Abdoulaye K. Kone

Blood group O protects against severe Plasmodium falciparum malaria through the mechanism of reduced rosetting

Malaria has been a major selective force on the human population, and several erythrocyte polymorphisms have evolved that confer resistance to severe malaria. Plasmodium falciparum rosetting, a parasite virulence phenotype associated with severe malaria, is reduced in blood group O erythrocytes compared with groups A, B, and AB, but the contribution of the ABO blood group system to protection against severe malaria has received little attention. We hypothesized that blood group O may confer resistance to severe falciparum malaria through the mechanism of reduced rosetting. In a matched case-control study of 567 Malian children, we found that group O was present in only 21% of severe malaria cases compared with 44–45% of uncomplicated malaria controls and healthy controls. Group O was associated with a 66% reduction in the odds of developing severe malaria compared with the non-O blood groups (odds ratio 0.34, 95% confidence interval 0.19–0.61, P < 0.0005, severe cases versus uncomplicated malaria controls). In the same sample set, P. falciparum rosetting was reduced in parasite isolates from group O children compared with isolates from the non-O blood groups (P = 0.003, Kruskal–Wallis test). Statistical analysis indicated a significant interaction between host ABO blood group and parasite rosette frequency that supports the hypothesis that the protective effect of group O operates through the mechanism of reduced P. falciparum rosetting. This work provides insights into malaria pathogenesis and suggests that the selective pressure imposed by malaria may contribute to the variable global distribution of ABO blood groups in the human population.

Serum Levels of the Proinflammatory Cytokines Interleukin-1 Beta (IL-1β), IL-6, IL-8, IL-10, Tumor Necrosis Factor Alpha, and IL-12(p70) in Malian Children with Severe Plasmodium falciparum Malaria and Matched Uncomplicated Malaria or Healthy Controls

ABSTRACT Inflammatory cytokines play an important role in human immune responses to malarial disease. However, the role of these mediators in disease pathogenesis, and the relationship between host protection and injury remains unclear. A total of 248 cases of severe Plasmodium falciparum malaria among children aged 3 months to 14 years residing in Bandiagara, Mali, were matched to cases of uncomplicated malaria and healthy controls. Using modified World Health Organization criteria for defining severe malaria, we identified 100 cases of cerebral malaria (coma, seizure, and obtundation), 17 cases of severe anemia (hemoglobin, <5 g/dl), 18 cases combined cerebral malaria with severe anemia, and 92 cases with hyperparasitemia (asexual trophozoites, >500,000/mm3). Significantly elevated levels (given as geometric mean concentrations in picograms/milliliter) of interleukin-6 (IL-6; 485.2 versus 54.1; P = <0.001), IL-10 (1,099.3 versus 14.1; P = <0.001), tumor necrosis factor alpha (10.1 versus 7.7; P = <0.001), and IL-12(p70) (48.9 versus 31.3; P = 0.004) in serum were found in severe cases versus healthy controls. Significantly elevated levels of IL-6 (485.2 versus 141.0; P = <0.001) and IL-10 (1,099.3 versus 133.9; P = <0.001) were seen in severe malaria cases versus uncomplicated malaria controls. Cerebral malaria was associated with significantly elevated levels of IL-6 (754.5 versus 311.4; P = <0.001) and IL-10 (1,405.6 versus 868.6; P = 0.006) compared to severe malaria cases without cerebral manifestations. Conversely, lower levels of IL-6 (199.2 versus 487.6; P = 0.03) and IL-10 (391.1 versus 1,160.9; P = 0.002) were noted in children with severe anemia compared to severe malaria cases with hemoglobin at >5 g/dl. Hyperparasitemia was associated with significantly lower levels of IL-6 (336.6 versus 602.1; P = 0.002). These results illustrate the complex relationships between inflammatory cytokines and disease in P. falciparum malaria.

A Field Trial to Assess a Blood-Stage Malaria Vaccine

BACKGROUND Blood-stage malaria vaccines are intended to prevent clinical disease. The malaria vaccine FMP2.1/AS02(A), a recombinant protein based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, has previously been shown to have immunogenicity and acceptable safety in Malian adults and children. METHODS In a double-blind, randomized trial, we immunized 400 Malian children with either the malaria vaccine or a control (rabies) vaccine and followed them for 6 months. The primary end point was clinical malaria, defined as fever and at least 2500 parasites per cubic millimeter of blood. A secondary end point was clinical malaria caused by parasites with the AMA1 DNA sequence found in the vaccine strain. RESULTS The cumulative incidence of the primary end point was 48.4% in the malaria-vaccine group and 54.4% in the control group; efficacy against the primary end point was 17.4% (hazard ratio for the primary end point, 0.83; 95% confidence interval [CI], 0.63 to 1.09; P=0.18). Efficacy against the first and subsequent episodes of clinical malaria, as defined on the basis of various parasite-density thresholds, was approximately 20%. Efficacy against clinical malaria caused by parasites with AMA1 corresponding to that of the vaccine strain was 64.3% (hazard ratio, 0.36; 95% CI, 0.08 to 0.86; P=0.03). Local reactions and fever after vaccination were more frequent with the malaria vaccine. CONCLUSIONS On the basis of the primary end point, the malaria vaccine did not provide significant protection against clinical malaria, but on the basis of secondary results, it may have strain-specific efficacy. If this finding is confirmed, AMA1 might be useful in a multicomponent malaria vaccine. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00460525.).

Differential var gene transcription in Plasmodium falciparum isolates from patients with cerebral malaria compared to hyperparasitaemia

The Plasmodium falciparum variant erythrocyte surface antigens known as PfEMP1, encoded by the var gene family, are thought to play a crucial role in malaria pathogenesis because they mediate adhesion to host cells and immuno-modulation. Var genes have been divided into three major groups (A, B and C) and two intermediate groups (B/A and B/C) on the basis of their genomic location and upstream sequence. We analysed expressed sequence tags of the var gene DBLα domain to investigate var gene transcription in relation to disease severity in Malian children. We found that P. falciparum isolates from children with cerebral malaria (unrousable coma) predominantly transcribe var genes with DBLα1-like domains that are characteristic of Group A or B/A var genes. In contrast, isolates from children with equally high parasite burdens but no symptoms or signs of severe malaria (hyperparasitaemia patients) predominantly transcribe var genes with DBLα0-like domains that are characteristic of the B and C-related var gene groups. These results suggest that var genes with DBLα1-like domains (Group A or B/A) may be implicated in the pathogenesis of cerebral malaria, while var genes with DBLα0-like domains promote less virulent malaria infections.

Impact of Trimethoprim-Sulfamethoxazole Prophylaxis on Falciparum Malaria Infection and Disease

BACKGROUND Trimethoprim-sulfamethoxazole (TS) prophylaxis is recommended for persons living with human immunodeficiency virus infection and acquired immunodeficiency syndrome in Africa. TS and the antimalarial combination sulfadoxine-pyrimethamine (SP) share mechanisms of action and resistance patterns, and concerns about the impact of TS resistance on SP efficacy have contributed to reluctance to implement TS prophylaxis in Africa. METHODS To determine whether TS prophylaxis impairs SP efficacy for treatment of uncomplicated falciparum malaria, we conducted a randomized, controlled, open-label study of TS prophylaxis. Two hundred and forty children 5-15 years old were randomized in a 2 : 1 fashion to receive either thrice-weekly TS for 12 weeks or no prophylaxis and were treated with SP for subsequent episodes of malaria. The incidence of malaria, SP efficacy, and the prevalence of parasite mutations that confer antifolate drug resistance were measured. RESULTS TS prophylaxis had a 99.5% protective efficacy against episodes of clinical malaria, with 97% efficacy against infection. Four SP treatment failures occurred in the control group, and none occurred in the TS group. No evidence was seen for selection by TS of antifolate resistance-conferring mutations in parasite dihydrofolate reductase or dihydropteroate synthase during subclinical infections. CONCLUSIONS In this setting of low antifolate resistance, TS was highly effective in preventing falciparum malaria infection and disease and did not appear to select for SP-resistant parasites.

Safety and immunogenicity of an AMA1 malaria vaccine in Malian children: results of a phase 1 randomized controlled trial.

Background The objective was to evaluate the safety, reactogenicity and immunogenicity of the AMA-1-based blood-stage malaria vaccine FMP2.1/AS02A in adults exposed to seasonal malaria. Methodology/Principal Findings A phase 1 double blind randomized controlled dose escalation trial was conducted in Bandiagara, Mali, West Africa, a rural town with intense seasonal transmission of Plasmodium falciparum malaria. The malaria vaccine FMP2.1/AS02A is a recombinant protein (FMP2.1) based on apical membrane antigen-1 (AMA-1) from the 3D7 clone of P. falciparum, adjuvanted with AS02A. The comparator vaccine was a cell-culture rabies virus vaccine (RabAvert). Sixty healthy, malaria-experienced adults aged 18–55 y were recruited into 2 cohorts and randomized to receive either a half dose or full dose of the malaria vaccine (FMP2.1 25 µg/AS02A 0.25 mL or FMP2.1 50 µg/AS02A 0.5 mL) or rabies vaccine given in 3 doses at 0, 1 and 2 mo, and were followed for 1 y. Solicited symptoms were assessed for 7 d and unsolicited symptoms for 30 d after each vaccination. Serious adverse events were assessed throughout the study. Titers of anti-AMA-1 antibodies were measured by ELISA and P. falciparum growth inhibition assays were performed on sera collected at pre- and post-vaccination time points. Transient local pain and swelling were common and more frequent in both malaria vaccine dosage groups than in the comparator group. Anti-AMA-1 antibodies increased significantly in both malaria vaccine groups, peaking at nearly 5-fold and more than 6-fold higher than baseline in the half-dose and full-dose groups, respectively. Conclusion/Significance The FMP2.1/AS02A vaccine had a good safety profile, was well-tolerated, and was highly immunogenic in malaria-exposed adults. This malaria vaccine is being evaluated in Phase 1 and 2 trials in children at this site. Trial Registration ClinicalTrials.gov NCT00308061

Dynamics of polymorphism in a malaria vaccine antigen at a vaccine-testing site in Mali.

Background Malaria vaccines based on the 19-kDa region of merozoite surface protein 1 (MSP-119) derived from the 3D7 strain of Plasmodium falciparum are being tested in clinical trials in Africa. Knowledge of the distribution and natural dynamics of vaccine antigen polymorphisms in populations in which malaria vaccines will be tested will guide vaccine design and permit distinction between natural fluctuations in genetic diversity and vaccine-induced selection. Methods and Findings Using pyrosequencing, six single-nucleotide polymorphisms in the nucleotide sequence encoding MSP-119 were genotyped from 1,363 malaria infections experienced by 100 children who participated in a prospective cohort study in Mali from 1999 to 2001. The frequencies of 14 MSP-119 haplotypes were compared over the course of the malaria transmission season for all three years, in three age groups, and in consecutive infections within individuals. While the frequency of individual MSP-119 haplotypes fluctuated, haplotypes corresponding to FVO and FUP strains of P. falciparum (MSP-119 haplotypes QKSNGL and EKSNGL, respectively) were most prevalent during three consecutive years and in all age groups with overall prevalences of 46% (95% confidence interval [CI] 44%–49%) and 36% (95% CI 34%–39%), respectively. The 3D7 haplotype had a lower overall prevalence of 16% (95% CI 14%–18%). Multiplicity of infection based on MSP-119 was higher at the beginning of the transmission season and in the oldest individuals (aged ≥11 y). Three MSP-119 haplotypes had a reduced frequency in symptomatic infections compared to asymptomatic infections. Analyses of the dynamics of MSP-119 polymorphisms in consecutive infections implicate three polymorphisms (at positions 1691, 1700, and 1701) as being particularly important in determining allele specificity of anti-MSP-119 immunity. Conclusions Parasites with MSP-119 haplotypes different from that of the leading vaccine strain were consistently the most prevalent at a vaccine trial site. If immunity elicited by an MSP-1-based vaccine is allele-specific, a vaccine based on either the FVO or FUP strain might have better initial efficacy at this site. This study, to our knowledge the largest of its kind to date, provides molecular information needed to interpret population responses to MSP-1-based vaccines and suggests that certain MSP-119 polymorphisms may be relevant to cross-protective immunity.

High Levels of Plasmodium falciparum Rosetting in All Clinical Forms of Severe Malaria in African Children

Plasmodium falciparum rosetting (the spontaneous binding of infected erythrocytes to uninfected erythrocytes) is a well-recognized parasite virulence factor. However, it is currently unclear whether rosetting is associated with all clinical forms of severe malaria, or only with specific syndromes such as cerebral malaria. We investigated the relationship between rosetting and clinical malaria in 209 Malian children enrolled in a case-control study of severe malaria. Rosetting was significantly higher in parasite isolates from severe malaria cases compared with non-severe hyperparasitemia and uncomplicated malaria controls (F(2,117) = 8.15, P < 0.001). Analysis of sub-categories of severe malaria (unrousable coma, severe anemia, non-comatose neurological impairment, repeated seizures or a small heterogeneous group with signs of renal failure or jaundice) showed high levels of rosetting in all sub-categories, and no statistically significant differences in rosetting between sub-categories (F(4,67) = 1.28, P = 0.28). Thus rosetting may contribute to the pathogenesis of all severe malaria syndromes in African children, and interventions to disrupt rosetting could be potential adjunctive therapies for all forms of severe malaria in Africa.

First detection of Leishmania major DNA in Sergentomyia (Spelaeomyia) darlingi from cutaneous leishmaniasis foci in Mali.

Background Leishmania major complex is the main causative agent of zoonotic cutaneous leishmaniasis (ZCL) in the Old World. Phlebotomus papatasi and Phlebotomus duboscqi are recognized vectors of L. major complex in Northern and Southern Sahara, respectively. In Mali, ZCL due to L. major is an emerging public health problem, with several cases reported from different parts of the country. The main objective of the present study was to identify the vectors of Leishmania major in the Bandiagara area, in Mali. Methodology/Principal Findings An entomological survey was carried out in the ZCL foci of Bandiagara area. Sandflies were collected using CDC miniature light traps and sticky papers. In the field, live female Phlebotomine sandflies were identified and examined for the presence of promastigotes. The remaining sandflies were identified morphologically and tested for Leishmania by PCR in the ITS2 gene. The source of blood meal of the engorged females was determined using the cyt-b sequence. Out of the 3,259 collected sandflies, 1,324 were identified morphologically, and consisted of 20 species, of which four belonged to the genus Phlebotomus and 16 to the genus Sergentomyia. Leishmania major DNA was detected by PCR in 7 of the 446 females (1.6%), specifically 2 out of 115 Phlebotomus duboscqi specimens, and 5 from 198 Sergentomyia darlingi specimens. Human DNA was detected in one blood-fed female S. darlingi positive for L. major DNA. Conclusion Our data suggest the possible involvement of P. duboscqi and potentially S. darlingi in the transmission of ZCL in Mali.

Extended Safety, Immunogenicity and Efficacy of a Blood-Stage Malaria Vaccine in Malian Children: 24-Month Follow-Up of a Randomized, Double-Blinded Phase 2 Trial

Background The FMP2.1/AS02A candidate malaria vaccine was tested in a Phase 2 study in Mali. Based on results from the first eight months of follow-up, the vaccine appeared well-tolerated and immunogenic. It had no significant efficacy based on the primary endpoint, clinical malaria, but marginal efficacy against clinical malaria in secondary analyses, and high allele-specific efficacy. Extended follow-up was conducted to evaluate extended safety, immunogenicity and efficacy. Methods A randomized, double-blinded trial of safety, immunogenicity and efficacy of the candidate Plasmodium falciparum apical membrane antigen 1 (AMA1) vaccine FMP2.1/AS02A was conducted in Bandiagara, Mali. Children aged 1–6 years were randomized in a 1∶1 ratio to receive FMP2.1/AS02A or control rabies vaccine on days 0, 30 and 60. Using active and passive surveillance, clinical malaria and adverse events as well as antibodies against P. falciparum AMA1 were monitored for 24 months after the first vaccination, spanning two malaria seasons. Findings 400 children were enrolled. Serious adverse events occurred in nine participants in the FMP2.1/AS02A group and three in the control group; none was considered related to study vaccination. After two years, anti-AMA1 immune responses remained significantly higher in the FMP2.1/AS02A group than in the control group. For the entire 24-month follow-up period, vaccine efficacy was 7.6% (p = 0.51) against first clinical malaria episodes and 9.9% (p = 0.19) against all malaria episodes. For the final 16-month follow-up period, vaccine efficacy was 0.9% (p = 0.98) against all malaria episodes. Allele-specific efficacy seen in the first malaria season did not extend into the second season of follow-up. Interpretation Allele-specific vaccine efficacy was not sustained in the second malaria season, despite continued high levels of anti-AMA1 antibodies. This study presents an opportunity to evaluate correlates of partial protection against clinical malaria that waned during the second malaria season. Trial Registration Clinicaltrials.gov NCT00460525 NCT00460525