Dynamics of PfEMP1 Antibody Profile From Birth to 12 Months of Age in Beninese Infants.

BACKGROUND: Plasmodium falciparum-infected erythrocytes bind to specific endothelial cell receptors via members of the PfEMP1 family exported onto the erythrocyte surface. These interactions are mediated by different types of cysteine-rich interdomain region (CIDR) domains found in the N-terminal region of all PfEMP1. CIDRα1 domains bind endothelial protein C receptor (EPCR), CIDRα2-6 domains bind CD36, whereas the receptor specificity of CIDRß/γ/δ domains is unknown. METHODS: In this study, we investigated the level of immunoglobulin (Ig)G targeting the different types of PfEMP1 CIDR during the first year of life. We used plasma collected longitudinally from children of pregnant women who had been followed closely through pregnancy. RESULTS: Antibodies to CIDRα1 domains were more frequent in cord blood compared with antibodies to CIDRα2-6 domains. Higher IgG levels to EPCR-binding CIDRα1 variants positively correlated with the timing of first infections. Antibodies to all PfEMP1 types declined at similar rates to the point of disappearance over the first 6 months of life. At 12 months, children had acquired antibody to all types of CIDR domains, mostly in children with documented P falciparum infections. CONCLUSIONS: These observations agree with the notion that the timing and phenotype of first P falciparum infections in life are influenced by the immune status of the mother.

Genome-wide association study of antibody responses to Plasmodium falciparum candidate vaccine antigens.

We conducted a genome-wide association study (GWAS) of antibody responses directed to three Plasmodium falciparum vaccine candidate antigens (MSP1, MSP2 and GLURP) previously associated with different patterns of protection against malaria infection in Senegalese children. A total of 174 950 single-nucleotide polymorphisms (SNPs) were tested for association with immunoglobulin G1 (IgG1) responses directed to MSP1 and to GLURP and with IgG3 responses to MSP2 FC27 and to MSP2 3D7. We first performed a single-trait analysis with each antibody response and then a multiple-trait analysis in which we analyzed simultaneously the three immune responses associated with the control of clinical malaria episodes. Suggestive associations (P<1 × 10(-4)) were observed for 25 SNPs in MSP1 antibody response analysis or in multiple-trait analysis. According to the strength of their observed associations and their functional role, the following genes are of particular interest: RASGRP3 (2p22.3, P=7.6 × 10(-6)), RIMS1 (6q13, P=2.0 × 10(-5)), MVB12B (9q33.3, P=8.9 × 10(-5)) and GNPTAB (12q23.2, P=7.4 × 10(-5)). Future studies will be required to replicate these findings in other African populations. This work will contribute to the elucidation of the host genetic factors underlying variable immune responses to P. falciparum.

Schistosoma haematobium infection affects Plasmodium falciparum-specific IgG responses associated with protection against malaria.

We have previously shown that antibody responses directed to Plasmodium falciparum merozoite surface protein (MSP)-1, MSP-2 and glutamate-rich protein (GLURP) are associated with anti-malarial protection in residents of the Niakhar area of Senegal. In the same area, urinary schistosomiasis is frequent and we therefore assessed the possible influence of Schistosoma haematobium infection on these protective anti-malarial IgG responses. After adjustment for confounders, we found that the levels of IgG1 directed to MSP1 and GLURP were significantly lower in helminth carriers. The higher circulating levels of interleukin (IL)-10 present in the plasma of co-infected individuals were associated with decreased anti-plasmodial IgG responses, particularly of those directed to MSP-2. Our data thus reveal a modulation of P. falciparum-specific immune responses in the presence of a trematode helminth infection, potentially increasing infected individuals' risk of plasmodial infection or disease.

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.

Immunologic responses to soluble exoantigens of Plasmodium falciparum in Gabonese children exposed to continuous intense infection.

The development of antidisease immunity in children infected with Plasmodium falciparum is thought to be related to their immunologic responses to certain soluble parasite-derived exoantigens. We have assessed both cellular and humoral responses to these antigens in a cross-sectional study of a cohort of Gabonese schoolchildren who live in an area where malaria is holoendemic and perenially transmitted, in an attempt to identify immunologic markers of this early developing protective immunity. Concurrent parasitemia was found to have a significant influence on lymphoproliferative and antibody responses to the exoantigens. Individuals with higher levels of parasitemia had significantly lower proliferative and IgG isotype responses. Higher concentrations of specific IgG1 and IgG3, in particular, were associated with lower or no parasitemia, suggesting a possible protective role for these isotypes, whereas the level of IgM antibodies showed a trend towards higher concentrations in those with parasitemia, perhaps indicative of an exoantigen-induced T cell-independent response. Cytokine responses were unaffected by either the presence or the intensity of parasitemia and were dissociated from both proliferative and antibody response to the exoantigens. However, the mitogen-stimulated production of tumor-necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL)-6 was positively correlated with the corresponding lymphoproliferative responses. At the individual level, mitogen-stimulated TNF-alpha, interferon-gamma, IL-2, and IL-6 responses were positively correlated, as were mitogen- and exoantigen-induced TNF-alpha. The results are discussed in the light of current knowledge of immune responses to the exoantigens and the development of protective immunity to P. falciparum.

Differential effects of human serum and cells on the growth of Plasmodium falciparum adapted to serum-free in vitro culture conditions.

A single Plasmodium falciparum isolate was adapted for growth in serum-free culture medium. The parasitemia increased from 0.5% to 20% on day 7 after thawing. The asexual forms of the parasites appeared morphologically normal and pigment formation was comparable with that seen under standard conditions with serum present. Parasites were coincubated in 96-well plates with serum, peripheral blood mononuclear cells (PBMC), and PBMC in the presence of autologous serum from healthy non-immune individuals (n = 12), healthy semi-immune individuals (n = 12), and malaria patients (n = 7). Growth was monitored for six days. The concentration of interleukin-6 and interferon-gamma (IFN-gamma) in supernatants from the continuous cultures were measured by a bioassay and an enzyme-amplified sensitivity immunoassay. The results of this study showed that parasites cultured in serum-free medium in the presence of PBMC develop more rapidly, particularly with cells from malaria patients, compared with parasites cultured alone. The growth of parasites was different if 10% autologous serum was added to the culture. Parasite growth with sera from acutely infected individuals was similar with that with sera from aparasitemic, nonimmune individuals, and both supported significantly higher parasite growth over the six-day culture period compared with sera from the uninfected semi-immune individuals. Production of IFN-gamma by cells from nonimmune individuals and malaria patients was higher when cultures did not contain autologous serum. Nonimmune donor cells produced high amounts of IFN-gamma, but cells from the semi-immune donors produced little of this cytokine. There was no marked inhibition of parasite growth with any combination of serum and cells over six days of culture. A difference between the groups was observed after two days of culture, when growth with cells and serum from the uninfected, semi-immune group was significantly lower than that from the nonimmune group, but this was not subsequently sustained. The results of the study show that continuous cultivation of P. falciparum in serum-free medium provides a novel in vitro model to study mechanisms of the interplay between components of the human immune system and the malarial parasite, in which any possible influence of human serum is removed.

Relationships between malaria prevalence and malaria-related morbidity in school children from two villages in central Africa.

To investigate the relationship between parasite prevalence and malaria-related morbidity, we carried out a comparative study among cohorts of school children from two villages, Dienga, Gabon, and Pouma, Cameroon, both located in malaria-endemic areas. Seven to 17 year-old children attending primary schools were similarly followed-up at each site to evaluate the frequency of malaria attacks. Follow-up involved daily temperature recording (and blood smears in the case of fever) and preparation of blood smears every two weeks. In Pouma, 186 children were followed-up for six months. In Dienga, 228 children were followed-up for nine months. The mean prevalence rate of Plasmodium falciparum infections (as assessed by the blood smears) was twice as high in Pouma compared with Dienga (45.2% versus 26.8%; P < 0.0001), whereas the monthly malaria attack rate (as assessed by the daily surveillance) was twice as high in Dienga compared with Pouma (21.5% versus 41.4%; P = 0.003). The possible implication of several parameters that may differ between the two areas, such as the malaria transmission level, the economical and social status of the inhabitants, the characteristics of infecting parasite strains, and the genetic background of the population, is discussed.

Antibody responses to Plasmodium falciparum: evolution according to the severity of a prior clinical episode and association with subsequent reinfection.

We measured sporozoite- and total parasite antigen-specific IgG and IgM antibodies before and after treatment in matched groups of Gabonese children who presented with either mild or severe Plasmodium falciparum malaria. We investigated the influence of various parameters on these antibody responses, including clinical presentation, age, and post-treatment reinfection profiles. IgG but not IgM responses were strongly influenced by both clinical and parasitological status. IgG responses to the repeat region of the circumsporozoite protein, which were low at admission, particularly so in those with severe anemia, increased after treatment but showed no association with either age or reinfection profiles. Total parasite antigen-specific IgG responses were strongly influenced by parasitological status, and also differed significantly when segregated according to clinical status at admission, age, and reinfection histories. Most notably, anti-parasite IgG responses measured when children were parasite-free were higher and a good indicator of recent reinfections in those who presented with mild rather than with severe malaria. The profile of responses in the latter group suggests some immune system dysfunction, which may reflect the induction of tolerance to parasite antigens.

Immune responses against Plasmodium falciparum asexual blood-stage antigens and disease susceptibility in Gabonese and Cameroonian children.

The frequency and level of cellular and humoral responses to seven synthetic peptides from asexual blood stages of Plasmodium falciparum were measured in two cohorts of children living in areas highly endemic for malaria in Gabon and Cameroon. A prospective longitudinal study was conducted for one year in these sites to examine the relationship between specific in vitro immune responses and susceptibility to clinical malaria. Clinical protection was related to high proliferative responses (merozoite surface antigen-1 [MSA-1] and MSA-2 peptides) as well as to elevated antibody levels (schizont extract, MSA-2, and rhoptry-associated protein-1 [RAP-1] peptides) in the village of Dienga, Gabon. Higher response rates of interferon-gamma but lower response rates of tumor necrosis factor-alpha to four and six peptides, respectively, were observed in Dienga than in Pouma that were independent of the older age of the Gabonese children. Age accounted only for the higher prevalence rate in Dienga of the antibody responders to the peptide from Pf155/ring-infected erythrocyte surface antigen (RESA). Our results support the inclusion of epitopes from MSA-1, MSA-2, RAP-1, and Pf155/RESA antigens in a subunit vaccine against malaria, but show that a longitudinal clinical, parasitologic, and immunologic study conducted according to identical criteria in two separate areas may lead to contrasting observations, demonstrating the geographic limitation of the interpretation of such results.

A community trial of ivermectin for onchocerciasis in Sierra Leone: clinical and parasitological responses to four doses given at six-monthly intervals.

Clinical and parasitological responses were studied in villagers receiving all 4 doses of treatment, at 6-monthly intervals, in a placebo-controlled community trial of ivermectin for onchocerciasis in Sierra Leone. Skin microfilarial loads were markedly lowered by ivermectin throughout and there were reductions in the severity, but not the prevalence, of skin lesions. Markers of general health and the prevalences of itching, Onchocerca nodules and visual loss were not significantly reduced during the study period. Despite our inability to demonstrate obvious clinical benefit, treatment with ivermectin was well accepted throughout the study. Simple clinical measures for evaluating the short to medium term impact of the mass distribution of ivermectin on populations with onchocerciasis need further development.

Immune response to Plasmodium falciparum liver stage antigen-1: geographical variations within Central Africa and their relationship with protection from clinical malaria.

Two populations of schoolchildren from Gabon and Cameroon were tested in 1995 for their immunological reactivity to synthetic peptides (LSA-Rep, LSA-J and LSA-CTL) from Plasmodium falciparum liver stage antigen-1 (LSA-1). The prevalence and levels of both cellular (lymphocyte proliferation, tumour necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma), and interleukin-10 (IL-10)) and humoral (immunoglobulin G) responses were determined. Protection from clinical malaria, determined after a prospective 1 year study in both sites, was associated with elevated proliferative responses to LSA-Rep and LSA-CTL in the Gabonese children, as well as with higher antibody levels to both schizont extract and LSA-Rep. The prevalence of peptide-stimulated TNF-alpha secretion was higher in the Cameroonian group, but higher levels of antibodies to LSA-Rep and LSA-J were found in the Gabonese children. The immunological differences observed between children in the 2 study sites are discussed in the context of both epidemiological and individual host factors.

Ivermectin does not reduce the burden of itching in an onchocerciasis endemic community.

Degrees of itching were estimated before and for 6 months after a fourth dose of ivermectin or placebo was given to 97 subjects in Sierra Leone. There was no reduction in itching attributable to ivermectin at any stage, but there were non-significant increases in the prevalence, severity and localization of itching within the first 2 months after ivermectin compared to placebo. We also found that cell-mediated immune responses to Onchocerca volvulus were significantly increased 4 weeks after a single dose of ivermectin compared to before treatment. A temporary reversal of the state of immunosuppression in people with onchocerciasis may counterbalance the reduction in skin microfilarial loads following ivermectin, with no consequent reduction in itching. The lack of effect of ivermectin on itching, a major symptom of onchocerciasis, while disappointing, need not detract from the success of mass distribution programmes.

Relationship between entomological inoculation rate, Plasmodium falciparum prevalence rate, and incidence of malaria attack in rural Gabon.

To assess the relationships between variations of Plasmodium falciparum transmission and those of peripheral parasitaemia prevalence or malaria attack incidence rates in regions with limited fluctuations of transmission, we conducted a follow-up in two Gabonese populations. Entomological surveys were carried out from May 1995 to April 1996 in Dienga, and from May 1998 to April 1999 in Benguia. In Dienga, malaria transmission was seasonal, being not detected during two 3-month periods. Mean entomological inoculation rate (EIR) was 0.28 infective bite/person/night. In Benguia, malaria transmission was perennial with seasonal fluctuations, mean EIR being 0.76 infective bite/person/night. In Dienga, 301 schoolchildren were followed from October 1995 to March 1996. Clinical malaria attack was defined as fever associated with >5000 parasites/microl of blood. P. falciparum prevalence varied from 28 to 42%, and monthly malaria attack incidence from 30 to 169 per thousand. In Benguia, the entire population (122 persons) was followed from November 1998 to April 1999. Prevalence varied from 22 to 50%, and monthly malaria attack incidence from 52 to 179 per thousand. In each area, entomological variations were not related to parasite prevalence, but preceded malaria attack incidence with 1- or 2-month time lag, corresponding to the pre-patency period that differs in the two populations, possibly according to differences in immunity related to parasite transmission.

Plasmodium falciparum liver-stage antigen-1 peptide-specific interferon-gamma responses are not suppressed during uncomplicated malaria in African children.

Liver-stage antigen (LSA)-1 is a candidate vaccine molecule for Plasmodium falciparum malaria, but knowledge of the evolution of naturally acquired immune responses to LSA-1 in African children is lacking. We therefore assessed cellular immune responses to two defined T cell epitopes of LSA-1, during and after uncomplicated P. falciparum malaria in a group of Gabonese children. In terms of their prevalence, interferon (IFN)-gamma responses of peripheral blood mononuclear cells (PBMC) to an LSA-1 N-terminal peptide, T1, were significantly higher when measured during the acute phase compared with convalescence. IFN-gamma responses to the LSA-J (hinge region) peptide showed a similar profile, but at a lower prevalence. Depletion experiments confirmed that CD8+ T cells are a major source of peptide-driven IFN-gamma, but both lymphoproliferation and the production of IL-10 in response to either of the peptides was low in all children at all times. PBMC from 25% of the children failed to produce IFN-gamma in response to either peptide at any time-point. The results suggest that lymphocytes producing IFN-gamma in response to at least one T cell epitope of LSA-1 are most frequent in the peripheral circulation during the acute phase of P. falciparum malaria. Thus, in this case, the generalised suppression of cell-mediated responses which characterises acute malaria does not affect liver-stage antigen-specific IFN-gamma production. These findings imply that measurements of the frequency of parasite antigen-specific cellular immune responses in clinically healthy individuals may represent significant underestimations, which has important implications for the design of field-based vaccine antigen-related studies.

Parasite antigen-specific interleukin-10 and antibody reponses predict accelerated parasite clearance in Plasmodium falciparum malaria.

Using strict inclusion criteria, we conducted a hospital-based, case-control study in which 100 Gabonese children with severe Plasmodium falciparum malaria were matched for age, gender and provenance with 100 children presenting with mild malaria. Parasite antigen-specific cellular and humoral immunological responses were measured and compared with post-treatment parasite clearance times in each group. Significantly faster parasite clearance times were associated with in vitro production of IL-10 by acute-phase peripheral blood mononuclear cells (PBMC) in response to both liver and asexual stage parasite antigens, but not with proliferative, IFN-gamma, or TNF responses to the same antigens. In addition, in those children with mild malaria, higher levels of acute-phase antibody responses to liver stage antigen-1 (LSA-1) were associated with faster parasite clearance times, and were correlated with the presence of IL-10 responses to the same antigen. No such associations were found for IL-10 or antibody responses to a range of asexual blood stage antigens. Those with severe malaria had significantly lower levels of anti-LSA-1 antibodies compared to their counterparts with mild malaria. In conclusion, the results of this study suggest that parasite antigen-specific IL-10-mediated antibody responses may play a role in the control of asexual stage parasite multiplication in P. falciparum malaria.

Site-based study on polymorphism of Plasmodium falciparum MSP-1 and MSP-2 genes in isolates from two villages in Central Africa.

We investigated Plasmodium falciparum genetic diversity in isolates collected from school-going residents aged from 5 to 15 years in the village of Pouma (Cameroon, Central Africa). Seventy-six children were grouped according to the clinical status. Asymptomatic status was defined as parasite carriage in the absence of any clinical symptom and malaria symptomatic status with patent parasitemia over 5000 parasites/microliter of blood and an axillary temperature > 37.5 degrees C. Parasite DNA was analysed prior to malaria treatment. Genotyping of the P. falciparum merozoite surface proteins (MSP) 1 and 2 was performed by polymerase chain reaction using allele-specific primers. K1, MAD20, Ro33 and 3D7/CAMP, FC27 allelic families were attributed to MSP-1 and MSP-2 genes, respectively. No association was found between P. falciparum MSP-1 and MSP-2 genotypes and the clinical status of children. Mixed P. falciparum infections were detected in 78% of overall samples and all isolates from symptomatic children contained more than 1 clone. The results obtained in the village of Pouma were compared to those of the village of Dienga in Gabon where a similar study, using the same genotyping methods, had been carried out in the same age group of schoolchildren. Data are interpreted in the context of malaria epidemiology in both settings.

[Allelic polymorphism of Plasmodium falciparum MSP-2 gene in blood samples from Gabonese children]. / Polymorphisme allélique du gène MSP-2 de Plasmodium falciparum analysé a partir d'échantillons sanguins d'enfants gabonais.

In this study we have undertaken the molecular analysis of the MSP-2 gene of R falciparum isolates collected from schoolchildren living in the village of Dienga (Gabon). Using conventional microscopy and the polymerase chain reaction, 61% of these children harboured parasites without any symptom of malaria (asymptomatic status). Children with a malaria episode were those with an axillary temperature > or = 37.5 degrees C and a parasitaemia > or =800 parasites/microl of blood. Comparisons of the allelic diversity and distribution of MSP-2 gene were carried out according to the clinical status at the time of sampling. Polymorphism of the MSP-2 gene was large in both clinical groups, both asymptomatic and symptomatic (11 identified alleles). The allele FC27/560bp (base pairs) was found significantly in clinical isolates. Prevalence of the 3D7 family was 68% and 44% in asymptomatic infections and clinical infections, respectively. Multiple P. falciparum genotypes were more predominant in clinical cases (2.96 clones/child with a malaria attack vs 2.01 clones/child with asymptomatic infections). We observed also a reduction of the complexity of infection beyond the age of 10 years. These results are discussed in regard to studies conducted in other areas in Africa.

Indications d'amygdalectomie à Lubumbashi : profil clinique

A l'ère où les indications d'amygdalectomie sont constamment discutées et face à l'absence des données dans notre milieu, nous avons mené cette première étude descriptive transversale multicentrique à Lubumbashi du 1er juillet 2013 au 31 décembre 2014, afin déterminer le profil clinique de l'amygdalectomie dans notre milieu. L'analyse statistique et le traitement des données ont été effectués à l'aide des logiciels Excel version 2010 et Epi info 7 version 7.1.1.14 de 2013.Sur 84 patients opérés durant la période de la présente étude, 68 cas d'amygdalectomie ou tonsillectomie ont été colligés, ce qui a représenté 81% des activités chirurgicales oto-rhino-laryngologiques (ORL). Le sexe féminin était plus représenté (53%) avec un sex-ratio H/F de 1:1,1 (0,9). L'âge médian était de 5 [1-37] ans (âge moyen 8,5 ans) et la majorité des opérés avait un âge ≤ 5 ans. Les principaux symptômes étaient dominés par la respiration bouche ouverte pendant le sommeil, la dysphagie et le ronflement pendant le sommeil. L'hypertrophie amygdalienne dans le cadre du syndrome d'apnée obstructive du sommeil et l'amygdalite récurrente étaient les indications les plus représentées avec respectivement 53% (n = 36) et 37% (n = 25). L'amygdalectomie a été couplée à l'adénoïdectomie dans 65% des cas (n = 44). L'hémorragie primaire a été la seule complication objectivée, chez un patient (1,5%). L'amygdalectomie a été réalisée en ambulatoire dans 49% des cas.L'hypertrophie amygdalienne dans le cadre du SOAS et l'amygdalite récurrente ont constitué les indications les plus fréquentes. La maîtrise de ces indications nous éviterait les interventions superfétatoires

Association of a new mannose-binding lectin variant with severe malaria in Gabonese children.

Mannose-binding lectin (MBL2) variants that decrease the plasma level of the protein or encode dysfunctional proteins are frequently associated with the severity of a number of infections and autoimmune disorders. The high frequencies of these variants in most populations of the world are probably maintained by some selective advantage against widespread diseases. We found 14 new MBL2 allelic haplotypes, two of them with non-synonymous variants, by screening 136 children with uncomplicated malaria, 131 children with severe malaria and 39 older healthy schoolchildren. We also found a significant association of a novel variant with susceptibility to severe malaria (P=0.010). Increased MBL plasma levels and corresponding MBL2 genotypes were associated with lower concentration of several cytokines and chemokines in plasma of malaria patients. We suggest that malaria could have been one of the evolutionary driving forces shaping the MBL2 polymorphism in the African population.