The laboratory confirmation of meningococcal disease and characterization of Neisseria meningitidis isolates was improved considerably in England and Wales by the Meningococcal Reference Unit between epidemiological years 1993/94 and 2003/04 to meet the challenge of increasing numbers of cases of clinical disease and the requirement for enhanced surveillance. Improved case ascertainment was made possible by the rapid introduction of an innovative centralized reference service for non-culture PCR-based DNA detection of meningococci utilizing the ctrA and siaD PCR assays, complemented by consistent phenotypic characterization of submitted isolates from culture-proven cases. This allowed the increased prevalence of serogroup C disease in specific age groups and the apparent associated increase in mortality from 1995/96 to 1999/00 to be defined, thereby prompting accelerated intervention with the newly licensed meningococcal serogroup C conjugate (MCC) vaccines into the under-25-year UK population (in November 1999). The continued increase in and predominance of serogroup B cases (1993/94 to 2000/01) were observed in conjunction with their diverse and changing phenotypic characteristics. Trends observed to be associated with the predominant phenotypic combinations of serogroup, serotype and sero-subtype were: a decline of both C : 2b and B : 2b meningococci, and a decline of B : 15 : P1.7,16 with a concomitant increase of B : 4 : P1.4 over the 11-year period. Detailed routine surveillance rapidly confirmed the introduction of W135 : 2a : P1.5,2 meningococci into the UK during 2000 and 2001. The importance of continued detailed surveillance of this important pathogen cannot be overestimated, both to monitor the effectiveness of the MCC vaccine and to identify changes within the meningococcal population that can inform the design of anti-serogroup B vaccines.

Epidemiology of meningococcal disease in England and Wales 1993/94 to 2003/04: contribution and experiences of the Meningococcal Reference Unit.

Background. Diagnostic polymerase chain reaction (PCR) detection of Neisseria meningitidis has enabled accurate quantification of the bacterial load in patients with meningococcal disease. Methods. Quantification of the N. meningitidis DNA level by real time-PCR was conducted on whole-blood samples obtained from patients presenting with meningococcal disease to hospitals throughout England and Wales over a 3-year period. Levels were correlated with clinical outcome, infecting serogroup, and host factors including,interleukin-1 genotype (IL-1). Results. Bacterial loads were available for 1045 patients and were not associated with the age of the patient, delay in sample submission, or administration of antibiotics prior to admission. The median log bacterial load was higher in 95 patients who died (5.29 log10copies/mL; interquartile range, 4.41-6.30 log10copies/mL) than in 950 patients who survived (3.79 log10copies/mL; interquartile range, 2.87-4.71 log10copies/mL). Logistic regression revealed that age (odds ratio, 1.04 per 1-year increase in age) and bacterial load (odds ratio, 2.04 per log10-copies/mL increase) had a statistically significant effect on the risk of death. Infection with N. meningitidis serogroup C was associated with increased risk of death and an increased bacterial load. Also associated with a higher bacterial load were prolonged hospitalization (duration, >10 days); digit, limb, or soft-tissue loss; and requirement of hemodialysis. Carriage of IL-1RN(+2018) was associated with increased mortality (odds ratio, 2.14; P=.07) but not with a higher bacterial load. Conclusions. In meningococcal disease, bacterial load is associated with likelihood of death, development of permanent disease sequelae, and prolonged hospitalization. The bacterial load was relatively higher in patients infected with N. meningitidis serogroup C than in those infected with other serogroups. The effects of age and IL-1 genotype on mortality are independent of a high genomic bacterial load.

Severity of Meningococcal Disease Associated with Genomic Bacterial Load

Twenty clinical samples (18 cerebrospinal fluid samples and 2 articular fluid samples) were sent to 11 meningococcus reference centers located in 11 different countries. Ten of these laboratories are participating in the EU-MenNet program (a European Union-funded program) and are members of the European Monitoring Group on Meningococci. The remaining laboratory was located in Burkina Faso. Neisseria meningitidis was sought by detecting several meningococcus-specific genes (crgA, ctrA, 16S rRNA, and porA). The PCR-based nonculture method for the detection of N. meningitidis gave similar results between participants with a mean sensitivity and specificity of 89.7 and 92.7%, respectively. Most of the laboratories also performed genogrouping assays (siaD and mynB/sacC). The performance of genogrouping was more variable between laboratories, with a mean sensitivity of 72.7%. Genogroup B gave the best correlation between participants, as all laboratories routinely perform this PCR. The results for genogroups A and W135 were less similar between the eight participating laboratories that performed these PCRs.

Interlaboratory Comparison of PCR-Based Identification and Genogrouping of Neisseria meningitidis

PCR-based assays for the identification of Neisseria meningitidis serogroups 29E, X, and Z by detection of specific regions of the ctrA gene are described. The specificities of these assays were confirmed using serogroups A, B, C, 29E, H, W135, X, Y, and Z and nongroupable meningococcal isolates.

https://jcm.asm.org/content/jcm/42/4/1764.full.pdf

PCR-Based Assay for Detection of Neisseria meningitidis Capsular Serogroups 29E, X, and Z

We developed two Neisseria meningitidis multiplex PCR assays to be used consecutively that allow determination of the serogroup and capsular status of serogroup A, B, C, 29E, W135, X, and Y cnl-3/cnl-1-like-containing N. meningitidis isolates by direct analysis of the amplicon size. These assays offer a rapid and simple method of serogrouping N. meningitidis.

Consecutive Use of Two Multiplex PCR-Based Assays for Simultaneous Identification and Determination of Capsular Status of Nine Common Neisseria meningitidis Serogroups Associated with Invasive Disease

Standard and fluorescence-based PCR assays were developed for the identification of serogroup A meningococci by detection of the mynA gene. This assay was evaluated using bacterial cultures but provides the sensitivity required for the detection of the mynA gene from bodily fluids during meningococcal disease.

E. K. Girvan

Papers

Evaluation of a Fluorescence-Based PCR Method for Identification of Serogroup A Meningococci