1. Andrew Lawrence
Microbiology and Infectious Diseases Dept.
SA Pathology at Women’s and Children’s Hospital
Adelaide, SA
Neisseria meningitidis:
Clinical Laboratory
Diagnosis - Plus

2. Neisseria and related spp. of
human origin
• Neissseria meningitidis
• Neisseria gonorrhoeae
• N. lactamica, N. cinerea, N.
polysacharea, N. subflava, N. sicca,
N.mucosa, N.flavescens, N.elongata
• Moraxella (Branhamella) catarrhalis –
coccoid form

3. Neisseria spp.
• Characteristic diplococcus (bean shaped)
• Inhabit mucous membranes of warm
blooded hosts
• Aerobic, non-motile, no spores, grow
optimally at 37°C, growth stimulated by
CO2 and NG has obligate requirement,
oxidase positive, produce acid from
carbohydrates oxidatively, most catalase
positive (cf Kingella –ox + but cat -)

4. Gram’s Stain of CSF Showing
GN Diplococci

5. Neisseria spp:Clinical Significance
• NG always considered a pathogen
irrespective of site of isolation
• Other Neiss. Inhabitants of upper
respiratory tract and most not pathogenic
• Only a few strains of meningococci are
pathogenic ie hypervirulent strains

6. Specimens and culture media
M
C
Meningitis/
bacteraemia
CSF, skin
lesions, blood,
nasopharynx
Selective
/non-
selective
M
cat
Pneumonia Sputum or BAL
etc
Non-
selective
Otitis media Tympanocentesis
(not routine)
Non-
selective
Sinus Sinus biopsy or
aspirate
Selective
/Non-
selective

7. Neisseria spp: ID
• Sugar fermentation (GC = glu pos only
while MC = glu, malt pos)
• Growth on selective agar
• Latex/ co-agglutination tests/slide
agglutination for serogrouping MC
(Pastorex/Murex)
• Vitek card
• NATs – serogroup specific

8. N.meningitidis cell wall
Protects against host defences
13 Serogroups (ctrA diagnostic test)
Porins (porA and porB) - serosubtype, serotype
Opacity proteins - adherence to leukocytes / host cells,
Fet A (Fe binding protein formally FrpB)
Potent endotoxin
13 Immunotypes
Adherence to epith and
RB cells

9. Meningococcal Disease. Editor Cartwright K 1995
Neisseria meningitidis showing blebs
50% lipooligosaccharide
50% OMPs, phospholipids and capsular
polysaccharide

10. Meningococcal purpuric rash

11. IMD and the Military
• 1812 US Civil War - meningitis outbreaks
• Turn of century - bacteriologic confirmation
• Increased rates in 1907 - Cuban
occupation and 1913 Mexican border
mobilisation
• New recruits = higher levels of disease cf
seasoned troops

12. IMD and the Military
Meningococcal meningitis in the US Army, 1910-1946
Evolution of the Meningococcus, Vedros editor

13. IMD and the Military
• WW1 (US)- IMD at all training camps
Most crowded had most disease
• During WW1 5839 cases of IMD and 2279
deaths (army) with fatality rate of 39% -
pre antibiotic era
• Between WW1 and WW2 - 2 epidemics
coincidental with epidemics as a whole in
US civilian population

14. IMD and the Military
• Throughout WW2 13,922 cases of IMD
• 559 deaths
• Cases fatality rate 4% - sulphadiazine
prophylaxsis
• 67% cases in troops who had been in the
army for less than 3 months

15. IMD and the Military
• Large epidemics in winters 1917-18, 1942-43
• Smaller secondary peaks approx 9 months later
• New recruits at greater risk than “regular” troops
• Both epidemics coincident with civilian population
epidemics
• Serogroup A accounted for approx. 90% cases
• Post demobilisation rates of disease returned to
“normal”
Summary

16. IMD and the Military
• Post WW2 sulphadiazine used for
prophylaxis but in 1963 (Vietnam era) sdz
resistance developed
• Serogroup B and C (1967) disease reported
and increased - sdz prophylaxis stopped
• 1969-1970 trials of C polysaccharide
vaccines - efficacy of 90%
• 1971 routine use of serogroup C ps vaccine
• 1982 all trainees given A/C/Y/W135 ps
vaccine

17. Typical meningococcal petechial rash

18. Meningococcal sepsis - digital gangrene

19. Meningococcal Disease in
Australia
• July 1900 - Feb 1901 large outbreak
Sydney 69% mortality
• Post WW1 increased rates
• 1915 Large outbreak in Victoria beginning
at Seymour military camp - 644 cases
mortality 52%
• 1939 – 1946 conflict saw increases but
mortality 23% - sulphonamide therapy

20. The annual number of deaths from meningococcal disease was
obtained from the compilation of mortality data collected by the
Australian Bureau of Statistics.
Annual notification rates and deaths from meningococcal disease for all
states/territories in Australia, 1915–2003
Australia’s century of meningococcal disease:
development and the changing ecology of an
accidental pathogen
Mahomed S Patel MJA 2007; 186 (3): 136-141

21. Typing Methods
• Phenotyping
• Genotyping
• Why type N.meningitidis?
Tracking infections - short term or long term
Epidemiological spread of NM across
households/countries/the world
Vaccine development
Evolution of the organism

22. Meningococci
Phenotypic Typing Methods
• Sero-grouping
• Sero-typing
• Sero-subtyping
• Multilocus Enzyme Electrophoresis (14-
20 enzyme mobilities) - ET Type
• Immunotyping

23. Meningococci Phenotyping
• Meningococcal Pheno-types
– Serogroups (Capsular polysaccharide)
• A, B, C, D, E, H, I, K, L, X, Y, Z, W135, 29E,
non-encapsulated
• Only B and C common currently
– Serotypes
• based on outer membrane protein
epitopes
Serotypes - Por B
Serosubtypes - Por A
• eg C:2a,15

24. Serogrouping
• Murex antisera (gpB is a monoclonal)
• Slide agglutination done on fresh organisms
(overnight incubation)
• Light inoculum
• Often need light box to read slide
• Must be done in a bio-safety cabinet
• group B strains sometimes stringy and
autoagglutinate
• non-encapsulate

25. 2D Model of Nm PorA
Meningococcal Disease. Editor Cartwright K 1995

26. Serotyping/sub-typing

27. Genotypic Typing Methods
• Pulse Field Gel Electrophresis - whole
chromosome
• porA PCR with RFLP analysis
• Random Amplified Polymorphic DNA PCR
• *porA/porB Sequencing
• *fetA sequencing
• *penA sequencing
• *Multi Locus Gene Sequencing
- house keeping genes
• Ribotyping
• VNTR

28. Genotyping
• Identification of capsule serotype (serogroup) by
PCR
• Identification of a clone or lineage by MLST and
PFGE
• Identification of serosubtype by porA sequencing
• Identification of serotype by porB gene
sequencing
• Penicillin binding protein 2 variable regions by
penA
• Iron binding protein variable regions by fetA

29. Genotyping
• Not dependent on gene expression
• Confirmation of IMD from culture negative
specimens
• Capsule and subtype determination
directly from clinical samples
• Electronic data portable and reproducible
• MLST aid to longer term epidemiological
studies. PFGE short term studies.

30. Genotyping
• MLST is more robust than MLEE
• porA/fetA/penA sequencing almost routine
– www. database
• Recombination events more easily picked
up by sequencing

31. Ponte Sant’Angelo

36. Strain Characterisation and Typing
• http://neisseria.org/nm/
• Martin Maiden
– Probably 20,000 MN sequence typed now
– ST-11 28x more likely to cause disease compared with
“ordinary NM”
– Different clonal complexes are associated with different
serogroups/particular antigens and reflect human immunity
– Capsule null locus carried in 16% NM
– NM are continually producing variants but these are
continuously cleaved = not much change
– Once vaccines have been introduced there is a major
change in population structure
– Future lies in using/interpreting sequence data now in
databases rather than new ST techniques
EMGM 2007

37. Strain Characterisation and Typing
Andrew Fox – EMGM Questionnaire
• Working group on strain characterisation
• Movement is toward genotyping but some GT data not
being submitted
• Phenotyping reagents still available and are supported
by NIBSC
• Want a hub system for smaller labs not doing
sequencing (Oxford can do sequence for 75p!)
• Recommendation from last EMGM was to do porA
sequencing
• MLST also needs to be maintained to monitor population
biology
• fetA probably helpful when combined with other
Ags
• http://neisseria.org/nm/ EMGM 2007

38. The diversity and dynamics of Neisseria meningitidis populations generate a
requirement for high resolution, comprehensive, and portable typing schemes for
meningococcal disease surveillance. Molecular approaches, specifically DNA
amplification and sequencing, are the methods of choice for various reasons,
including: their generic nature and portability, comprehensive coverage, and ready
implementation to culture negative clinical specimens. The following target genes
are recommended: (1) the variable regions of the antigen-encoding genes porA and
fetA and, if additional resolution is required, the porB gene for rapid investigation
of disease outbreaks and investigating the distribution of antigenic variants; (2) the
seven multilocus sequence typing loci–these data are essential for the most
effectivenational, and international management of meningococcal disease, as well
as being invaluable in studies of meningococcal population biology and evolution.
These targets have been employed extensively in reference laboratories
throughout the world and validated protocols have been published. It is further
recommended that a modified nomenclature be adopted of the form:
serogroup: PorA type:FetA type: sequence type (clonal complex)
Molecular typing of meningococci: recommendations for target
choice and nomenclature
Keith A. Jolley, Carina Brehony & Martin C.J. Maiden
Department of Zoology, University of Oxford, Oxford, UK

39. Antigenic Formula
• Based on -
Serogroup (SiaD gene detection)
Serotype (porB gene sequencing)
Serosubtype (porA gene sequencing)
C:2a:P1.7-2,4
fetA (gene sequencing) eg F1-5
Sequence Type
• B: P1.19,15: F5-1: ST-33 (cc32).
Angelo Zaia NNN 2008

40. Fet A (ferric enterobactin binding
protein)
• Protein expressed in response to iron limitation
• Expressed during invasive infection but clear role in
the bloodstream not defined
• Receptor function in iron acquisition pathways =
attractive vaccine candidate as expression essential
for growth in vivo
• FetA antibodies found in convalescent antisera
• mAbs raised in mice are bactericidal and specific for
the parent isolate

41. FetA Sequence Typing
• Work on FetA done in the context of
vaccine development for SG B MC
• Thompson etal 2003 – 107 MC isolates -
60 fetA alleles encoding 56 protein
sequences
• Variability due to point mutations as well
as horizontal genetic exchange
• Nomenclature established with 6 FetA
variant families identified
• Concluded that FetA unlikely to be a
singular vaccine candidate

42. Urwin etal 2004
• This combination of PorA and FetA
variants would potentially protect against
95 (89%) of the 107 diverse
meningococcal isolates used to develop
MLST

43. Claus et al 2007
(Deletion of the Meningococcal fetA Gene Used for Antigen Sequence
Typing of Invasive and Commensal Isolates from Germany:
Frequencies and Mechanisms)
• fetA negative isolates in Germany identified from
2201 invasive isolates during 2001-2007
• 11 (0.5%) found
• Randomly distributed geographically and no
association with a particular ST observed
• Due to deletions in fetA gene (similar to the
situation with porA)
• 12/821 carrier strains also contained these
deletions

44. Claus et al 2007
• Mutation in fetA is non-lethal
• Iron uptake not affected
• Serum sensitivity mildly enhanced
• The fact that fetA negative strains are
present has implications for vaccine
development
• There is a trend to carrier strains having
fetA deletions at a higher rate

45. fetA typing at WCH 2008
F1-5 8 B
F1-7 1 W135
F3-1 1 B
F3-3 1 B
F5-1 2 B
F5-5 1 B
• 14 isolates tested and
all could be typed
• 5 clinical samples
tested with 2 giving
weak PCR product
only and 3 no product

46. # FetA VR SG ST SST Year Site
NM 638 F1-5 B NT P1.4 2008 CSF
NM 641 F1-5 B NT P1.4 2008 Blood
NM 646 F1-5 B NT P1.4 2008 Blood
NM 648 F1-5 B NT P1.4 2008 Blood
NM 649 F1-5 B NT P1.4 2008 Blood
NM 651 F1-5 B NT P1.4, 1.1 2008 CSF
NM 653 F1-5 B NT NST 2008 Blood
NM 656 F1-5 B NT P1.4 2008 Blood
NM 645 F1-7 W135 NT P1.4 2008 Blood
NM 640 F3-1 B NT NST 2008 Blood
NM 637 F3-3 B 15 NST 2008 Blood
NM 639 F5-1 B 15 NST 2008 Blood
NM 655 F5-1 B NT P1.4 2008 Blood
NM 652 F5-5 B NT P1.4 2008 Blood
fetA summary 2008

47. Conclusions
• fetA typing works well for isolates but we
require some modification to the protocol
for clinical samples
• fetA can help with strain differentiation
• F1-5 most common type in SA
• Await report from EMGM 2009 for
recommendations on standard
nomenclature for Australian isolates

48. Antimicrobial
Susceptibility/Resistance

49. Mechanisms of resistance to β-
Lactams in Nm
• Production of β-Lactamases -rare 5
isolates
• Alteration of the Structure of Penicillin
Binding Proteins (PBP2 particularly) due to
acquisition of DNA from other organisms -
major mechanism
• Alteration in the permeability of cellular
membranes possibly by decreased
expression of class 3 porin

50. Mechanisms of resistance to β-
Lactams in Nm
• Lability of β-lactamase-β-Lactam
interaction determines resistance
• Stability of the high molecular weight
PBP-β-lactam interaction determines
susceptibility
• There is however a range of interactions
and rates of enzyme turnover which
leads to variations of susceptibility

51. Antibiotic Resistance
• Move towards molecular testing for penicillin
susceptibility
• Paper recently published outlining a European
study and methods for testing penA gene
sequences (result of initiative from EMGM
2005 Dublin)
• Standard methods for penA sequencing
• penA sequencing can also be used to
differentiate strains which have the same ST

52. Penicillin Binding Proteins
• Alterations to PBP’s structure (mostly PBP
2) lead to increases in MIC ranging from
0.125-1 mg/L
• Alterations to PBP 1 can lead to higher
level MIC increases
• Accumulations of these plus other PBPs
may lead to further increases in MIC and
treatment failure as in S.pneumoniae and
gonococci

53. Figure 1
Phenotype distribution among the tested meningococcal
isolates with known minimal inhibitory concentration (MIC mg/L)
(n=1644, 98% of total isolates).
Target gene sequencing to characterize penicillin G susceptibility of N.meningitidis TAHA M et al personal communication 2007
Reduced Pen susc

54. Antibiotic Resistance- penA sequencing Project
• Reduced susceptibility = alterations in PBP2 binding due to
mutations in 3’ half of the penA gene
• 402 bp fragment of penA sequenced
• 1670 strains from 60 years and 22 countries tested
• 808 csf; 585 blood (ie 83% were invasive isolates-55%B,
29%C)
• Phenotyping, genotyping and penicillin G MIC tests
performed
• 65% had reduced susc to penicillin
• 139 different penA alleles found (penA1-penA139)
• 38 of these were highly related and clustered to correspond
to susceptible strains
• Remaining 101 were diverse and accounted for 38% of total
isolates

55. Antibiotic Resistance- penA sequencing Project
• No clonal expansion observed
• Sequence of 5 amino acid residues were
always altered in these strains
• Correlation not 100% which suggests other
mechanisms may be involved
• Evidence of mosaic structures through
interspecies recombination were detected
• Data argues for use of penA sequencing to
identify isolates with reduced susc. To
penicillin
• Database now available on web

56. PBP 2
• Alteration of the gene encoding PBP2
(penA) leads to this structural change
• Occurs by horizontal exchange of genetic
material from other organisms eg
N.flavescens via transformation

57. penA translation
← Increasing MIC

58. Aims
• To determine the correlation of penA
polymorphisms and penicillin MIC
• To determine if the polymorphisms are a
result of the interspecies transfer of genetic
material
• To discover if any unique penA alleles
exist within our isolates.

59. Background:
Definitions of Penicillin Susceptibility:
CSLI Guidelines:
Susceptible: MIC ≤ 0.06ug/mL
Intermediate MIC: 0.12-0.25ug/mL
Resistant: ≥0.5ug/mL
Proposed EMGM Guidelines:
Intermediate: MIC 0.06-1ug/mL
Resistant ≥1ug/mL

60. Penicillin Susceptibility in South
Australian Isolates

61. Method:
• Using protocols described by Taha et al
2007.
• All isolates with MIC>0.094 selected for
sequencing.
• 10 isolates with MIC≤0.094 randomly
selected.

62. • 57 isolates retrieved for penA sequecing
• Of these, 23 did not yield the expected
product on initial PCR.
• 34 yielded the expected product and were
then sequenced.
• 23 different alleles were found amongst
these isolates
• 11 of these alleles were unique.
• Excluding allele 1, the most common
alleles were two unique alleles, each found
in 3 isolates.

63. Conclusions:
• A subpopulation of penicillin less-susceptible
isolates have alterations to the transpeptidase
region of the penA gene
• In these isolates, substitutions F504L , A510V
and I515V are always present.
• Additional mutations in amino acid positions 485-
552 often correlate with a higher MIC
(>0.125ug/mL).
• A subpopulation of penicillin less-susceptible
isolates have a more significantly altered gene,
and require further study.

64. Antimicrobial Susceptibility
• E Test (CLSI guidelines) or agar dilution

65. ETest vs Agar Dilution

68. Australian Meningococcal
Surveillance Programme (AMSP
1994- continuing)
(National Neisseria Network)

69. AMSP Requirements
• Serogrouping within 48 hours
• Phenotyping
• Antibiotic susceptibility data
• Genotyping
• Methodology harmonisation

70. Number of laboratory confirmed cases of invasive meningococcal disease,
Australia, 2007, by State or Territory and serogroup.
State/Territo
ry
Serogroup Total
B C A Y W135 NG*
ACT 4 0 1 5
NSW 78 7 5 1 10 101
NT 1 1 2
Qld 30 3 1 1 1 36
SA 11 1 1 1 14
Tas 3 0 1 1 5
Vic 46 2 4 3 4 59
WA 19 0 1 20
Australia 192 14 0 12 8 16 242
* not serogrouped

71. Australian Meningococcal
Surveillance Program : 2007
• Seasonality
Jan 1- Mar 31 16.5 %
April 1 - June 30 18.1%
July 1 - Sept 30 31.9%
Oct 1 - December 31 34.5 %
• M/F Ratio (all) 1.08:1
Mortality 4.2%
(higher for gp C strains)
Tapsall JW CDI 2008

72. AMSP Data :
Antibiotic Susceptibility
• penicillin 76/177 (21%) fully sensitive
101/177 (79%) less sensitive
(MIC 0.06 - 0.5 ug/mL)
• ciprofloxacin (1 isolate 0.6 –slightly raised) Sensitive (0.002ug/mL)
• ceftriaxone Sensitive (<0.002 ug/mL)
• Rifampicin (1 isolate slightly raised) Sensitive (0.004 - 0.38 ug/mL)
• chloramphenicol Sensitive (038 - 1.0 ug/mL)

73. 0
10
20
30
40
50
60
70
80
0-4y 5-14y 15-19 20-24 25-44 45+
B
C
Figure 1 Number of serogroup B and C cases of IMD
confirmed by all methods, by age, Australia, 2007
AMSP Report CDI 2007

74. Anatomical source of samples positive for a laboratory confirmed case of IMD Australia 2007
Specimen type Isolate of MC PCR positive* Total
Blood 93 63 156
CSF +/- Blood 29 45 74
Other+ 5 2 7
Serology alone** 5
Total 127 110 242
*PCR positive in the absence of a positive culture;
**serology positive in the absence of positive culture or PCR.
+ Joint and fluid samples (4 isolates from joints and 2 by PCR of joint fluid;
1 culture from peritoneal fluid)

75. Lab Diagnosis IMD (CSF/Blood)
: Overview
• Cell count
• Gram’s Stain
• Culture
• Antigen Detection
• Serology
• Nucleic Acid Amplification Assays
• RDTs

76. Diagnostic PCR
• Manchester PHLS Ref Lab method
• ctrA gene (WCH 20 cfu/mL)
• siaDB and siaDC gene (WCH 200cfu/mL)
• (porA gene)
• Samples
CSF, whole blood, serum, tissue
• Sensitivity (Poritt 2001) 94% (csf)
• Specificity (Poritt 2001) 100% (csf)

77. Diagnostic PCR
• Manchester PHLS Ref Lab method (in 1998
additional 56% cases confirmed by using NAA)
• ctrA gene (capsular biosynthesis locus – transport
of capsular polysaccharide)
• siaDB and siaDC gene (polymerisation of sialic
acid to polysialic acid chain and regions of the
gene are serogroup specific)
• porA gene (porB gene for typing)

78. Diagnostic meningococcal PCR
ctrA ---
--- IS1106
siaD B/C ---
--- siaD WY

79. Corbett Rotor-Gene 6000

81. Materials & Methods
• Samples - clinical samples (blood/CSF)
- cultures of N.meningitidis
- cultures of other bacteria
• WCH PCR protocol using ctrA primers developed for ELISA
PCR (Manchester UK) with gel detection of PCR product
• Real-time PCR using CtrA primers developed for Taqman
assays
- FAM/ BHQ-1 dual-labelled 20mer probe with ctrA specific
sequence for product detection

82. Organisms other than
N.meningitidis
Staph.aureus Mycoplasma hominis
Strep. agalactiae Moraxella catarrhalis
Strep. pneumoniae Ur. Urealyticum B.
Strep. Pyogenes Burk.cepacia
B.pertussis Legionella pneumophila
Ps.Aeruginosa Haemophilus influenzae
Proteus mirabilis Chlamydia trachomatis
M.pneumoniae Chl. pneumoniae
N.gonorrhea

83. DNA from Bacterial Cultures
Tested by RotorGene
Rotorgene
Pos
Rotorgene
Neg
Total
N.meningo 81 2* 83
Other#
0 18 18
•# Organisms other than N.meningitidis
•*These subsequently identified as N.lactamica

84. Clinical Sample (blood/csf) Tested by
WCH Current Method and Rotorgene
RotorGene
Pos
RotorGene
Neg
WCH PCR
Pos
32 1#
WCH PCR
Neg
2* 68
*36 cycles before positive (total 39 cycles)
# non repeatable ctrA, siaD neg and pos gp B isolate
from pharynx
Total
33
70

85. Conclusions
• rapid test time (approx. 2 hours cf > 4 hours)
• sensitive & specific
• early active management of patient and patient’s
contacts

86. Recent Case
Limb Necrosis
Intracellular diplococci

87. Positive patient

88. Avantages of Molecular
Techniques
• Can perform assays despite treatment
and/or reluctance to lumbar puncture
• No requirement for culture
genotyping possible (porA/B, MLST)
Some resistance genes detectable
• Rapid (diagnostics, typing)
• Sensitive and specific
• Automation

89. Disadvantages
• Unable to perform antimicrobial
susceptibilities
• Specialised equipment and laboratories
• Cost
• Interpretation difficulties
• Trained staff

90. Bibliography-Diagnostics
• Chakrabarti P Indian J Med Res. 2009 Feb;129(2):182-8.
Application of 16S rDNA based seminested PCR for diagnosis of acute bacterial meningitis
• The overall sensitivity, specificity, positive
predictive value and negative predictive value of
16S rDNA PCR were 79.24, 97.6, 89.36 and
94.88 per cent respectively when culture was
considered as gold standard. The detection limit
of 16S rDNA PCR was determined to be 1000
cfu/ml of E. coli and 4000 cfu/ml of S.
pneumoniae

91. Loop-mediated isothermal amplification (LAMP)
of gene sequences and simple visual detection of
products
Norihiro Tomita, Yasuyoshi Mori, Hidetoshi Kanda & Tsugunori Notomi
• Loop-mediated isothermal amplification
(LAMP) is a simple, rapid, specific and
cost-effective nucleic acid amplification
method when compared to PCR, nucleic
acid sequence-based amplification, self-
sustained sequence replication and strand
displacement amplification. This protocol
details an improved simple visual
detection system for the results of the
LAMP reaction.

92. Boving J Clin Microbiol. 2009 Apr;47(4):908-13.
Eight-plex PCR and liquid-array detection of bacterial and viral pathogens in
cerebrospinal fluid from patients with suspected meningitis
• Eight-plex PCR and liquid-array detection
of bacterial and viral pathogens in
cerebrospinal fluid from patients with
suspected meningitis.
• Luminex 100 suspension array system
• N. meningitidis (sens 100% and spec
99.7%)

93. Sampling methods to detect carriage of Neisseria meningitidis;
literature review.
J Infect. 2009 Feb;58(2):103-7 Roberts J, Greenwood B, Stuart J
• The evidence to date suggests that
meningococcal carriage should be
assessed by swabbing the posterior
pharyngeal wall through the mouth, and
that swabs should be plated directly on
site or placed in transport medium for <5h.

94. Application of atmospheric pressure matrix-assisted laser
desorption/ionization mass spectrometry for rapid identification of
Neisseria species.
J Biomol Tech. 2008 Jul;19(3):200-4 Gudlavalleti SK, et al
• five serogroups (A, B, C, W135, and Y) of
Neisseria meningitidis subjected to on
probe/peptide extraction and tryptic digestion
followed by AP-MALDI-Tandem MS
• Amino acid sequences derived from three
protonated peptides were used to probe a
database yielding 3 of neisserial proteins which
are potential biomarkers for neisserial species
identification

95. Simultaneous detection of Haemophilus influenzae type b
polysaccharide-specific antibodies and Neisseria meningitidis serogroup
A, C, Y, and W-135 polysaccharide-specific antibodies in a fluorescent-
bead-based multiplex immunoassay.
Clin Vaccine Immunol. 2009 Mar;16(3):433-6
de Voer RM, Schepp RM, Versteegh FG, van der Klis FR, Berbers GA.
• meningococcal serogroup A, C, Y, and W-
135 multiplex immunoassay (MIA)
• Used for serological analysis for childhood
vaccine studies

96. Trop Med Int Health. 2009 Jan;14(1):111-7. Epub 2008 Nov 12.
Field evaluation of rapid diagnostic tests for meningococcal
meningitis in Niger.
Boisier P, Mahamane AE, Hamidou AA, Sidikou F, Djibo S, Nato F,
Chanteau S.
• Rapid Diagnostic Tests (RDTs) used for detecting antigen to NM in
CSF in Niger, Africa
• Using RDTs, health facilities reported 382 negative results (73.9%),
114 NmA (22.1%), 12 NmW135 (2.3%)
• CONCLUSION: We confirmed that dipstick RDTs to identify N.
meningitidis serogroups A, C, W135 and Y can be reliably operated
by non-specialized staff in basic health facilities. RDTs proved very
useful to recommend vaccination in NmA epidemics, and also to
avoid vaccination in epidemics due to serogroups not included in
vaccines (NmX).

97. FEMS Immunol Med Microbiol. 2008 Jul;53(2):178-82.
Simultaneous single-tube PCR-based assay for the direct identification of
the five most common meningococcal serogroups from clinical samples.
Drakopoulou Z et al
• Neisseria meningitidis serogroups (A, B,
C, W-135 and Y) in 530 clinical samples
obtained from 428 patients (271 blood and
259 cerebrospinal fluid). The sensitivity
and the specificity was calculated to 100%
[positive predictive value 100% (95%, CI
99.0-100%) and negative predictive value
100% (95% CI 99.0-100%)].

98. Arch Dermatol. 2008 Jun;144(6):770-3.
.
Value of a novel Neisseria meningitidis--specific polymerase chain
reaction assay in skin biopsy specimens as a diagnostic tool in chronic
meningococcemia.
Parmentier L, et al
• In 2 patients with CM, we established the
diagnosis by a newly developed PCR-
based approach performed on skin biopsy
specimens

99. Acknowledments
• Prof John Tapsall and his laboratory at
POW Hospital
• NNN Laboratories
• Kathryn Whetter
• Stuart McKessar and WCH Laboratory
• SA Pathology RAH Site Molecular
Pathology Unit