A principal target of human immunity to malaria identified by molecular population genetic and immunological analyses.

New strategies are required to identify the most important targets of protective immunity in complex eukaryotic pathogens. Natural selection maintains allelic variation in some antigens of the malaria parasite Plasmodium falciparum. Analysis of allele frequency distributions could identify the loci under most intense selection. The merozoite surface protein 1 (Msp1) is the most-abundant surface component on the erythrocyte-invading stage of P. falciparum. Immunization with whole Msp1 has protected monkeys completely against homologous and partially against non-homologous parasite strains. The single-copy msp1 gene, of about 5 kilobases, has highly divergent alleles with stable frequencies in endemic populations. To identify the region of msp1 under strongest selection to maintain alleles within populations, we studied multiple intragenic sequence loci in populations in different regions of Africa and Southeast Asia. On both continents, the locus with the lowest inter-population variance in allele frequencies was block 2, indicating selection in this part of the gene. To test the hypothesis of immune selection, we undertook a large prospective longitudinal cohort study. This demonstrated that serum IgG antibodies against each of the two most frequent allelic types of block 2 of the protein were strongly associated with protection from P. falciparum malaria.

Management of severe malarial anaemia in Gambian children.

The optimum management of children with severe malarial anaemia is still uncertain. Hence, we have undertaken a study to determine whether iron treatment is as effective at restoring haemoglobin levels one month after presentation as blood transfusion without iron treatment in children with moderately severe malarial anaemia. Two hundred and eighty-seven children with a packed cell volume (PCV) < 15% and malaria infection were recruited into the study; 173 children were assigned to receive blood transfusion because they had a PCV < 12% and/or signs of respiratory distress and the remaining 114 children were allocated at random to receive either blood transfusion (58) or treatment with oral iron (56) for 28 d. Twenty-four children died, 23 in the most severely anaemic group. Fifteen children (65%) died before transfusion was given and most deaths occurred within the first 4 h of admission. One child died in the iron treatment group and 10 subsequently required transfusion. Among the severely anaemic children, those with respiratory distress were at greater risk of death than those without respiratory distress. After 28 d, haematological restoration was significantly better in children who had received iron than in those treated by blood transfusion (P = 0.02). Children who received malaria chemoprophylaxis after discharge from hospital had fewer episodes of malaria and subsequent admissions to a hospital or health centre than those who did not. Children with severe anaemia and clinical signs of respiratory distress must be identified quickly and transfused as soon as possible. However, for less severely anaemic children who are clinically stable, iron therapy offers an alternative to transfusion provided such children can be kept under surveillance and transfused subsequently should this become necessary.

A trial of Fansidar plus chloroquine or Fansidar alone for the treatment of uncomplicated malaria in Gambian children.

Chloroquine can no longer be recommended as the first-line treatment for uncomplicated malaria in several parts of Africa because of the increasing prevalence of chloroquine resistance. However, chloroquine was a highly effective treatment for malaria not only because of its ability to kill parasites quickly but also because it is an anti-inflammatory drug. Therefore, we have investigated whether Fansidar (pyrimethamine/sulfadoxine) plus chloroquine is a more effective treatment for uncomplicated malaria than Fansidar alone. Four hundred and five Gambian children with uncomplicated Plasmodium falciparum malaria were studied in a randomized controlled trial. Significantly more children treated with Fansidar alone, compared to those treated with Fansidar plus chloroquine (19/203 vs. 2/202; P < 0.001), returned to the clinic with persistent symptoms during the first 3 d after treatment. Three children who had received Fansidar alone had fits, but none of the children treated with Fansidar plus chloroquine did so. At the day 7 follow-up, the parasite failure rate in the Fansidar alone group was 3/198 (1.5%), whilst in the Fansidar plus chloroquine group it was 3/201 (1.5%). At the day 28 follow-up, there was still no significant difference between the parasite failure rate in the Fansidar alone group (15/150; 10.0%) and the Fansidar plus chloroquine group (7/141; 5.0%) and the mean packed cell volume (PCV) in the 2 groups was similar. Thus, a combination of Fansidar plus chloroquine was a more effective symptomatic treatment than Fansidar given alone, but neither the parasite cure rate nor the PCV was enhanced by use of the combination.

The diagnosis of Plasmodium falciparum infection in Gambian children, by field staff using the rapid, manual, ParaSight-F test.

A rapid immunodiagnostic test for Plasmodium falciparum, the ParaSight-F test, was evaluated in the diagnosis of malaria in 139 children with uncomplicated malaria, who presented at the Medical Research Council's clinic at Basse in Upper River division, The Gambia. The aim was to evaluate the performance and usefulness of the test as a diagnostic method in a malaria-endemic area, when performed by a field worker. Compared with microscopy, the test had a sensitivity of 96.5%, a specificity of 90.5%, a negative predictive value of 94.2% and a positive predictive value of 94.3%. Because of its sensitivity, specificity and simplicity, the ParaSight-F test will be of value in situations where microscopy is not possible.

A prospective evaluation of a clinical algorithm for the diagnosis of malaria in Gambian children.

Diagnosis of clinical malaria remains difficult, especially in areas where a high proportion of the asymptomatic population have parasitaemia, for the symptoms and signs of malaria overlap with those of other common childhood diseases, such as acute lower respiratory tract infections. However, a study of symptoms and signs in a group of children who presented to Farafenni Health Centre, The Gambia with a history of recent fever identified a group of signs and symptoms which were strong predictors of malaria as opposed to other febrile illnesses. Using these predictors, an algorithm was developed which could be used by fieldworkers and which had a similar sensitivity and specificity for the diagnosis of malaria as that of an experienced paediatrician working without laboratory support. This algorithm has been validated prospectively on 518 children who presented to the Medical Research Council clinic at Basse, The Gambia with fever or a history of recent fever during a 10-month period. A fieldworker obtained a detailed history from the parent or guardian of each child and performed a clinical examination which included measurement of axillary temperature and respiratory rate. Packed cell volume was measured and a thick smear was examined for malaria parasites. A malaria score, based on the presence or absence of malaria-related signs and symptoms, was determined for 382 children who were seen at the clinic during the high transmission season. Using the cut-off score which was optimal during the previous retrospective study, a sensitivity of 70% and a specificity of 77% for a diagnosis of malaria was obtained. The optimal cut-off score for the Basse population was a score of 7; this gave a sensitivity of 88% and a specificity of 62%, figures comparable to those obtained by an experienced paediatrician without laboratory support.

Predictors of mortality in Gambian children with severe malaria anaemia.

Severe malaria anaemia is a frequent cause of admission to hospital in tropical Africa and about 10% of children with this condition die. To determine ways in which mortality might be reduced we have studied risk factors for a fatal outcome in 173 children with severe malaria anaemia who were assigned to receive blood transfusion because they had a packed cell volume of less than 12% and/or signs of respiratory distress. Twenty-three children died (13%); in 15 cases (65%) death occurred before blood transfusion was given. The presence of respiratory distress was found to be the most important predictor of death. Children with severe malaria anaemia and signs of respiratory distress must therefore be transfused as soon as possible.

PIP: Severe anemia is a major cause of morbidity and mortality among children in Africa. In areas of moderate seasonal transmission, cerebral malaria is the dominant form of severe malaria. Severe malaria anemia is a frequent cause of admission to hospital in tropical Africa, and results in the death of about 10% of children with the condition. While children with severe malaria anemia can often be saved from death by blood transfusion, deaths from the condition continue to occur even in centers in which transfusion is available. Findings are presented from a study conducted at Royal Victoria Hospital, Banjul, the main referral hospital in The Gambia, to determine how such mortality may be reduced. The authors studied risk factors for a fatal outcome in 173 children of mean age 25.3 months with severe malaria anemia who were assigned to undergo blood transfusion because they had a packed cell volume of less than 12% and/or signs of respiratory distress. 15 of the 23 children who died did so before receiving transfused blood. The presence of respiratory distress was found to be the most important predictor of death. These findings suggest that children with severe malaria anemia and signs of respiratory distress should be transfused as soon as possible.

Follow-up of Gambian children recruited to a pilot safety and immunogenicity study of the malaria vaccine SPf66.

A pilot safety and immunogenicity trial of the malaria vaccine SPf66 was undertaken in The Gambia in 1993. One hundred and fifty infants aged 6-11 months were immunized with either 0.5 mg or 1.0 mg of SPf66 produced either in Colombia or in the USA or with a control vaccine. Children who received SPf66 experienced more clinical attacks of malaria than did children in the control group during the first period of surveillance and the difference in incidence between children who had received high dose Colombian vaccine and the control children was statistically significant at the 5% level. During the 1995 malaria transmission season, 127 children from the original cohort of 150 were observed. During 18 weeks of intensive surveillance, the incidence of clinical malaria was again higher among children who had received SPf66 than among children who had received inactivated polio vaccine (6.23 vs 4.89 clinical attacks per 1000 days at risk), the effect being most marked among children who were in the high dose groups, but differences between groups were now no longer statistically significant.

PIP: 150 human subjects aged 6-11 months were involved in a pilot safety and immunogenicity trial of the malaria vaccine SPf66 conducted in The Gambia in 1993. The infants were immunized with either 0.5 mg or 1.0 mg of the vaccine produced in either Colombia or the US, or with a control vaccine. Children who received SPf66 experienced more clinical attacks of malaria than did children in the control group during the first period of surveillance, with the difference in incidence between children who had received high dose Colombian vaccine and the control children being statistically significant. 127 children from the original cohort of 150 were observed during the 1995 malaria transmission season. During 18 weeks of intensive surveillance, the incidence of clinical malaria was again higher among children who had received SPf66 than among children who had received inactivated polio vaccine. The effect was most marked among children in the high dose groups, although the intergroup differences were statistically insignificant. The SPf66 vaccine may have induced an immune response which made the immunized children more susceptible to malaria. It is also possible that the increased susceptibility to malaria among children who received SPf66 was a chance event following the randomization process. No enhancement of either disease frequency or severity was found in a much larger efficacy trial of Colombian SPf66 conducted among Gambian children during a 2-year follow-up period.

An efficacy trial of the malaria vaccine SPf66 in Gambian infants--second year of follow-up.

In 1994, 630 Gambian infants were immunized with three doses of the synthetic polypeptide malaria vaccine SPf66 or with a control vaccine. No significant protection against first or total attacks of malaria was observed among the children who received SPf66. However, the period of follow-up was short. Thus, 532 children were followed for a second malaria transmission season during which 291 episodes of malaria were detected. Protective efficacies of SPf66 against first attacks of malaria and against all attacks of malaria were 8% [95% CI-20%, 30%] and 2% [95% CI-26% 24%] respectively. SPf66 did not provide any significant degree of protection to Gambian infants during a second year of follow-up.

Unique T cell effector functions elicited by Plasmodium falciparum epitopes in malaria-exposed Africans tested by three T cell assays.

Natural immunity to malaria is characterized by low level CD4 T cell reactivity detected by either lymphoproliferation or IFN-gamma secretion. Here we show a doubling in the detection rate of responders to the carboxyl terminus of circumsporozoite protein (CS) of Plasmodium falciparum by employing three T cell assays simultaneously: rapid IFN-gamma secretion (ex vivo ELISPOT), IFN-gamma secretion after reactivation of memory T cells and expansion in vitro (cultured ELISPOT), and lymphoproliferation. Remarkably, for no individual peptide did a positive response for one T cell effector function correlate with any other. Thus these CS epitopes elicited unique T cell response patterns in malaria-exposed donors. Novel or important epitope responses may therefore be missed if only one T cell assay is employed. A borderline correlation was found between anti-CS Ab levels and proliferative responses, but no correlation was found with ex vivo or cultured IFN-gamma responses. This suggested that the proliferating population, but not the IFN-gamma-secreting cells, contained cells that provide help for Ab production. The data suggest that natural immunity to malaria is a complex function of T cell subgroups with different effector functions and has important implications for future studies of natural T cell immunity.

Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomised trial.

BACKGROUND: RTS,S/AS02 is a pre-erythrocytic malaria vaccine based on the circumsporozoite surface protein of Plasmodium falciparum fused to HBsAg, incorporating a new adjuvant (AS02). We did a randomised trial of the efficacy of RTS,S/AS02 against natural P. falciparum infection in semi-immune adult men in The Gambia. METHODS: 306 men aged 18-45 years were randomly assigned three doses of either RTS,S/AS02 or rabies vaccine (control). Volunteers were given sulfadoxine/pyrimethamine 2 weeks before dose 3, and kept under surveillance throughout the malaria transmission season. Blood smears were collected once a week and whenever a volunteer developed symptoms compatible with malaria. The primary endpoint was time to first infection with P. falciparum. Analysis was per protocol. FINDINGS: 250 men (131 in the RTS,S/AS02 group and 119 in the control group) received three doses of vaccine and were followed up for 15 weeks. RTS,S/AS02 was safe and well tolerated. P. falciparum infections occurred significantly earlier in the control group than the RTS,S/AS02 group (Wilcoxon's test p=0.018). Vaccine efficacy, adjusted for confounders, was 34% (95% CI 8.0-53, p=0.014). Protection seemed to wane: estimated efficacy during the first 9 weeks of follow-up was 71% (46-85), but decreased to 0% (-52 to 34) in the last 6 weeks. Vaccination induced strong antibody responses to circumsporozoite protein and strong T-cell responses. Protection was not limited to the NF54 parasite genotype from which the vaccine was derived. 158 men received a fourth dose the next year and were followed up for 9 weeks; during this time, vaccine efficacy was 47% (4-71, p=0.037). INTERPRETATION: RTS,S/AS02 is safe, immunogenic, and is the first pre-erythrocytic vaccine to show significant protection against natural P. falciparum infection.