Person:

Valim, Clarissa

Background: Cerebral malaria, a severe form of Plasmodium falciparum infection, is an important cause of mortality in sub-Saharan African children. A Taqman 24 Single Nucleotide Polymorphisms (SNP) molecular barcode assay was developed for use in laboratory parasites which estimates genotype number and identifies the predominant genotype. Methods The 24 SNP assay was used to determine predominant genotypes in blood and tissues from autopsy and clinical patients with cerebral malaria. Results: Single genotypes were shared between the peripheral blood, the brain, and other tissues of cerebral malaria patients, while malaria-infected patients who died of non-malarial causes had mixed genetic signatures in tissues examined. Children with retinopathy-positive cerebral malaria had significantly less complex infections than those without retinopathy (OR = 3.7, 95% CI [1.51-9.10]).The complexity of infections significantly decreased over the malaria season in retinopathy-positive patients compared to retinopathy-negative patients. Conclusions: Cerebral malaria patients harbour a single or small set of predominant parasites; patients with incidental parasitaemia sustain infections involving diverse genotypes. Limited diversity in the peripheral blood of cerebral malaria patients and correlation with tissues supports peripheral blood samples as appropriate for genome-wide association studies of parasite determinants of pathogenicity.

Molecular interactions between male and female factors during mating profoundly affect the reproductive behavior and physiology of female insects. In natural populations of the malaria mosquito Anopheles gambiae, blood-fed females direct nutritional resources towards oogenesis only when inseminated. Here we show that the mating-dependent pathway of egg development in these mosquitoes is regulated by the interaction between the steroid hormone 20-hydroxy-ecdysone (20E) transferred by males during copulation and a female Mating-Induced Stimulator of Oogenesis (MISO) protein. RNAi silencing of MISO abolishes the increase in oogenesis caused by mating in blood-fed females, causes a delay in oocyte development, and impairs the function of male-transferred 20E. Co-immunoprecipitation experiments show that MISO and 20E interact in the female reproductive tract. Moreover MISO expression after mating is induced by 20E via the Ecdysone Receptor, demonstrating a close cooperation between the two factors. Male-transferred 20E therefore acts as a mating signal that females translate into an increased investment in egg development via a MISO-dependent pathway. The identification of this male–female reproductive interaction offers novel opportunities for the control of mosquito populations that transmit malaria.

Malaria surveillance and interventions in endemic countries often target young children at highest risk of malaria morbidity and mortality. We aimed to determine whether school-age children and adults not captured in surveillance serve as a reservoir for malaria infection and may contribute to malaria transmission. Cross-sectional surveys were conducted in one rainy and one dry season in southern Malawi. Demographic and health information was collected for all household members. Blood samples were obtained for microscopic and PCR identification of Plasmodium falciparum. Among 5796 individuals aged greater than six months, PCR prevalence of malaria infection was 5%, 10%, and 20% in dry, and 9%, 15%, and 32% in rainy seasons in Blantyre, Thyolo, and Chikhwawa, respectively. Over 88% of those infected were asymptomatic. Participants aged 6–15 years were at higher risk of infection (OR=4.8; 95%CI, 4.0–5.8) and asymptomatic infection (OR=4.2; 95%CI, 2.7–6.6) than younger children in all settings. School-age children used bednets less frequently than other age groups. Compared to young children, school-age children were brought less often for treatment and more often to unreliable treatment sources. Conclusion: School-age children represent an underappreciated reservoir of malaria infection and have less exposure to antimalarial interventions. Malaria control and elimination strategies may need to expand to include this age group.

Background: Malaria treatment efforts are hindered by the rapid emergence and spread of drug resistant parasites. Simple assays to monitor parasite drug response in direct patient samples (ex vivo) can detect drug resistance before it becomes clinically apparent, and can inform changes in treatment policy to prevent the spread of resistance. Methods: Parasite drug responses to amodiaquine, artemisinin, chloroquine and mefloquine were tested in approximately 400 Plasmodium falciparum malaria infections in Thiès, Senegal between 2008 and 2011 using a DAPI-based ex vivo drug resistance assay. Drug resistance-associated mutations were also genotyped in pfcrt and pfmdr1. Results: Parasite drug responses changed between 2008 and 2011, as parasites became less sensitive to amodiaquine, artemisinin and chloroquine over time. The prevalence of known resistance-associated mutations also changed over time. Decreased amodiaquine sensitivity was associated with sustained, highly prevalent mutations in pfcrt, and one mutation in pfmdr1 – Y184F – was associated with decreased parasite sensitivity to artemisinin. Conclusions: Directly measuring ex vivo parasite drug response and resistance mutation genotyping over time are useful tools for monitoring parasite drug responses in field samples. Furthermore, these data suggest that the use of amodiaquine and artemisinin derivatives in combination therapies is selecting for increased drug tolerance within this population.

Background: One of the main causes of mortality from severe malaria in Plasmodium falciparum infections is cerebral malaria (CM). An important host genetic component determines the susceptibility of an individual to develop CM or to clear the infection and become semi-immune. As such, the identification of genetic loci associated with susceptibility or resistance may serve to modulate disease severity. Methodology The Plasmodium berghei mouse model for experimental cerebral malaria (ECM) reproduces several disease symptoms seen in human CM, and two different phenotypes, a susceptible (FVB/NJ) and a resistant mouse strain (DBA/2J), were examined. Results: FVB/NJ mice died from infection within ten days, whereas DBA/2J mice showed a gender bias: males survived on average nineteen days and females either died early with signs of ECM or survived for up to three weeks. A comparison of brain pathology between FVB/NJ and DBA/2J showed no major differences with regard to brain haemorrhages or the number of parasites and CD3+ cells in the microvasculature. However, significant differences were found in the peripheral blood of infected mice: For example resistant DBA/2J mice had significantly higher numbers of circulating basophils than did FVB/NJ mice on day seven. Analysis of the F2 offspring from a cross of DBA/2J and FVB/NJ mice mapped the genetic locus of the underlying survival trait to chromosome 9 with a Lod score of 4.9. This locus overlaps with two previously identified resistance loci (char1 and pymr) from a blood stage malaria model. Conclusions: Survival best distinguishes malaria infections between FVB/NJ and DBA/2J mice. The importance of char1 and pymr on chromosome 9 in malaria resistance to P. berghei was confirmed. In addition there was an association of basophil numbers with survival.

Background: The sequestration of Plasmodium falciparum-infected erythrocytes in brain microvasculature through cytoadherence to endothelium, is the hallmark of the definitive diagnosis of cerebral malaria and plays a critical role in malaria pathogenesis. The complex pathophysiology, which leads each patient to the final outcome of cerebral malaria, is multifaceted and thus, metrics to delineate specific patterns within cerebral malaria are needed to further parse patients. Methods: A method was developed for quantification utilizing counts of capillary contents (early-stage parasites, late-stage parasites and fibrin) from histological preparations of brain tissue after death, and compared it to the standard approach, in which the percentage of parasitized vessels in cross-section is determined. Results: Within the initial cohort of 50 patients, two different observers agreed closely on the percentage of vessels parasitized, pigmented parasites and pigment globules (ICC = 0.795-0.970). Correlations between observers for correct diagnostic classification were high (Kendall’s tau-b = 0.8779, Kappa = 0.8413). When these methods were applied prospectively to a second set of 50 autopsy samples, they revealed a heterogeneous distribution of sequestered parasites in the brain with pigmented parasites and pigment globules present in the cerebellum > cortex > brainstem. There was no difference in the distribution of early stages of parasites or in the percentage of vessels parasitized across the same sites. The second cohort of cases was also used to test a previously published classification and regression tree (CART) analysis; the quantitative data alone were able to accurately classify and distinguish cerebral malaria from non-cerebral malaria. Classification errors occurred within a subclassification of cerebral malaria (CM1 vs CM2). A repeat CART analysis for the second cohort generated slightly different classification rules with more accurate subclassification, although misclassification still occurred. Conclusions: The traditional measure of parasite sequestration in falciparum malaria, the percentage of vessels parasitized, is the most reliable and consistent for the general diagnosis of cerebral malaria. Methods that involve quantitative measures of different life cycle stages are useful for distinguishing patterns within the cerebral malaria population; these subclassifications may be important for studies of disease pathogenesis and ancillary treatment.

Plasmodium falciparum infected erythrocytes (IE) accumulate in the placenta through the interaction between Duffy-binding like (DBL) domains of parasite-encoded ligand VAR2CSA and chondroitin sulphate-A (CSA) receptor. Polymorphisms in these domains, including DBL2X and DBL3X, may affect their antigenicity or CSA-binding affinity, eventually increasing parasitemia and its adverse effects on pregnancy outcomes. A total of 373 DBL2X and 328 DBL3X sequences were obtained from transcripts of 20 placental isolates infecting Mozambican women, resulting in 176 DBL2X and 191 DBL3X unique sequences at the protein level. Sequence alignments were divided in segments containing combinations of correlated polymorphisms and the association of segment sequences with placental parasite density was tested using Bonferroni corrected regression models, taking into consideration the weight of each sequence in the infection. Three DBL2X and three DBL3X segments contained signatures of high parasite density (P<0.003) that were highly prevalent in the parasite population (49–91%). Identified regions included a flexible loop that contributes to DBL3X-CSA interaction and two DBL3X motifs with evidence of positive natural selection. Limited antibody responses against signatures of high parasite density among malaria-exposed pregnant women could not explain the increased placental parasitemia. These results suggest that a higher binding efficiency to CSA rather than reduced antigenicity might provide a biological advantage to parasites with high parasite density signatures in VAR2CSA. Sequences contributing to high parasitemia may be critical for the functional characterization of VAR2CSA and the development of tools against placental malaria.

Through rapid genetic adaptation and natural selection, the Plasmodium falciparum parasite—the deadliest of those that cause malaria—is able to develop resistance to antimalarial drugs, thwarting present efforts to control it. Genome-wide association studies (GWAS) provide a critical hypothesis-generating tool for understanding how this occurs. However, in P. falciparum, the limited amount of linkage disequilibrium hinders the power of traditional array-based GWAS. Here, we demonstrate the feasibility and power improvements gained by using whole-genome sequencing for association studies. We analyzed data from 45 Senegalese parasites and identified genetic changes associated with the parasites’ in vitro response to 12 different antimalarials. To further increase statistical power, we adapted a common test for natural selection, XP-EHH (cross-population extended haplotype homozygosity), and used it to identify genomic regions associated with resistance to drugs. Using this sequence-based approach and the combination of association and selection-based tests, we detected several loci associated with drug resistance. These loci included the previously known signals at pfcrt, dhfr, and pfmdr1, as well as many genes not previously implicated in drug-resistance roles, including genes in the ubiquitination pathway. Based on the success of the analysis presented in this study, and on the demonstrated shortcomings of array-based approaches, we argue for a complete transition to sequence-based GWAS for small, low linkage-disequilibrium genomes like that of P. falciparum.

BACKGROUND: Case fatality rates among African children with cerebral malaria remain in the range of 15 to 25%. The key pathogenetic processes and causes of death are unknown, but a combination of clinical observations and pathological findings suggests that increased brain volume leading to raised intracranial pressure may play a role. Magnetic resonance imaging (MRI) became available in Malawi in 2009, and we used it to investigate the role of brain swelling in the pathogenesis of fatal cerebral malaria in African children. METHODS: We enrolled children who met a stringent definition of cerebral malaria (one that included the presence of retinopathy), characterized them in detail clinically, and obtained MRI scans on admission and daily thereafter while coma persisted. RESULTS: Of 348 children admitted with cerebral malaria (as defined by the World Health Organization), 168 met the inclusion criteria, underwent all investigations, and were included in the analysis. A total of 25 children (15%) died, 21 of whom (84%) had evidence of severe brain swelling on MRI at admission. In contrast, evidence of severe brain swelling was seen on MRI in 39 of 143 survivors (27%). Serial MRI scans showed evidence of decreasing brain volume in the survivors who had had brain swelling initially. CONCLUSIONS: Increased brain volume was seen in children who died from cerebral malaria but was uncommon in those who did not die from the disease, a finding that suggests that raised intracranial pressure may contribute to a fatal outcome. The natural history indicates that increased intracranial pressure is transient in survivors.

Purpose: Adverse drug events (ADEs) are a common complication of medical care resulting in high morbidity and medical expenditure. Population level estimates of outpatient ADEs are limited. Our objective was to provide national estimates and characterizations of outpatient ADEs and determine risk factors associated with these events. Methods: Data are from the National Center for Health Statistics which collects information on patient visits to outpatient clinics and emergency departments throughout the United States. We examined visits between 1995 and 2005 and measured the national annual estimates of and risk factors for outpatient ADEs requiring medical treatment. Results: The national annual number of ADE-related visits was 4,335,990 (95%CI, 4,326,872–4,345,108). Visits for ADEs to outpatient clinics increased over the study period from 9.0 to 17.0 per 1000 persons (P value for trend<0.001). In multivariate analyses, factors associated with ADE visits included patient age (OR 2.13; 95%CI 1.63–2.79 for 65 years and older), number of medications taken by patient (OR, 1.88; 95%CI, 1.58–2.25 for five medications or more), and female gender (OR, 1.51; 95%CI, 1.34–1.71). Overall, outpatient ADEs resulted in 107,468 (95%CI, 89,011–125,925) hospital admissions annually, with older patients at highest risk for hospitalization (P value for trend<0.001). Conclusions: Both patient age and polypharmacy use are risk factors for ADE-related healthcare visits, which have substantially increased in outpatient clinics between 1995 and 2005. The incidence of ADEs has particularly increased among patients 65 years and older with as many as 1 in 20 persons seeking medical care for an ADE.