A controlled trial of lambda-cyhalothrin-impregnated bed nets and/or dapsone/pyrimethamine for malaria control in Sierra Leone.

A randomized controlled trial investigated the impact of community-wide use of mosquito nets impregnated with lambda-cyhalothrin alone or with dapsone/pyrimethamine (d/p) prophylaxis on clinical malaria due to perennially transmitted Plasmodium falciparum in children in the Bo district of Sierra Leone. The 17 study communities were pair-matched and randomly allocated to receive treated mosquito nets or no nets and the children (age range = 3 months-6 years) in each community were randomly allocated to receive d/p or placebo individually every two weeks. This resulted in each of the approximately 2,000 children recruited being in one of four study groups (impregnated mosquito nets and d/p prophylaxis, impregnated mosquito nets, d/p prophylaxis, and controls). The intervention phase of the study lasted 12 months. A total of 1,800 children attended more than 25% of the 48 total weekly morbidity surveillance surveys and were included in the analysis. The effects of the exclusive use of either treated mosquito nets or d/p prophylaxis on protection against clinical malaria due to P. falciparum was significantly similar (49% and 42%, respectively), while in combination this protective efficacy was significantly increased to 72% (95% confidence interval = 67-76%). Children in the control group had an average of 1.3 clinical malaria episodes per child annually compared with 0.65 episodes or 0.78 episodes for those using treated mosquito nets and d/p, respectively. Children using both treated mosquito nets and d/p prophylaxis had an average of 0.37 episodes per child. The interventions significantly reduced spleen rates and increased hematocrit values, and reduced the duration of episodes of clinical malaria.

Hyperpigmented dermal macules in children following the administration of Maloprim for malaria chemoprophylaxis.

The occurrence of an unexpected side effect following the use of Maloprim (pyrimethamine/dapsone) for malaria chemosuppression in 3-59 months old children in Sierra Leone is reported. As part of a trial of chemoprophylaxis and insecticide-impregnated bed nets, 2000 children received either Maloprim or placebo; 4% of children who received Maloprim fortnightly for more than 3 months developed hyperpigmented macules, whereas none of the children who received placebo did so. Histopathological examination of full thickness skin biopsies showed macrophages containing melanin in the dermal layer. Clustering of cases was noted among siblings, suggesting the possible involvement of genetic factors in the pathogenesis of these skin reactions. One child was accidentally re-exposed to Maloprim after the drug had been withdrawn and he developed a severe reaction. No other serious side effect was noted. Hyperpigmented lesions similar to those reported in this study have been described previously in patients with leprosy treated with dapsone, and the dapsone component of Maloprim is the likely cause of the skin reactions seen in children given this drug for malaria chemoprophylaxis.

Effects of community-wide use of lambdacyhalothrin-impregnated bednets on malaria vectors in rural Sierra Leone.

The effect of community-wide use of bednets treated with lambdacyhalothrin 10 mg/m2 on the malaria vector Anopheles gambiae (forest form) was evaluated in Sierra Leone. Sixteen similar villages near the town of Bo were randomly allocated either to remain without nets or to receive treated bednets for all inhabitants, with effect from June 1992. Mosquitoes were sampled using human biting catches on verandas, light-trap catch (beside an occupied untreated bednet), window exit-trap catch and pyrethrum spray collections. During the first year of intervention (June 1992 to July 1993) the treated bednets provided personal protection for people sleeping under them, but had very little impact on densities of An.gambiae collected on human bait. The human blood index (HBI) of An.gambiae was not affected (HBI = 99% in villages with and without nets). An.gambiae parous rates were significantly reduced in all intervention villages, but malaria sporozoite rates fell in only some of the villages. These results are intermediate between those obtained from other projects in Tanzania and Burkina Faso, where treated bednets reduced man-biting, parity and sporozoite rates, versus The Gambia where treated bednets had no significant impact on any of these factors. Possible reasons for these contrasted findings are discussed.

Comparison of two methods for enumerating malaria parasites in thick blood films.

Calculation of parasite densities is important for estimating herd immunity to malaria, and for determining end points in field trials for interventions such as malaria vaccines, impregnated bed nets, and chemosuppression. Two methods of enumeration were compared: method 1, in which 100 consecutive high-power fields (HPFs) are examined, and if they all contain at least one parasite, the number per field is then counted in 10-100 of these fields according to density; and method 2, in which the actual number of parasites present in 100 consecutive fields are counted. The first method significantly underestimates parasite density in samples in which less than all high-power fields are parasite-positive. A correction of method 1 is suggested, which results in a parasite density, which is comparable with that obtained using method 2. The correction factor estimated was 2(-In(1 - p)), where p is the proportion of positive HPFs. The correction factor presented will allow accurate estimate of parasite densities per volume of blood even if only the proportion of parasite-positive high-power fields containing at least one parasite are counted.

Relationship between maternally derived anti-Plasmodium falciparum antibodies and risk of infection and disease in infants living in an area of Liberia, west Africa, in which malaria is highly endemic.

In areas where Plasmodium falciparum is endemic, immunoglobulin G is acquired by the fetus in utero, mainly during the third trimester of pregnancy. The potential protective effect of transferred anti-P. falciparum maternal antibodies was examined in a longitudinal study of 100 infants from birth to 1 year of age. The probability of acquiring a P. falciparum infection and developing an episode of clinical malaria was determined in relation to the P. falciparum-specific antibody level of the infant at birth against P. falciparum schizont antigen or recombinant merozoite surface protein MSP1(19) antigen. The risk of acquiring an episode of clinical malaria increased from birth to 6 months of age, after which it decreased. The overall prevalence of P. falciparum parasitemia was highest (48.9%) in the 6-month-old infants. The age-specific hematocrit value showed the lowest mean value (30.2) from 6 to 9 months, and the spleen rate was the highest (69.8%) at the same age. There was a lower risk of developing an episode of clinical malaria during the first year of life in the infants with high levels of anti-MSP1(19) antibodies at birth. The level of maternally derived overall anti-schizont antigen antibodies did not seem to play a role in the relative risk of developing malaria infection or disease during the first year of life, though the level of specific anti-MSP1(19) antibodies may be associated with protection.

Classification of clinical falciparum malaria and its use for the evaluation of chemosuppression in children under six years of age in Liberia, west Africa.

The possible role of malaria as cause of morbidity was assessed during one year in 262 children aged 6 months to 6 years living in two villages in a rural area of Liberia. The study population was followed by weekly clinics and three-monthly surveys and the children were randomly allocated to receive either chloroquine or placebo every 3 weeks. The morbidity of the children was evaluated by criteria based on the history and the clinical condition into four different stages, in order to describe the probability that an observed clinical event could be attributed to malaria infection, based on the presence of detectable parasites in the blood, the history the previous week, and the clinical status of the child. The level of anaemia, splenomegaly and measured body temperature supported that malaria was the major contributor to the overall morbidity observed. Based on the stage classification of clinical illness, children were classified as having 'possible clinical malaria' or 'probable clinical malaria'. Malaria appeared to be an important cause of febrile episodes during both dry and rainy seasons. During the rainy season more than 60% of the children experienced at least one clinical malaria episode, and during the dry season more than 50% of the children experienced at least one clinical attack of malaria. Children receiving chemosuppression had overall fewer clinical malaria attacks, and the effect of the chemosuppression was most pronounced in the dry season, the odds ratio comparing children receiving regular chemosuppression with children receiving presumptive treatment only was estimated to 0.39 (0.25-0.62).

Controlled trial of lambda-cyhalothrin impregnated bed nets and Maloprim chemosuppression to control malaria in children living in a holoendemic area of Sierra Leone, west Africa. Study design and preliminary results.

The effect of lambda-cyhalothrin impregnated bed nets and maloprim/placebo was studied in approximately 1,500 children living in 17 villages in a rural area of Sierra Leone, approximately 150 miles south east of Freetown, 30 miles north of the town of Bo. Villages were selected randomly amongst villages with impregnated bed nets and villages with no nets at all. Within these villages, children with ages ranging between 3 months to 6 years were chosen to receive maloprim or a double-blind distributed placebo fortnightly. In the villages randomised to receive nets, all beds have received nets. Malaria morbidity is estimated from weekly active case detection, and the impact on the Anopheles vector is being estimated by indoor spray catching, exit trap catching, human night landing catches and light trap catches. During the first 8 weeks of the intervention there was a significant reduction in slide positive rates, reported fever rates and children with temperature > or = 37.5 degrees C in the villages with impregnated bed nets.

The antibody response to well-defined malaria antigens after acute malaria in individuals living under continuous malaria transmission.

The IgG and IgM antibody responses to the C-terminal 783 amino acids of the P. falciparum glutamate-rich protein, GLURP489-1271, expressed as an E. coli fusion protein, the IgG response to a 18-mer synthetic peptide EDKNEKGQHEIVEVEEIL (GLURP899-916) representing the C-terminal repeats of GLURP, and a synthetic peptide (EENV)6 representing the C-terminal repeats from Pf155/RESA, were investigated longitudinally in 13 children and 7 adults living under conditions of continuous, intense malaria transmission. Some subjects did not recognize the antigens after malaria infection, and in subjects recognizing the antigens, the responses were often short-lived. In adults, the antibody responses to the GLURP489-1271 fusion protein and the (EENV)6 peptide peaked after 2 weeks, and not all individuals responded to all antigens. The antibody response, even against large fragments of conserved antigens, is not uniformly elicited by natural malaria infection in previously primed donors.

Genetic regulation of human anti-malarial antibodies in twins.

Immune responses to defined antigens may differ between individuals in a population as the reflection of differences in genetic regulation. In experimental animals, variation in responsiveness to a given epitope may be due to major histocompatibility complex (HLA, in humans) class II restrictions, implying serious limitations for the development of subunit vaccines. For human populations, knowledge of the relative importance of genetic as opposed to environmental factors affecting the immune response is scarce. We have compared antibody levels after immunization through repeated infections to a major malarial antigen (Pf155/RESA) in monozygotic twins with those in dizygotic twins, siblings, or unrelated controls. Antibody responses to the intact antigen and to some of its immunodominant epitopes were found to be more concordant within monozygotic twin pairs than in dizygotic pairs or age- and sex-matched siblings living under similar environmental conditions. The results support the conclusion that the antibody responses were genetically regulated. When the responses were assessed for possible associations with different HLA class II DRB, DQA, and DQB alleles had haplotypes, no associations were found. This suggests that the regulation of the Pf155/RESA antibody responses seen in this study reflects the impact of factors encoded by genes outside the HLA class II regions.

The effect of splenectomy on immunity to Plasmodium malariae and P. falciparum in a malaria immune donor.

A 55 year old Liberian male was splenectomized after an abdominal trauma. A few days after splenectomy he experienced a pure Plasmodium malariae infection with high fever. He was later followed for 12 months with monthly blood films and temperature measurements, and did never show any signs of clinical malaria. The parasite densities observed during the longitudinal follow after splenectomy did not differ from parasite densities in villagers with intact spleens.

Clinical and parasitological studies on malaria in Liberian adults living under intense malaria transmission.

Occurrence of fevers and chills, headaches and body and joint pains, and body temperature and malaria parasitaemias were recorded monthly for a year for 121 Liberian adults. There was no apparent correlation between any of the symptoms and the presence or density of blood parasites; it was therefore not possible to define a case of clinical malaria in the study population, which was probably highly immune to infection. Only a few people with patent blood infections had elevated blood temperatures and these were below 37.5 degrees C. Malaria prevalence and levels of parasitaemia declined with age and indicated that immunity continues to develop well into adult age. The data did not support the view that adults experience symptoms at lower parasitaemias than children. Pregnant and non-pregnant women had similar levels of symptoms, but high levels of parasitaemia were found more frequently in the pregnant group.

Development of immunity against Plasmodium falciparum malaria: clinical and parasitologic immunity cannot be separated.

A total of 1622 individuals of all ages living under conditions of continuous malarial transmission in Liberia were enrolled in a cross-sectional study of parasite rates, positive parasite densities, and body temperatures. The age-specific Plasmodium falciparum-positive parasite densities were greatest at ages 0.5-1.0 year, then slowly declined into adulthood. The age-specific mean body temperature at parasite isodensity showed a steady decline even in the oldest age group. The results do not support the hypothesis that adults have higher body temperatures at a given parasite density than do children with the same parasite density. The age-specific P. falciparum parasite density for specific isotemperatures showed that a subgroup of children in the age group 0.5-1.0 year had low temperatures (less than 36.5 degrees C) despite high parasite densities. This indicates that low body temperature should be investigated further as a possible indicator of serious malaria in young children. Parasitologic and clinical immunity develops concomitantly and cannot be separated. The findings do not support the hypothesis that a special "anti-disease" immunity exists independently of parasitologic immunity.

A longitudinal study of seroreactivities to Plasmodium falciparum antigens in infants and children living in a holoendemic area of Liberia.

Investigators studied 348 children age 0-10 years, living in a holoendemic area of Liberia, for parasitological, serological and clinical parameters. The age-specific parasite rate increased towards the 7-10 year-old age group in which it was 86.8%. The geometrical mean parasite density decreased from the 3-4 year-old age group, in which fewer episodes of clinical malaria were observed. Antibodies to crude Plasmodium falciparum parasite antigens were detected in all children. The (EENV)6 seropositive rate was a maximum of 67.9% in the 3-11 month-old age group. It declined to a minimum of 31.7% in the 5-6 years age group after which it increased slowly in the 7-10 years age group. Antibodies to the synthetic peptide (NANP)6 showed a steady seropositive rate after the age of 3 months, between 30.0% and 39.3% in all the age groups up to 10 years. No statistically significant correlation was found between seropositivity to (EENV)6 and malarial parasitemia. In contrast, a statistically significant positive correlation was found between seropositivity to (NANP)6 and parasite rates. The antibody response for the individual child was transient to both Pf155/RESA, measured by immunofluorescence, and to (EENV)6 and (NANP)6, measured by ELISA, especially in the younger age groups of this study population. Parasitological and clinical immunity developed before a stable antibody response to these defined malaria antigens was established. These antibodies may still contribute to the immune protection against malaria, but they were not reliable parameters for protective immunity in the population we studied.

Characterization of regulatory T cell responses to defined immunodominant T cell epitopes of the Plasmodium falciparum antigen Pf155/RESA.

Several immunodominant B and T cell epitopes of the P. falciparum blood stage antigen Pf155/RESA, a vaccine candidate, are located in the central (5') and C-terminal (3') invariant repeat regions of the molecule. Here we have attempted to functionally analyze human T cell responses to some of the T cell epitopes. For this purpose short synthetic peptides corresponding to these epitopes were used to study the induction of in vitro expression of IL-4 mRNA, IFN-gamma secretion, proliferation and B cell help for antibody production. In individual malaria immune donors these different T cell activities were not correlated. The findings emphasize the importance of examining multiple parameters of T cell activation when estimating the total proportion of individuals responding to a defined antigen. IL-4 mRNA was expressed in activated T cells of donors who had elevated serum concentrations of antibodies to the peptide used for T cell activation. These results suggest the involvement of IL-4 producing T helper cells in the induction of Pf155/RESA specific antibody production in individuals in which immunity has been induced by natural infection. Taken together, these findings also suggest that functionally distinct CD4+ T cells occur in humans similarly to what has been described in mice. In further experiments, we have also attempted to establish MHC class II restriction of the immune response to these epitopes at the level of the donor populations. When studying monozygotic twins, antibody responses to Pf155/RESA derived peptides and some of the T cell responses could be paired within the twin pairs, indicating a genetic regulation of their B cell responses. Whether or not this regulation reflects MHC class II restriction, or other factors needs to be elucidated.

A longitudinal study of antibodies to the Plasmodium falciparum antigen Pf155/RESA and immunity to malaria infection in adult Liberians.

118 adult Liberians from 2 villages were studied prospectively for one year with monthly blood examinations for malaria parasites. The crude parasite rate was 41.5% and the crude gametocyte rate was 6.1%. The inoculation rate varied between 0.075 in the dry season and almost 0.4 in the rainy season, which is in accordance with other data from holoendemic areas. 47.5% (56) had a titre to the Pf155/RESA antigen less than or equal to 1/50 ('low responders') and 52.5% (62) had a titre of greater than or equal to 1/250 ('high responders'). The response was not age-dependent in this adult population, which may suggest that genetic factors are determining whether the individual become a high or low responder. Antibodies against the Pf155/RESA antigen were measured in 2 surveys 8 months apart, and the mean antibody response to Pf155/RESA and its EENV sequence was constant without seasonal variation. Pf155/RESA high responders had lower parasite densities during all 3 seasons surveyed, and Pf155/RESA high responders, with high antibody reactivity against the (EENV)6 sequence from the 3' repeat region of Pf155/RESA, had significantly lower parasite densities in the rainy season of 1987. The data suggest that high titres of antibodies to the Pf155/RESA antigen, and especially to its EENV sequence, might play a role in protective immunity in adults.

An epidemiological study of humoral and cell-mediated immune response to the Plasmodium falciparum antigen PF155/RESA in adult Liberians.

We investigated the seroreactivity and T cell reactivity against the Plasmodium falciparum antigen Pf155/RESA, different oligopeptides from the 3' and 5' repeat regions of the Pf155/RESA antigen, and crude Plasmodium falciparum antigens in 164 adult Liberians. We compared 2 long-term residential groups with high and low exposure to malaria. The seropositive rate to the peptides was significantly higher with increased exposure. There was no significant difference in response rates to the Pf155/RESA. This may indicate the level of persistent T cell memory in previously primed donors. The seropositive rates to 3 Pf155/RESA peptides and the rates measured by either 3H-thymidine incorporation or IFN-gamma release after stimulation with Pf155/RESA and the peptides were all lower in parasite positive individuals. Even low grade, asymptomatic parasitemia can impair the T cell response in vitro. The lower antibody response in parasite positive subjects may be explained by either antibody consumption or lower protection against malaria parasitemia in subjects with low concentrations of antibodies against the Pf155/RESA antigen.