1. DEVELOPMENT OF A MOLECULAR BIOLOGY TOOL
BASED ALGORITHM TO IMPROVE THE DIAGNOSIS OF
MENINGITIS IN THE AFRICAN MENINGITIS BELT
Kanny Diallo
M.Sc. Biochemistry
CVD-Mali

2. MENINGITIS IN THE
AFRICAN BELT
• Meningitis is an inflammation of the meninges that affects the brain
membranes:
- causes: bacteria, viruses or other micro-organisms
- consequences: life threatening damages, complicated sequelaes
•In the African belt:
-Recurrent epidemic
-2009 epidemic season: 14 African countries  enhanced surveillance
 88,199 suspected cases, 5,352 deaths, Largest number since 1996
epidemic
• Importance of rapid and cost efficient Diagnosis and Intervention tool
Source: Control of epidemic meningococcal disease, WHO
practical guidelines, World Health Organization, 1998, 2nd
edition, WHO/EMC/BAC/98.3

3. MENINGITIS IN THE AFRICAN BELT
 Major cause of meningitis in the African Belt is Neisseria meningitidis
serogroup A (N.m.A)
 ..DocumentsCVD-MaliMRF projectmeetingsMRF
visitBulletinMeningite2012_S31_35.pdf 1996
188,345
1978
80,743
1989
88,939
2001
68,089
(WHO Epidemiological Review 2007)

4. MENAFRIVAC
 Neisseria meningitidis is a bacteria that is commensal to the internal
flora of the throat
- healthy carriage is the most common stage
- there are 12 capsulated serogroups (A, B, C, W, Y,E,H, Z, I K, L X)
 Polysaccharide vaccines have been previously developed
- short lasting immunity
- not immunogenic in kids and infant
 In December 2010, a new conjugate vaccine was introduced in the
meningitis belt, against N.m.A:
- supposed to elicit long term immunity
- can be used on children
- very low cost
- claim to be able to eliminate carriage of N.m.A

5. MENAFRICAR
 MenAfriCar is a global research effort to determinate the level of
meningococcal carriage in 7 countries of the Meningitis African Belt and to
evaluate the impact of the MenAfriVac vaccine on carriage and immunity.
 Traditional microbiology and biochemistry methods have been used to
identify the different strains of N.m in a cohort of participants in all selected
countries
 Immunologic status of ~ 1000 participants will be evaluated before
vaccination by MenAfriVac in selected countries using ELISA and SBA
techniques
 Molecular biology was introduced in all participating centres via a simple
traditional PCR to confirm the serogroups of N.m suspected strains

6. VISIT IN A PARTICIPANT FAMILY

7. TRADITIONAL MICROBIOLOGY
• Neisseria meningitidis: Oxidase positive, Gram negative, diploccocus,
• GGT positive, ONPG negative, tributyrin negative
• Sero-agglutination
• minimum 3 days of testing
• Not always efficient (cross-reaction, subjectivity of visual results…)
• BETTER DIAGNOSIS TOOLS ARE NEEDED

8. MENINGITIS RESEARCH FOUNDATION GRANT
 “Development of an African adapted PCR algorithm
to characterize non-groupable isolates of Neisseria
meningitidis”
 Written by Dr Olivier Manigart in 2009 with help of
Prof Samba Sow, Prof Martin Maiden and Prof Ray
Borrow
 Accepted in 2010 but started only in 2012 due to
difficulty to find good candidate to work both on this
project and MenAfriCar

9. MOLECULAR BIOLOGY
• most of the N.m genome
sequences are available
• Using the species and
serogroups specific genes
allows direct targeting of the
bacteria
• PCR allows amplification of the
bacterial DNA using specific
primers and luminescent
probes
• Can be done in 1 to 2 days
depending on DNA extraction
techniques
• Requires good GCLP practices
to avoid contaminations

10. REAL TIME PCR
• Similar to gel base PCR, but risks of contaminations are reduces (closed system)
• Semi-automated, we use the ABI 7500 fast machine, that allow us to visualise
amplification on a computer screen
• Fast and accurate: our program run for 2 hours
• Primers and probes designs and optimisation of the technique

11. REAL TIME PCR OPTIMISATION
oIn this paper they used real time PCR to differentiate between
N.meningitidis, S. pneumoniae and H. influenzae
o They have designed primers specific for N.m but also for
serogroups A, W, X, Y, B, C

12. AKNOWLEDGEMENT
o MRF
oMenAfriCar: Dr Olivier
Manigart and all the team
oCVD-Mali: Prof Samba Sow,
Dr Tamboura, and all the team
oLondon School of Hygiene
and Tropical Medicine (Pr
Brian Greenwood)
oCDC- Atlanta: Dr Xin Wang
oOxford University Zoology
department: Pr Maiden, Dr
Odile Harisson
oVEU, Health Protection
Agency, Manchester: Pr
Borrow