Molecular Analysis of MSP1 and MSP2
Genotypes of Plasmodium falciparum
Obtained from Adults Participating in the P.
Falciparum Malaria Protein 10 (FMP10)
Vaccine Trial in Kombewa Division,
Western Kenya.
Richard Forde.
M.Sc. Immunology and Global
Health
Overview of Presentation:
• Background – Life Cycle / Genetic Diversity /
Multiplicity of Infection/ Capillary Electrophoresis
• Objectives
• Results:
1. Plasmodium falciparum genotype diversity
2.Temporal Distribution of P. falciparum Genotypes
• Conclusions
• Questions
Background: Malaria Burden
• 300 million clinical cases reported every year
- African Region (85%)
• Annual death toll of 0.7 – 2.7 million
• Majority = Children. Each 30 seconds, a child
dies from malaria
Plasmodium Falciparum
• Most common - 2.2 billion people worldwide
• Most pathogenic - kills over 1 million children
annually in sub - Saharan Africa alone
P. falciparum: Life Cycle
• Bite of an infected
Anopheles female
mosquito.
• Sporozoites invade
hepatocytes = the preerythrocytic stage (Liver)
• Asexual multiplication
• Merozoites rupture from
the infected hepatocytes
• Infect the red blood cells
= erythrocyitic stage
• Associated with the
clinical manifestation of
the disease due to
rosetting and
cytoadherance.
P. Falciparum : Genetic Diversity
• The P. falciparum genome sequence is
comprised of 5,268 predicted protein-encoding
genes - 60% of which have not been assigned
function
• Extensive polymorphism - particularly those
expressed on the parasite’s surface
• This study - polymorphism that exists within
merozoite surface proteins (MSP) 1 and 2
• Markers to determine the genotype of the
parasite and to describe the dynamics of
parasite infection.
MSP 1+2 – Genetic Diversity
MSP1 allelic variants fall under three major types
– K1, MAD20 and RO33
MSP2 allelic variants fall under two dimorphic
allelic families, designated FC27 and IC3D7
Multiplicity of Infection (MOI)
• The extensive genetic diversity displayed by P.
falciparum results in complex infections
consisting of multiple parasite clones
• Endemic areas - infected individuals can harbor
several different parasite clones with distinct
genotypes
• Distinguishing between parasitic clones based
on their genotype allows for the calculation of
Multiplicity of Infection (MOI).
Traditionally…
• Discrimination of P. falciparum genotypes was
performed using agarose-gel electrophoresis to
make size comparisons of the amplified
products of these genes.
• ease of use and low cost
However,
• provides inadequate discrimination of alleles.
• subjective results may lead to a number of
genotyping misclassifications.
Capillary Electrophoresis
• Capillary Electrophoresis (CE) = high-resolution
genotyping
• Allows for the recognition of changes between allele
types down to 1bp in size.
Studies show:
CE detected more MSP 1 and 2 alleles with more precise sizing
compared to gel electrophoresis (Liljander et al 2009)
Direct comparison of gel and capillary electrophoresis for two anti malarial
drug trials showed CE to detect more alleles and provide higher
discriminatory power than gel elctrophoresis (Gupta et al 2010)
Objectives:
1.To genotype P. falciparum obtained from
volunteers participating in the FMP10 vaccine
trial through Capillary Electrophoresis analysis
of the MSP1 and MSP2 allelic variants.
1.To analyse the temporal changes in MSP1 and
MSP2 allelic frequency and MOI during the
course of the FMP10 vaccine trial.
Study Cohort: FMP-10 Vaccine Trial Volunteers
1. P. falciparum genotype diversity
• A total of 285 MSP1
and MSP2 alleles
were detected.
•
•
•
•
•
76 (26.7%) K1
36 (12.6%) MAD 20
33 (11.5%) R033
50 (17.5%) FC27
90 (31.7%) IC3D7.
2. Temporal
Distribution of
P. falciparum
genotypes and MOI
Conclusions:
• Our findings corroborate previous studies that
show that CE is a highly sensitive technique that
allows for the precise sizing and discrimination
of MSP1 and MSP2 alleles
• The observed temporal changes of P. falciparum
infection dynamics were susceptible to the
effects of season and the clearance of parasitic
infections through anti-malarial treatment.
Future Research:
• Further assessment of this data comparing
vaccinated and non-vaccinated populations is
suggested, which would allow an exploration of
the workings of the FMP10 vaccine at the
molecular level.
• It is recommended that further genotyping of
volunteers associated with clinical malaria be
undertaken.
Acknowledgements:
• Dr. Noel Murphy
• Combat Diseases of Poverty Consortium
• Dr. John Waitumbi and all KEMRI staff in
Kisumu
Questions????