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

<p>Abstract</p> <p>Background</p> <p>Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of <it>P. falciparum </it>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.</p> <p>Conclusions</p> <p>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.</p

Cord blood IgG and the risk of severe Plasmodium falciparum malaria in the first year of life

Young infants are less susceptible to severe episodes of malaria but the targets and mechanisms of protection are not clear. Cord blood antibodies may play an important role in mediating protection but many studies have examined their association with the outcome of infection or non-severe malaria. Here, we investigated whether cord blood IgG to Plasmodium falciparum merozoite antigens and antibody-mediated effector functions were associated with reduced odds of developing severe malaria at different time points during the first year of life. We conducted a case-control study of well-defined severe falciparum malaria nested within a longitudinal birth cohort of Kenyan children. We measured cord blood total IgG levels against five recombinant merozoite antigens and antibody function in the growth inhibition activity and neutrophil antibody-dependent respiratory burst assays. We also assessed the decay of maternal antibodies during the first 6months of life. The mean antibody half-life range was 2.51months (95% confidence interval (CI): 2.19-2.92) to 4.91months (95% CI: 4.47-6.07). The rate of decline of maternal antibodies was inversely proportional to the starting concentration. The functional assay of antibody-dependent respiratory burst activity predicted significantly reduced odds of developing severe malaria during the first 6months of life (Odds ratio (OR) 0.07, 95% CI: 0.007-0.74, P=0.007). Identification of the targets of antibodies mediating antibody-dependent respiratory burst activity could contribute to the development of malaria vaccines that protect against severe episodes of malaria in early infancy

Assessment of exposure in three-monthly visits from birth until admission^a in 61 children admitted with severe malaria.

<p>a. samples until 2 years of age at most.</p><p>b. cases with impaired consciousness with or without other syndromes.</p><p>c. including respiratory distress and severe malaria anemia and not impaired consciousness.</p><p>d. ever detected in all samples.</p><p>e. total number of clones detected in an individual including all visits.</p><p>f. exposed (parasite positive and/or antibodies to schizont extract at least once) n (%).</p><p>g. not exposed as determined by PCR and antibodies to schizont extract.</p

Matched case-control study profile.

<p>Matched case-control study profile.</p

Characteristics of the study population.

<p>a. age at case admission of the cases and respective controls.</p><p>b. restricted to the 42 risk sets where case was younger than 2 years and 3 months at the severe malaria admission and thus had complete follow up periods before admission,</p><p>c. including PCR and microscopy results.</p><p>d. antibodies to schizont extract detected in at least one of the three most recent visits before admission in the cases and corresponding time in the controls.</p

Risk of impaired consciousness in relation to parasite exposure in three-monthly samples among 61 cases with severe malaria.

<p>Unmatched case-control analysis performed with exact logistic regression adjusted for age and number of samples, defining patients with impaired consciousness as “cases” (n = 32) and those with non-cerebral severe malaria as “controls” (n = 29).</p><p>a. including PCR and/or microscopy results.</p><p>b. including only PCR results.</p><p>c. antibodies to schizont extract detected in at least one of the three most recent visits before admission in the cases and corresponding time in the controls.</p

Syndromes overlap and mortality among the 61 cohort children admitted with severe malaria to Kilifi District Hospital.

<p>Four children died (marked *).</p

Risk of severe malaria associated with parasite positivity, number of clones and antibodies to schizont extract in three-monthly visits before admission.

<p>Conditional logistic regression adjusted for log number of visits including 42 risk sets where the case was no older than 2 years and 3 months at the time of severe malaria episode and thus had samples until three months before the admission^a.</p><p>a. excluding 13 risk sets with children with longer time since their last visit (4–47 months).</p><p>b. adjusted for log number of visits in the period before the severe admission of the cases and respective periods in the controls.</p><p>c. including PCR and microscopy results.</p><p>d. including only risk sets where PCR results were available (n = 41).</p><p>e. antibodies to schizont extract detected in at least one of the three most recent visits before admission in the cases and corresponding time in the controls, including 40 risk sets where samples for antibody analysis were available.</p

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