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

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

MENINGITIS IN THE AFRICAN BELT

Major cause of meningitis in the African Belt is Neisseria meningitidis serogroup A (N.m.A)

..\Documents\CVD-Mali\MRF project\meetings\MRF visit\BulletinMeningite2012_S31_35.pdf

1996

188,345

1978

80,743

1989

88,9392001

68,089

(WHO Epidemiological Review 2007)

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

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

VISIT IN A PARTICIPANT FAMILY

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

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

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

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

REAL TIME PCR OPTIMISATION

o In 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

AKNOWLEDGEMENTo MRF

o MenAfriCar: Dr Olivier Manigart and all the team

o CVD-Mali: Prof Samba Sow, Dr Tamboura, and all the team

o London School of Hygiene and Tropical Medicine (Pr Brian Greenwood)

o CDC- Atlanta: Dr Xin Wang

o Oxford University Zoology department: Pr Maiden, Dr Odile Harisson

o VEU, Health Protection Agency, Manchester: Pr Borrow