Patients and samples
Samples were taken from patients with suspected meningococcal disease in Scotland and sent by area diagnostic microbiology laboratories to the SMPRL in Glasgow. Organisms were sent for characterisation whereas body fluids were sent for agglutination, PCR, and antibody testing. All samples sent for meningococcal investigations were included for analysis in our study.
Meningococcal disease notification database
The notified meningococcal disease database is held in Scotland by the SMPRL. Information is gained through the strategic notification scheme between the SMPRL, Information and Statistics Division, Public Health Consultants, and SCIEH.
10 The database uses a system of classification for each notification depending on whether the case is a clinical or laboratory notification (fig 1
).
 | Figure 1 Diagnostic categories for the confirmation of meningococcal disease in Scotland. CSF, cerebrospinal fluid; PCR, polymerase chain reaction. |
Culture confirmation of meningococcal disease from blood, CSF, eye, or other normally sterile site
Culture confirmation of meningococcal disease was performed on presumed isolates of
Neisseria meningitidis using standard procedures for the biochemical identification, serogrouping, and serotyping/subtyping of meningococci.
15–17 Culture confirmation was assigned to diagnostic category I as shown in fig 1
.
Microscopy of cerebrospinal fluid (CSF)
Microscopy of CSF was performed in area laboratories. The presence of Gram negative diplococci in a CSF sample with other laboratory findings consistent with bacterial infection, and in a patient with symptoms suggestive of meningococcal meningitis, was recorded as a laboratory confirmed case of clinical meningitis under diagnostic category II.
Microscopy of skin aspirate/scrapings
Microscopy was performed in local laboratories on skin aspiration/scrapings taken from patients with a rash and other symptoms suggestive of meningococcal septicaemia. When Gram negative diplococci were present the case was recorded as a laboratory confirmed case of clinical septicaemia under diagnostic category III.
PCR testing of CSF, blood, or other normally sterile fluid
PCR was introduced in 1995 and is performed primarily on CSF, serum, and plasma to detect the insertion element IS1106, as described previously.
18,19 Serum and plasma samples were diluted 1/2 in sterile water and boiled for five minutes. Undiluted samples of CSF samples were boiled for five minutes. The thermocyling conditions were 27 cycles of 95°C for 25 seconds, 61°C for 40 seconds, and 72°C for 60 seconds, followed by extension for 60 seconds at 72°C. PCR products were run on a 1.5% agarose gel stained with ethidium bromide and visualised under an ultraviolet transilluminator. IS1106 positive samples were serogrouped by amplification and restriction fragment length polymorphism analysis of the siaD gene when disease was caused by serogroups B or C.
16 A positive PCR result was recorded as a laboratory confirmed case of meningococcal septicaemia and/or meningitis under diagnostic category IV.
Latex agglutination and co-agglutination on CSF
Latex agglutination was performed on CSF using the commercially available Wellcogen kit (Bio-stat Diagnostic Systems, Stockport, UK) for serogroups A, B, C, Y, and W135. Co-agglutination was performed as described previously using in house reagents for serogroups A, B, C, Y, W135, X, and Z
1.
15 A positive result was recorded as a laboratory confirmed case of meningococcal septicaemia and/or meningitis under diagnostic category V.
Throat swab culture
Throat swabs were taken from patients with suspected meningococcal disease. The isolation of
N meningitidis from the throat was recorded as a laboratory confirmed case of meningococcal septicaemia and/or meningitis under diagnostic category VI.
Antibody confirmation
An indirect enzyme linked immunosorbent assay method was performed on serum to detect antibodies directed against the outer membrane protein (OMP) of the infecting meningococcus.
20,21 Briefly, OMPs were purified from common meningococcal strains circulating in the UK and pooled as the antigen source. The antigen was coated on to the wells of a flat bottomed 96 well microtitre plate by drying overnight. The plate was then washed with phosphate buffered saline (PBS) and blocking was performed by the addition of 200 μl of 3% bovine serum albumin in PBS and incubation for one hour at 37°C. The plate was washed again with PBS and 100 μl of patient's serum or positive/negative control diluted 1/1000 was added to the appropriate wells. The plate was then incubated at 37°C for 1.5 hours. Aliquots of 100 μl of antihuman immunoglobulin–enzyme IgM and IgG conjugates (Sigma, Poole, Dorset, UK) were diluted 1/1000, added to the appropriate wells, and incubated for one hour at 37°C. The plate was washed again with PBS, 100 μl of OPD substrate was added to all wells, followed by incubation at room temperature for 0.5 hours. The reaction was stopped with 50 μl H
2SO
4 and the aborbance read at 492 nm. Positive and negative cut off values were calculated by (negative/(positive − negative)) and the result for each test was calculated by ((test − negative)/positive − negative)) × 100. Raised titres of IgM and/or IgG were recorded as a laboratory confirmed case of meningococcal septicaemia under diagnostic category VII after appropriate interpretation depending on the date of onset and dates of sera taken for testing.
Data analysis
Data from each test were analysed for each year and tabulated or presented in graphical form. Data comparisons were made between the culture confirmed and non-culture confirmed cases using the χ
2 test to show the significance of using non-culture based methods.
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