Poster Presentation 25th International Pathogenic Neisseria Conference 2026

Targeted sequencing of Neisseria meningitidis directly from clinical specimens, South Africa, 2023-2026 (#072)

Keabetswe Motsepe 1 , Rito Mikhari 1 2 , Fahima Moosa 1 , Linda de Gouveia 1 , Rivionia Nero 1 , Susan Meiring 3 , Sibongile Walaza 1 , Vanessa Quan 3 , Cheryl Cohen 1 4 , Anne von Gottberg 1 2 5 , Mignon du Plessis 1 2
  1. National Institute For Communicable Diseases, Johannesburg, GAUTENG, South Africa
  2. School of Pathology, Faculty of Health Sciences , University of the Witwatersrand, Johannesburg, Gauteng, South Africa
  3. Division of Public Health Surveillance and Response, National Institute For Communicable Diseases, Johannesburg, Gauteng, South Africa
  4. School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, Gauteng, South Africa
  5. Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town , Western Cape , South Africa

Introduction

Limited availability of viable meningococcal isolates poses a challenge for strain characterisation. We evaluated a culture-independent, whole-genome sequencing approach to enrich and amplify meningococcal DNA directly from clinical specimens.

 

Methods

A total of 262 cerebrospinal fluid (CSF) PCR-positive meningococcus specimens were collected from 2023 to 2026. Of these, 35/262 (13%) with sufficient residual volume (≥100µl) were included in the study (cycle threshold [Ct] range:23–38), comprising genogroups NmB (20/35;57%), NmY (5/35;14%), NmC (4/35;11%), NmW (4/35;11%), NmE (1/35;3%), and one with genogroup not determined. Selective whole-genome amplification was used to enrich meningococcal DNA. All enriched specimens positive for sodC PCR underwent whole-genome sequencing using Illumina. Sequence data were checked for quality and analysed using the Galaxy pipeline with the PubMLST Neisseria database for species confirmation, genogrouping, multilocus sequence typing (MLST), fine typing (PorA VR1/VR2, FetA, PorB), antigen profiling (nadA, nhbA, fHbp), and identification of penA alleles.

 

Results

Following enrichment and repeat sodC PCR, 9/35 (26%) specimens (pre-enrichment Ct >35) were PCR-negative and were not sequenced. Twenty-three percent (8/35) showed no change in Ct value (post-enrichment Ct:26–38) and yielded no sequence data. The remaining 18/35 (51%) demonstrated reduced Ct values post-enrichment, 7/18 (39%) (Ct:14–18) produced high-quality assemblies with >1400 core genome MLST (cgMLST) loci, indicating >95% locus-calling accuracy, and had concordant genogroup assignments with PCR-based results. Among these, 6/7 were fully typed across seven MLST loci (ST23, ST213, ST884, ST4244, ST4977) with complete fine-typing profiles. Nhba was identified in all 7 genomes, fHbp in 6 and nadA in 3. All seven had penA identified and one was associated with penicillin resistance (penA7). For the remaining 11/18 (61%, Ct range:18-26), 6/11 (55%) were partially typed, with ≤5 housekeeping genes and <1400 cgMLST loci identified, and 2/6 were assigned genogroups concordant with PCR-based results. Partial fine typing, antigen profiles, and AMR loci were identified in 7/11 (64%), while 4/11 (36%) yielded no results.   

 

Conclusion

High-quality sequence data were available for 20% of culture-negative specimens, providing adequate genetic detail to characterise strains and assess relatedness during outbreaks. Increased CSF volumes (≥200µl) and adequate bacterial load, post-enrichment (Ct<20), will likely increase this yield.