to be comparable to the ctrA-directed real-time PCR assay currently used to confirm invasive disease diagnoses from submitted clinical specimens. A panel of 96 diverse, patient-matched clinical specimen/isolate pairs from invasive disease cases was used to illustrate the breadth of strain coverage for the assay. All fHbp alleles sequenced from the isolates matched those derived from previous whole genome analyses. The first-round PCR primer binding sites are highly conserved, however an exceptional second-round PCR primer site mismatch in one validation isolate prevented amplification. In this case, amplification from the corresponding clinical specimen was achieved, suggesting that the use of a nested PCR procedure may compensate for any minor mismatches in round-two primer sites. The assay was successful at typing 91/96 (94.8%) of the non-culture clinical specimens in this study and exhibits sufficient sensitivity to type fHbp from the vast majority of non-culture clinical specimens received by the Meningococcal Reference Unit, Public Health England.

Meningococcal B vaccine antigen FHbp variants among disease-causing Neisseria meningitidis B isolates, Italy, 2014–2017

“…org/neisseria/). For culture negative samples, seven housekeeping genes (abcZ, adk, aroE, fumc, pgm, pdhc and gdh) together with porA, fetA and fHbp genes were amplified referring to PCR conditions available on the PubMLST.org database or using primers and amplification parameters as already described [21]. PCR was performed using Veriti 96 well instrument (Applied Biosystem, Foster City, USA) or Mastercycler personal (Eppendorf, Hamburg, Germany).…”

Differences between culture & non-culture confirmed invasive meningococci with a focus on factor H-binding protein distribution

“…DNA extraction and typing of fHBP from nonculture specimens was carried out as previously described. 16 fHBP nomenclature Differing nomenclatures are currently used for fHBP protein variants. In the system established by Fletcher et al, individual variants are given unique alphanumeric identifiers based upon the subfamily to which each variant belongs.…”

“…In order to tackle this significant epidemiological knowledge gap, a PCR sequencing assay was developed to sequence fHBP from non-culture clinical specimens. 16 Here we present fHBP typing data from non-culture specimens submitted to the MRU over three calendar years (2011e2013). For the first time we can compare these data with those derived from isolates received during the same period to assess the representativeness of viable isolates among all invasive meningococcal strains in E&W.…”

Clustered intergenic region sequences as predictors of factor H Binding Protein expression patterns and for assessing Neisseria meningitidis strain coverage by meningococcal vaccines

“…A key potential use for the fHbp expression cluster is to predict vaccine coverage for strains that have caused disease in individuals subject to immunization with an fHbp-containing vaccine and where case confirmation was by PCR with no viable culture. All PCR-only confirmed IMD cases routinely have their fHbp gene sequenced as part of the Public Health England enhanced surveillance of 4C-MenB [ 26 ]. The fHbp PCR product spans both the CDS and IGR and the routine sequencing of the fHbp CDS results in sequence data for the IGR (Borrow et al ., personal communication).…”