Long-lived Plasmodium falciparum specific memory B cells in naturally exposed Swedish travelers.

Antibodies (Abs) are critical for immunity to malaria. However, Plasmodium falciparum specific Abs decline rapidly in absence of reinfection, suggesting impaired immunological memory. This study determines whether residents of Sweden that were treated for malaria following international travel maintained long-lasting malaria-specific Abs and memory B cells (MBCs). We compared levels of malaria-specific Abs and MBCs between 47 travelers who had been admitted with malaria at the Karolinska University Hospital between 1 and 16 years previously, eight malaria-naïve adult Swedes without histories of travel, and 14 malaria-immune adult Kenyans. Plasmodium falciparum-lysate-specific Ab levels were above naïve control levels in 30% of the travelers, whereas AMA-1, merozoite surface protein-142 , and merozoite surface protein-3-specific Ab levels were similar. In contrast, 78% of travelers had IgG-MBCs specific for at least one malaria antigen (59, 45, and 28% for apical merozoite antigen-1, merozoite surface protein-1, and merozoite surface protein-3, respectively) suggesting that malaria-specific MBCs are maintained for longer than the cognate serum Abs in the absence of re-exposure to parasites. Five travelers maintained malaria antigen-specific MBC responses for up to 16 years since the diagnosis of the index episode (and had not traveled to malaria-endemic regions in the intervening time). Thus P. falciparum can induce long-lasting MBCs, maintained for up to 16 years without reexposure.

Genetic diversity of Plasmodium falciparum infections in mild and severe malaria of children from Kampala, Uganda.

Diversity in parasite virulence is one of the factors that contribute to the clinical outcome of malaria infections. The association between the severity of Plasmodium falciparum malaria and the number of distinct parasite populations infecting the host (multiplicity of infection) or polymorphism within any of the specific antigen genes was investigated. The study included 164 children presenting with mild and severe malaria from central Uganda where malaria is meso-endemic. The polymorphic regions of the circumsporozoite protein (csp), merozoite surface proteins 1 and 2 (msp1 and msp2), and glutamate-rich protein (glurp) were genotyped by polymerase chain reaction methods and fragment analysis by gel electrophoresis. In a subset of samples fragment analysis was also performed by fluorescent PCR genotyping followed by capillary electrophoresis. The multiplicity of infection (MOI), determined as the highest number of alleles detected within any of the four genetic loci, was significantly higher in severe than in mild malaria cases (mean 3.7 and 3.0, respectively, P=0.002). No particular genotype or allelic family of msp1 or msp2 was associated with severity of malaria, and nor did the genotyping method reveal any significant difference in MOI when only assessed by msp2 genotyping. Severity of malaria was not linked to the predominance of any particular msp1 or msp2 allelic types, independent of methods used for genotyping. Monitoring the dynamics of multiple clone infections in relation to disease outcome, host immune status and genetic factors will provide more insight into parasite virulence mechanisms.

Plasmodium falciparum infection patterns since birth and risk of severe malaria: a nested case-control study in children on the coast of Kenya.

Children in malaria endemic areas acquire immunity to severe malaria faster than to mild malaria. Only a minority of children suffers from severe malaria and it is not known what determines this. The aim of this study was to establish how P. falciparum infections during the first years of life affect the risk of severe malaria. A matched case-control study was nested within a large birth cohort set up to study the immunoepidemiology of pneumococci on the Kenyan coast. Infection patterns in three-monthly blood samples in cohort children admitted to hospital with severe malaria were compared to controls matched on age, residential location and time of sampling. P. falciparum detected at least once from birth conferred an increased risk of severe malaria and particularly if multiclonal infections, as characterized by genotyping of a polymorphic antigen gene, were ever detected. The results show for the first time that children with severe malaria have more infections early in life compared to community controls. These findings provide important insights on the immunity to severe disease, knowledge essential for the development of a vaccine against severe malaria.

High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA.

BACKGROUND: Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. METHODS: High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/µl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. RESULTS: High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. CONCLUSIONS: High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots.