Developing a novel risk prediction model for severe malarial anemia.

As a pilot study to investigate whether personalized medicine approaches could have value for the reduction of malaria-related mortality in young children, we evaluated questionnaire and biomarker data collected from the Mother Offspring Malaria Study Project birth cohort (Muheza, Tanzania, 2002-2006) at the time of delivery as potential prognostic markers for pediatric severe malarial anemia. Severe malarial anemia, defined here as a Plasmodium falciparum infection accompanied by hemoglobin levels below 50 g/L, is a key manifestation of life-threatening malaria in high transmission regions. For this study sample, a prediction model incorporating cord blood levels of interleukin-1ß provided the strongest discrimination of severe malarial anemia risk with a C-index of 0.77 (95% CI 0.70-0.84), whereas a pragmatic model based on sex, gravidity, transmission season at delivery, and bed net possession yielded a more modest C-index of 0.63 (95% CI 0.54-0.71). Although additional studies, ideally incorporating larger sample sizes and higher event per predictor ratios, are needed to externally validate these prediction models, the findings provide proof of concept that risk score-based screening programs could be developed to avert severe malaria cases in early childhood.

Correction: Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein.

[This corrects the article DOI: 10.1371/journal.pone.0159449.].

Antigens for pre-erythrocytic malaria vaccines: building on success.

Immunization with attenuated pre-erythrocytic malaria parasites can confer sterile protection against malaria in humans and rodents, and a single pre-erythrocytic antigen incorporated in a subunit vaccine has substantially reduced clinical Plasmodium falciparum malaria episodes in African infants during phase 2 trials. Building upon this success has been hindered by technical obstacles that limit research on pre-erythrocytic parasites, especially the liver stage (LS) parasites, and by an incomplete understanding of the immune mechanisms that confer protection in humans. Recent improvements in growing and isolating LS parasites have allowed progress in defining the transcriptome and proteome of the LS parasite, although more work remains to be done particularly for the early LS parasite of P. falciparum. Next generation pre-erythrocytic antigens can be assessed and prioritized based on immunization studies in animals, and on models of immunity such as attenuated parasite vaccines that confer sterile protection or naturally acquired LS-specific immune responses that correlate with protection in endemic areas. Although mechanisms of protection in humans remain poorly understood, the availability of a human malaria challenge model for early clinical testing of candidate vaccines is a valuable tool to confirm which immunogens should move forward to larger field trials.

Competitive facilitation of drug-resistant Plasmodium falciparum malaria parasites in pregnant women who receive preventive treatment.

Intermittent preventive treatment in pregnancy (IPTp) is used to prevent Plasmodium falciparum malaria. However, parasites resistant to the IPTp drug sulfadoxine-pyrimethamine (SP) have emerged worldwide, and infections with mixed resistant and susceptible parasites are exacerbated by pyrimethamine in mice. In a prospective delivery cohort in Muheza, Tanzania, we examined the effects of SP IPTp on parasite resistance alleles, parasite diversity, level of parasitemia, and inflammation in the placenta. IPTp use was associated with an increased fraction of parasites carrying the resistance allele at DHPS codon 581, an increase in the level of parasitemia, and more intense placental inflammation. The lowest mean level of parasite diversity and highest mean level of parasitemia occurred in women after recent IPTp use. These findings support a model of parasite release and facilitation, whereby the most highly resistant parasites out-compete less fit parasite populations and overgrow under drug pressure. Use of partially effective anti-malarial agents for IPTp may exacerbate malaria infections in the setting of widespread drug resistance.

Malaria in the pregnant woman.

Women become more susceptible to Plasmodium falciparum malaria during pregnancy, and the risk of disease and death is high for both the mother and her fetus. In low transmission areas, women of all parities are at risk for severe syndromes like cerebral malaria, and maternal and fetal mortality are high. In high transmission areas, where women are most susceptible during their first pregnancies, severe syndromes like cerebral malaria are uncommon, but severe maternal anemia and low birth weight are frequent sequelae and account for an enormous loss of life. P. falciparum-infected red cells sequester in the intervillous space of the placenta, where they adhere to chondroitin sulfate A but not to receptors like CD36 that commonly support adhesion of parasites infecting nonpregnant hosts. Poor pregnancy outcomes due to malaria are related to the macrophage-rich infiltrates and pro-inflammatory cytokines such as tumor necrosis factor-alpha that accumulate in the intervillous space. Women who acquire antibodies against chrondroitin sulfate A (CSA)-binding parasites are less likely to have placental malaria, and are more likely to deliver healthy babies. In areas of stable transmission, women acquire antibodies against CSA-binding parasites over successive pregnancies, explaining the high susceptibility to malaria during first pregnancy, and suggesting that a vaccine to prevent pregnancy malaria should target placental parasites. Prevention and treatment of malaria are essential components of antenatal care in endemic areas, but require special considerations during pregnancy. Recrudescence after drug treatment is more common during pregnancy, and the spread of drug-resistant parasites has eroded the usefulness of the few drugs known to be safe for the woman and her fetus. Determining the safety and effectiveness of newer antimalarials in pregnant women is an urgent priority. A vaccine that prevents pregnancy malaria due to P. falciparum could be delivered before first pregnancy, and would have an enormous impact on mother-child health in tropical areas.

The immunology and pathogenesis of malaria during pregnancy.

Women in endemic areas become highly susceptible to malaria during first and second pregnancies, despite immunity acquired after years of exposure. Recent insights have advanced our understanding of pregnancy malaria caused by Plasmodium falciparum, which is responsible for the bulk of severe disease and death. Accumulation of parasitized erythrocytes in the blood spaces of the placenta is a key feature of maternal infection with P. falciparum. Placental parasites express surface ligands and antigens that differ from those of other P. falciparum variants, facilitating evasion of existing immunity, and mediate adhesion to specific molecules, such as chondroitin sulfate A, in the placenta. The polymorphic and clonally variant P. falciparum erythrocyte membrane protein 1, encoded by var genes, binds to placental receptors in vitro and may be the target of protective antibodies. An intense infiltration of immune cells, including macrophages, into the placental intervillous spaces, and the production of pro-inflammatory cytokines often occur in response to infection, and are associated with low birth weight and maternal anemia. Expression of alpha and beta chemokines may initiate or facilitate this cellular infiltration during placental malaria. Specific immunity against placental-binding parasites may prevent infection or facilitate clearance of parasites prior to the influx of inflammatory cells, thereby avoiding a cascade of events leading to disease and death. Much less is known about pathogenic processes in P. vivax infections, and corresponding immune responses. Emerging knowledge of the pathogenesis and immunology of malaria in pregnancy will increasingly lead to new opportunities for the development of therapeutic and preventive interventions and new tools for diagnosis and monitoring.

PhD: a web database application for phenotype data management.

A database application has been developed for phenotype data management employing the Entity-Attribute-Value (EAV) model. By applying the EAV model, this application allows users to manage arbitrary phenotypes and customize data entry forms; therefore, it is suitable for different and multi-center projects.

The laparoscopic reoperation of failed Heller myotomy.

BACKGROUND: Laparoscopic Heller myotomy for achalasia has a 10-20% failure rate and may require reoperation to control persistent, or recurrent symptoms of dysphagia. We report our experience with laparoscopic reoperation for failed Heller myotomy. METHODS: Between 1996 and 2001, 5 patients underwent reoperative laparoscopic Heller myotomy. The mean age was 39 years. The presenting symptoms were persistent dysphagia ( n = 3), recurrent dysphagia ( n = 1), and weight loss ( n = 1). The mean duration between 1st surgery and recurrence of symptoms was 2 months and the mean duration between surgeries was 27.5 months. All operations were completed laparoscopically. RESULTS: There were no intraoperative or postoperative complications. Incomplete gastric myotomy was the cause of all 5 primary surgical failures. The mean hospital stay was 2 days. Mean follow-up was 12.8 months. Results were excellent in 2 patients who reported no dysphagia postoperatively (dysphagia grade 0) and good in 3 patients (60%) who all reported improvement in swallowing (dysphagia grade I-II). CONCLUSION: Laparoscopic reoperation for failed Heller myotomy is feasible with encouraging preliminary results.

Pre-erythrocytic immunity to Plasmodium falciparum: the case for an LSA-1 vaccine.

A vaccine is urgently needed to stem the global resurgence of Plasmodium falciparum malaria. Vaccines targeting the erythrocytic stage are often viewed as an anti-disease strategy. By contrast, infection might be completely averted by a vaccine against the liver stage, a pre-erythrocytic stage during which the parasite multiplies 10000-fold within hepatocytes. Sterilizing immunity can be conferred by inoculating humans with irradiated pre-erythrocytic parasites, and a recombinant pre-erythrocytic vaccine partially protects humans from infection. Liver-stage antigen-1, one of a few proteins known to be expressed by liver-stage parasites, holds particular promise as a vaccine. Studies of naturally exposed populations have consistently related immune responses against this antigen to protection.

Human resistance to Plasmodium falciparum increases during puberty and is predicted by dehydroepiandrosterone sulfate levels.

Immunity to Plasmodium falciparum develops slowly in areas of endemicity, and this is often ascribed to poorly immunogenic or highly variant parasite antigens. However, among populations newly exposed to malaria, adults acquire immunity more rapidly than children. We examined the relationship between pubertal development and resistance to P. falciparum. During two transmission seasons in western Kenya, we treated the same cohort of young males to eradicate P. falciparum and then obtained blood smears each week for 4 months. We determined pubertal development by Tanner staging and by levels of dehydroepiandrosterone sulfate (DHEAS) and testosterone in plasma. In multivariate and age-stratified analyses, we examined the effect of pubertal development on resistance to malaria. In both seasons (n = 248 and 144 volunteers, respectively), older males were less susceptible than younger males. Age-related decreases in the frequency and density of parasitemia were greatest during puberty (15- to 20-year-olds). DHEAS and testosterone were significant independent predictors of resistance to P. falciparum parasitemia, even after accounting for the effect of age. Fifteen- to 20-year-old males with high DHEAS levels had a 72% lower mean parasite density (P<0.01) than individuals with low DHEAS levels. Similarly, 21- to 35-year-old males with high DHEAS levels had a 92% lower mean parasite density (P<0.001) and 48% lower frequency of parasitemia (P<0.05) than individuals with low DHEAS levels. These data suggest that the long period needed to attain full immunity could be explained as a consequence of host development rather than as the requirement to recognize variant or poorly immunogenic parasite antigens.

Nitric oxide, malaria, and anemia: inverse relationship between nitric oxide production and hemoglobin concentration in asymptomatic, malaria-exposed children.

The cause of the anemia associated with chronic, intermittent, asymptomatic, low-level parasitemia in children in malaria-endemic endemic areas is not well understood. Nitric oxide (NO) decreases erythropoiesis, and it is likely an important mediator of anemia of chronic disease. Production of NO is decreased in acute uncomplicated and cerebral malaria, but it is increased in asymptomatic Tanzanian children (with or without parasitemia). We hypothesized that chronic overproduction of NO in these asymptomatic children contributes to the anemia associated with subclinical/subpatent malaria. In 44 fasting, asymptomatic, malaria-exposed, Tanzanian children, NO production (measured using fasting urine NOx excretion) was inversely associated with hemoglobin concentration (P = 0.03, controlling for age and gender). Using multiple linear regression, hemoglobin concentration was negatively associated with parasitemia (P = 0.005). After controlling for age and parasitemia, NO was no longer an independent predictor of anemia. One of the mechanisms of parasite-related anemia in such children may be through the adverse hematologic effects of parasite-induced NO production.

Effects of age and parasitemia on nitric oxide production/leukocyte nitric oxide synthase type 2 expression in asymptomatic, malaria-exposed children.

Age appears to influence not only the acquisition of clinical immunity to malaria but also the susceptibility to and clinical manifestations of severe malaria. Asymptomatic malaria-exposed Tanzanian children have high production of nitric oxide (NO) and universal expression of leukocyte NO synthase type 2 (NOS2), which may protect against disease. To determine the effects of age and parasitemia on NO production, we measured urine and plasma NO metabolites and leukocyte NOS2 expression in 45 fasting, asymptomatic, malaria-exposed children of different ages, stratifying parasitemia by thick film and polymerase chain reaction (PCR) analysis. Although NO production was significantly higher in thick film-positive children than in thick film-negative children, after adjusting for age and gender, we were unable to detect a difference in NO production in thick film-negative children between those who were PCR positive and PCR negative. The relationship between age and NO production was determined using a generalized additive model adjusted for the effects of gender and parasitemia. Production of NO using all three measures was highest in infancy, decreasing after the first year of life, and then increasing again after 5 years of age. This pattern of age-related NO production is the reverse of the pattern of age-related morbidity from cerebral malaria in coastal Tanzanian children. Elevated production of NO in both infants and older children may be related to age per se and malaria infection respectively, and may be one of the mediators of the anti-disease immunity found most commonly in these two age groups.

Interleukin-10 responses to liver-stage antigen 1 predict human resistance to Plasmodium falciparum.

The design of an effective vaccine against Plasmodium falciparum, the most deadly malaria parasite of humans, requires a careful definition of the epitopes and the immune responses involved in protection. Liver-stage antigen 1 (LSA-1) is specifically expressed during the hepatic stage of P. falciparum and elicits cellular and humoral immune responses in naturally exposed individuals. We report here that interleukin-10 (IL-10) production in response to LSA-1 predicts resistance to P. falciparum after eradication therapy. Resistance was not related to gamma interferon or tumor necrosis factor alpha production. This is the first report that human IL-10 responses are associated with resistance after eradication therapy, and our findings support the inclusion of LSA-1 in a vaccine against malaria.

Malaria during pregnancy: parasites, antibodies and chondroitin sulphate A.

CSA-binding forms of P. falciparum appear uncommonly in non-pregnant hosts but are selected by the human placenta for growth. Parasites are presumably selected by adherence to CSA within the vascular compartment of the placenta, allowing IRBCs to sequester and multiply to high density. Chondroitin sulphate appears on the surface of placental syncytiotrophoblasts, and CSA is a component of PGs found in the placenta [42], but the identification of the CSA-containing PG(s) mediating IRBC adhesion in vivo requires further study. Anti-adhesion antibodies against CSA-binding parasites are associated with protection from maternal malaria, but these antibodies develop only over successive pregnancies, accounting for the susceptibility of primigravidas to infection. PfCSA-L, the parasite ligand mediating adhesion to CSA, has not yet been identified but is known to be antigenically conserved among isolates from around the world. An anti-adhesion vaccine delivered to women before first pregnancy could confer protection from maternal malaria and might be globally effective.

Immunization with SPf66 and subsequent infection with homologous and heterologous Plasmodium falciparum parasites.

In an area of intense transmission, a malaria vaccine could reduce infection due to the parasite types represented in the vaccine, but have no detectable effect on the overall frequency of infection if it did not protect against infection with heterologous parasites. These studies were performed to determine whether immunization with SPf66 decreased infection with homologous parasites containing the 11 amino acid peptide from merozoite surface protein-1 (MSP-1) in SPf66, or increased infection due to heterologous parasites containing heterologous (alternative) MSP-1 sequences. Based on this 11 amino acid peptide (YSLFQKEKMVL), three forward primers (S,Q,V) were designed to amplify the MSP-1 sequence present in SPf66, and 3 additional forward primers (G,H,I) to amplify the alternative MSP-1 sequence (YGLFHKEKMIL). This strategy was validated by polymerase chain reaction (PCR) amplification and dideoxy sequencing with 14 cloned laboratory isolates, which demonstrated that each primer amplified one MSP-1 sequence or the other, but not both. The technique was then used to examine filter paper blots from an SPf66 vaccine study of 69 subjects in Saradidi, Kenya. In that study, the prevalence of infection with YSLFQKEKMVL or YGLFHKEKMIL type parasites was unaffected by immunization with SPf66 (based on PCR amplification with the S, Q, V, G, H and I primers, respectively). These results suggest that immunization with SPf66 does not produce a selective effect in vivo. They demonstrate a molecular method to test for selection in vivo as an indirect measure of vaccine efficacy.