Levels Of Malaria Transmission Research Articles

Evidence for Multiple B- and T-Cell Epitopes inPlasmodium falciparumLiver-Stage Antigen 3

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n = 143) and Ndiop (n = 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.

Plasmodium falciparum gametocyte dynamics in areas of different malaria endemicity

BackgroundThe aim of this study was to identify and compare factors associated with Plasmodium falciparum gametocyte carriage in three regions of differing malaria endemicity.MethodsRetrospective data from Thailand, The Gambia and Tanzania were used. The data came from large prospective field-based clinical trials, which investigated gametocyte carriage after different anti-malarial drug treatments.ResultsGametocytaemia was detected during the observation period in 12% of patients (931 out of 7548) in Thailand, 34% (683 out of 2020) in The Gambia, and 31% (430 out of 1400) in Tanzania (p < 0.001). Approximately one third (33%, 680/2044) of the patients with gametocytaemia during the observation period, already had patent gametocytaemia at enrolment (day 0 or day 1): 35% (318/931) in Thailand, 37% (250/683) in The Gambia, 26% (112/430) in Tanzania. Maximum gametocytaemia was usually observed on or before the seventh day after starting treatment (93% in Thailand, 70% in Tanzania and 78% in The Gambia). Lowest gametocyte carriage rates were observed following treatment with artemisinin derivatives, while sulphadoxine-pyrimethamine (SP) was associated with significantly greater development of gametocytaemia than other drug treatments (p < 0.001). The duration of gametocyte carriage was shorter in Thailand by 86% and Tanzania by 65% than in The Gambia. Gametocyte carriage was 27% longer among people presenting with anaemia, and was shorter in duration among patients who received artemisinin derivatives, by 27% in Thailand and by 71% in Tanzania and The Gambia.ConclusionThis study confirms the independent association of gametocytaemia with anaemia, and the significantly lower prevalence and duration of gametocyte carriage following treatment with an artemisinin derivative. The large differences in gametocyte carriage rates between regions with different levels of malaria transmission suggest that drug interventions to prevent transmission will have different effects in different places.

Trends in malaria morbidity following the introduction of artesunate plus amodiaquine combination in M'lomp village dispensary, south-western Senegal

BackgroundIn Thailand, South Africa and Zanzibar, a decrease in malaria morbidity was observed following the introduction of artemisinin-based combination therapy (ACT). In Senegal, therapeutic trials supervised the in vivo efficacy of artesunate plus amodiaquine from 1999 to 2005 at the M'lomp village dispensary. The trends in malaria morbidity in this village were evaluated from 2000 to 2002.MethodsEach year, between July and December inclusive, fevers treated with antimalarials and slide-proven, uncomplicated malaria cases were collected from dispensary health records. Data were also collected in 1998, just prior to ACT introduction. Pearson's chi square tests and Student tests were used to compare two percentages or two means respectively (α = 0.05).ResultsBetween 1998 and 2002, the total number of fevers treated with antimalarials and their repetitiveness progressively decreased: From 2824 to 945 fevers and from 17.6% to 9.7% (RR1998–2002 = 0.55; [0.44–0.69]; p < 0.0001) respectively. Considering uncomplicated malaria cases only, a decrease was observed in their total number between 2001 and 2002, from 953 to 570 cases. The incidence rate and repetitiveness also decreased. The incidence rate fell from 46.1% in 2001 to 37.5% in 2002 (p < 0.0001) and the repetitiveness decreased from 13.0% in 2000 to 6.6% in 2002 (RR2000–2002 = 0.51; [0.35–0.72]; p = 0.0001).ConclusionThe percentage of uncomplicated malaria cases treated with ACT increased, from 18.9% in 2000 to 64.0% in 2002, making it tempting to conclude an impact on malaria morbidity. Nonetheless, the decline in incidence rate of uncomplicated malaria was slight and a lower recorded rainfall was reported in 2002 which could also explain this decline. The context in which ACT is introduced affects the impact on malaria morbidity. In M'lomp, in contrast to studies in Thailand, South Africa and Zanzibar, ACT coverage of malaria cases was low and no vector control measure was deployed. Moreover, the malaria transmission level is higher. In sub-Saharan countries, in order to optimize the impact on malaria morbidity, ACT deployment must be supported, on the one hand, by a strengthening of public health system to ensure a high ACT coverage and, on the other hand, by others measures, such vector control measures.

Low Prevalence of Antibodies to Preerythrocytic but Not Blood-StagePlasmodium falciparumAntigens in an Area of Unstable Malaria Transmission Compared to Prevalence in an Area of Stable Malaria Transmission

In areas where levels of transmission of Plasmodium falciparum are high and stable, the age-related acquisition of high-level immunoglobulin G (IgG) antibodies to preerythrocytic circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) has been associated with protection from clinical malaria. In contrast, age-related protection from malaria develops slowly or not at all in residents of epidemic-prone areas with unstable low levels of malaria transmission. We hypothesized that this suboptimal clinical and parasitological immunity may in part be due to reduced antibodies to CSP or LSA-1 and/or vaccine candidate blood-stage antigens. Frequencies and levels of IgG antibodies to CSP, LSA-1, thrombospondin-related adhesive protein (TRAP), apical membrane antigen 1 (AMA-1), erythrocyte binding antigen 175 (EBA-175), and merozoite surface protein 1 (MSP-1) were compared in 243 Kenyans living in a highland area of unstable transmission and 210 residents of a nearby lowland area of stable transmission. Levels of antibodies to CSP, LSA-1, TRAP, and AMA-1 in the oldest age group (>40 years) in the unstable transmission area were lower than or similar to those of children 2 to 6 years old in the stable transmission area. Only 3.3% of individuals in the unstable transmission area had high levels of IgG (>2 arbitrary units) to both CSP and LSA-1, compared to 43.3% of individuals in the stable transmission area. In contrast, antibody levels to and frequencies of MSP-1 and EBA-175 were similar in adults in areas of stable and unstable malaria transmission. Suboptimal immunity to malaria in areas of unstable malaria transmission may relate in part to infrequent high-level antibodies to preerythrocytic antigens and AMA-1.

Antimalarial drug use in general populations of tropical Africa

BackgroundThe burden of Plasmodium falciparum malaria has worsened because of the emergence of chloroquine resistance. Antimalarial drug use and drug pressure are critical factors contributing to the selection and spread of resistance. The present study explores the geographical, socio-economic and behavioural factors associated with the use of antimalarial drugs in Africa.MethodsThe presence of chloroquine (CQ), pyrimethamine (PYR) and other antimalarial drugs has been evaluated by immuno-capture and high-performance liquid chromatography in the urine samples of 3,052 children (2–9 y), randomly drawn in 2003 from the general populations at 30 sites in Senegal (10), Burkina-Faso (10) and Cameroon (10). Questionnaires have been administered to the parents of sampled children and to a random sample of households in each site. The presence of CQ in urine was analysed as dependent variable according to individual and site characteristics using a random – effect logistic regression model to take into account the interdependency of observations made within the same site.ResultsAccording to the sites, the prevalence rates of CQ and PYR ranged from 9% to 91% and from 0% to 21%, respectively. In multivariate analysis, the presence of CQ in urine was significantly associated with a history of fever during the three days preceding urine sampling (OR = 1.22, p = 0.043), socio-economic level of the population of the sites (OR = 2.74, p = 0.029), age (2–5 y = reference level; 6–9 y OR = 0.76, p = 0.002), prevalence of anti-circumsporozoite protein (CSP) antibodies (low prevalence: reference level; intermediate level OR = 2.47, p = 0.023), proportion of inhabitants who lived in another site one year before (OR = 2.53, p = 0.003), and duration to reach the nearest tarmacked road (duration less than one hour = reference level, duration equal to or more than one hour OR = 0.49, p = 0.019).ConclusionAntimalarial drug pressure varied considerably from one site to another. It was significantly higher in areas with intermediate malaria transmission level and in the most accessible sites. Thus, P. falciparum strains arriving in cross-road sites or in areas with intermediate malaria transmission are exposed to higher drug pressure, which could favour the selection and the spread of drug resistance.

A mathematical model for the dynamics of malaria in mosquitoes feeding on a heterogeneous host population

We describe and develop a difference equation model for the dynamics of malaria in a mosquito population feeding on, infecting and getting infected from a heterogeneous population of hosts. Using the force of infection from different classes of humans to mosquitoes as parameters, we evaluate a number of entomological parameters, indicating malaria transmission levels, which can be compared to field data. By assigning different types of vector control interventions to different classes of humans and by evaluating the corresponding levels of malaria transmission, we can compare the effectiveness of these interventions. We show a numerical example of the effects of increasing coverage of insecticide-treated bed nets in a human population where the predominant malaria vector is Anopheles gambiae.

HIV and malaria co-infection: interactions and consequences of chemotherapy

The global epidemiology of HIV/AIDS and malaria overlap because a significant number of HIV-infected individuals live in regions with different levels of malaria transmission. Although the consequences of co-infection with HIV and malaria parasites are not fully understood, available evidence suggests that the infections act synergistically and together result in worse outcomes. The importance of understanding chemotherapeutic interactions during malaria and HIV co-infection is now being recognized. We know that some antimalarial drugs have weak antiretroviral effects; however, recent studies have also demonstrated that certain antiretroviral agents can inhibit malaria-parasite growth. Here, we discuss recent findings on the impact of HIV/AIDS and malaria co-infection and the possible roles of chemotherapy in improving the treatment of these diseases.

Differences in Human Antibody Reactivity to Plasmodium falciparum Variant Surface Antigens Are Dependent on Age and Malaria Transmission Intensity in Northeastern Tanzania

Plasmodium falciparum variant surface antigens (VSA) are involved in the pathogenesis of malaria. Immunoglobulin G (IgG) with specificity for VSA (anti-VSA IgG) is therefore considered important for acquired immunity. To better understand the nature and dynamics of variant-specific IgG responses at population level, we conducted an immunoepidemiological study in nearby communities in northeastern Tanzania, situated at different altitudes and therefore exposed to different levels of P. falciparum transmission intensity. Samples of plasma and infected red blood cells (IRBC) were collected from 759 individuals aged 0 to 19 years. Plasma levels of IgG with specificity for VSA expressed by a panel of different parasite isolates were measured by flow cytometry, while the ability of plasma to inhibit IRBC adhesion to CD36 was examined in cellular assays. The level and repertoire of the heterologous anti-VSA IgG response developed dramatically in individuals at 1 to 2 years of age in the high-transmission area, reaching a maximum level at around 10 years of age; only a modest further increase was observed among older children and adults. In contrast, at lower levels of malaria transmission, anti-VSA IgG levels were lower and the repertoire was more narrow, and similar age- and transmission-dependent differences were observed with regard to the ability of the plasma samples to inhibit adhesion of IRBC to CD36. These differences indicate a strong and dynamic relationship between malaria exposure and functional characteristics of the variant-specific antibody response, which is likely to be important for protection against malaria.

Do antibody responses to malaria vaccine candidates influenced by the level of malaria transmission protect from malaria?

To examine whether the humoural response to malaria vaccine candidate antigens, Plasmodium falciparum [circumsporozoite repetitive sequence (NANP)(5) GLURP fragments (R0 and R2) and MSP3] varies with the level of malaria transmission and to determine whether the antibodies (IgG) present at the beginning of the malaria transmission season protect against clinical malaria. Cross-sectional surveys were conducted to measure antibody response before, at the peak and at the end of the transmission season in children aged 6 months to 10 years in two villages with different levels of malaria transmission. A cohort study was performed to estimate the incidence of clinical malaria. Antibodies to these antigens showed different seasonal patterns. IgG concentrations to any of the four antigens were higher in the village with high entomological inoculation rate. Multivariate analysis of combined data from the two villages indicated that children who were classified as responders to the selected antigens were at lower risk of clinical malaria than children classified as non-responders [(NANP)(5) (incidence rate ratio (IRR) = 0.65, 95% CI: 0.46-0.92; P = 0.016), R0 (IRR = 0.69, 95% CI: 0.48-0.97; P = 0.032), R2 (IRR = 0.73, 95% CI: 0.50-1.06; P = 0.09), MSP3 (IRR = 0.52, 95% CI: 0.32-0.85; P = 0.009)]. Fitting a model with all four antibody responses showed that MSP3 looked the best malaria vaccine candidate (IRR = 0.63; 95% CI: 0.38-1.05; P = 0.08). Antibody levels to the four antigens are affected by the intensity of malaria transmission and associated with protection against clinical malaria. It is worthwhile investing in the development of these antigens as potential malaria vaccine candidates.

The impact of response to the results of diagnostic tests for malaria: cost-benefit analysis

Objective Rapid diagnostic tests for malaria seem cost effective in standard analyses, but these do not take account of clinicians’ response to test results. This study tested the impact of...

Social marketing of insecticide-treated bed net for malaria control: an experience in a semi-urban community in south-south Nigeria

Background: The effectiveness of the insecticide-treated bed net in reducing the morbidity and mortality associated with malaria has been proved at all levels of malaria transmission. Several models on how to achieve massive coverage have been suggested, but social marketing of the nets is highly favoured for its sustainability. Aim: To report the experience of a small-scale social marketing project for insecticide-treated bed net in a semi-urban community in south-south Nigeria. Methods: The social marketing project was established in 2003 in Egbema, a semi-urban community in Rivers State, with a population of 47,000. A descriptive cross-sectional study design was used for the study, with the sales records of the project and a structured, interviewer-administered questionnaire as study tools. The sales records were analysed to assess the performance of the project, while the questionnaire was used to collect data on the socio-economic characteristics of buyers of the net. Results: In six months, the project achieved an uptake rate of 1.10 ITN per month, per 1000 population, and a 6.5% coverage of the target population. Most, 208 (67%), of the paid up sales were achieved at the well-child clinic and the antenatal clinic of the health facilities that serve the community. Buyers in the two lower socio-economic quartiles bought only about one third of the nets. Members of the community were predominantly farmers/fishermen, but only 19 (10%) of the buyers of the nets identified themselves as such. Conclusion: The study shows that the use of social marketing for promoting the use of ITN for malaria control is slow in a poor community. Port Harcourt Medical Journal Vol. 1 (3) 2007: pp. 145-150

Discrimination of Seven Anopheles Species from San Pedro de Urabá, Antioquia, Colombia, by Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Analysis of Its Sequences

Accurate identification of anopheline species is essential for vector incrimination and implementation of appropriate control strategies. Several anopheline species are considered important malaria vectors in Colombia; however, species determination is complicated by cryptic morphology and intra-individual variation. We describe polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer 2 (ITS2) sequences for differentiation of seven Anopheles species collected in a locality in Antioquia, Colombia, with high levels of malaria transmission. Each of these seven species can be identified by unique AluI PCR-RFLP restriction patterns. Comparisons of morphologic identification with molecular identification of voucher specimens confirmed species designation for 886 wild-caught anophelines. This new method can be used as a diagnostic tool for discrimination of anopheline species of medical importance in this region, some of which have overlapping morphologic characters and for conducting complementary studies where rapid and accurate identification of large numbers of specimens is needed.

Field-based evidence for the linkage of pfcrt and pfdhfr drug-resistant malaria genotypes and clinical profiles of severe malaria in Niger

Drug resistance has been shown to increase malaria mortality and morbidity in both community- and hospital-based studies. We investigated the association between two Plasmodium falciparum drug resistance-related molecular markers and clinical profiles of severe malaria in children hospitalised in Niger. PCR-RFLP analysis showed that the codon 108 mutation of the pfdhfr gene was positively linked to severe malarial anaemia. These findings are consistent with persistent parasite infection leading to unbalanced anaemia in young children. No significant relationship was found between the molecular markers and hypoglycaemia or hyperparasitaemia. Conversely, the pfcrt T76 mutation was found to be negatively associated with cerebral malaria and neurological symptoms, such as convulsions and coma. These results have implications for the strain-specific virulence hypothesis and for parasite fitness and evolution. Our findings are discussed in regard to the local malaria transmission level.

The relationship between malaria parasitaemia and availability of healthcare facility in Mpwapwa District, central Tanzania

A study was carried out in six villages located at different altitudes in Mpwapwa district of central Tanzania to determine malaria parasitaemia and transmission levels in villages with or without health care facilities. A total of 1119 schoolchildren (age = 5.9-12.3 years) were examined for malaria parasitaemia. Plasmodiumfalciparum was the predominant malaria species accounting for 92.8% of all species. The average malaria prevalence rate among schoolchildren was 25.8% (range 1.5-53.8%). The geometric mean parasite densities for P.falciparum was 361 (N = 286). Higher malaria prevalence was observed in villages at lower (< 1000 m) than at intermediate (1000-1500m) or higher (> 1500m) altitudes. Schoolchildren in areas with health care facilities were less at risk of acquiring malaria by 33.4% as compared with those living in areas without health facilities. Mean packed cell volume in schoolchildren was 38.5% (range = 35.2-41.0%). Splenomegaly was observed in 18.1% (0-40.2%) of the schoolchildren examined and it was higher among those in villages without health care facilities. Anopheles gambiae sensu lato was the only malaria vector found in the district and was found in all villages and at all altitudes. Sporozoite rate in An. gambiae s.l. ranged from 0-10.5%, with the lowland villages recording the highest rates. This study indicates that altitude and geographical accessibility to healthcare service are important determinants of malaria infection among rural communities in Tanzania.

ANTIBODIES TO PLASMODIUM VIVAX APICAL MEMBRANE ANTIGEN 1: PERSISTENCE AND CORRELATION WITH MALARIA TRANSMISSION INTENSITY

The antibody responses to the apical membrane antigen 1 of the Plasmodium vivax (PvAMA-1) were investigated in subjects living in areas of Brazil with different levels of malaria transmission. The prevalence and the levels of IgG to PvAMA-1 increased with the time of exposure. The frequency of a positive response and the mean IgG level were higher in areas where malaria prevalence was more intense, especially among non-infected subjects exposed to moderate transmission over a period of 20 years. The proportions and levels of IgG1and IgG3 isotypes were significantly higher among those subjects with long-term exposure. Antibodies, mainly IgG1, to PvAMA-1 persisted for seven years among subjects briefly exposed to malaria in an outbreak outside the Brazilian malaria-endemic area. These data show the highly immunogenic properties of PvAMA-1 and emphasize its possible use as a malaria vaccine candidate.

THE EFFECT OF MALARIA TRANSMISSION INTENSITY ON NEONATAL MORTALITY IN ENDEMIC AREAS

Estimates of the impact of Plasmodium falciparum infections during pregnancy on neonatal mortality have not taken into account how this varies with the level of malaria endemicity and thus do not indicate the possible effects of malaria control strategies that reduce transmission. We now review the relevant literature, and propose a mathematical model for the association between P. falciparum transmission and neonatal death. The excess risk of neonatal mortality in malaria-endemic areas appears to be insensitive to the intensity of P. falciparum transmission over a wide range of endemicity. Moderate reductions in the overall level of malaria transmission in endemic areas are therefore unlikely to significantly reduce neonatal mortality. The magnitude of the excess risk is very uncertain because existing estimates are heavily dependent on the questionable assumption that the effects are mediated by birth weight. Accurate prediction of the impact of malaria control measures targeted at pregnant women requires direct estimates of malaria-attributable neonatal mortality rates.

Review: Intermittent preventive treatment – a new approach to the prevention of malaria in children in areas with seasonal malaria transmission

Intermittent preventive treatment, the administration of a full course of an anti-malarial treatment to a population at risk at specified time points regardless of whether or not they are known to be infected, is now a recommended approach to the prevention of malaria in pregnancy and is being explored as a potential way of preventing malaria in infants. However, in many malaria endemic areas, the main burden of malaria is in older children and increasing use of insecticide treated bednets is likely to increase further the proportion of episodes of malaria that occur in older children. Recently, it has been shown in Senegal and in Mali that intermittent preventive treatment given to older children during the malaria transmission season can be remarkably effective in preventing malaria. This approach to malaria control is likely to be most effective in areas with a high level of malaria transmission concentrated in a short period of the year. However, several issues need to be addressed before intermittent preventive treatment in children can be advocated for use in malaria control programmes. These include: (1) determination of whether intermittent preventive treatment adds to the protection afforded by other control measures such as insecticide-treated bednets; (2) whether an effective and sustainable delivery system can be found; (3) choice of drug to be used; (4) optimum timing of drug administration; (5) the requisite interval between treatments. The potential benefits of intermittent preventive treatment in children are substantial; more research is needed to determine if this is a practical approach to malaria control.

Measuring malaria drug efficacy and transmission intensity

Recently Damon Francis and colleagues reported on clinical parasitological and molecular measures of antimalarial drug resistance at six sites in Uganda in which malaria transmission intensity ranged from three to 1564 infective bites per person a year. Malaria patients were randomised to receive chloroquine plus sulfadoxine-pyrimethamine or amodiaquine plus sulfa doxine-pyrimethamine and followed-up for 28 days according to a standard protocol. In addition the researchers characterised mutations associated with resistance to chloroquine amodiaquine and sulfadoxine-pyrimethamine at six locations on the Plasmodium falciparum genome in samples taken at enrolment. Francis and colleagues document a strong association between the rate of treatment failure and geographic site. Patients from sites with lower levels of malaria transmission failed treatment more often than those from sites with higher levels. This association remained significant in patients with all six mutations and when controlled for age and multiple infection. They conclude that geographic differences in response to antimalarial treatment in Uganda are mediated more by host factors--acquired immunity through exposure to intense malaria transmission--than by parasite factors. (excerpt)

ANTI–PLASMODIUM VIVAX DUFFY BINDING PROTEIN ANTIBODIES MEASURE EXPOSURE TO MALARIA IN THE BRAZILIAN AMAZON

Plasmodium vivax Duffy binding protein (DBP) is functionally important in the erythrocyte invasion process and provides a logical target for vaccine-mediated immunity. In the current study, we demonstrated that DBP is naturally immunogenic in different populations of the Brazilian Amazon, and the proportions of DBP IgG positive subjects increased with exposure to malaria, reaching a peak in those subjects with long-term exposure (> 15 years) in the Amazon area. This profile of antibody response was significantly different from the one observed for the P. vivax merozoite surface protein 1 (MSP1(19)), which was relatively uniform in areas with markedly different levels of malaria transmission. In a small sample of adults with symptomless P. vivax infection, we could not detect any significant correlation between antibodies against these P. vivax proteins and asymptomatic infection. Our study provided an additional insight by demonstrating cumulative exposure as a determinant that acts independently of host age in generation of anti-DBP IgG response.

Exploring 30 years of malaria case data in KwaZulu‐Natal, South Africa: Part II. The impact of non‐climatic factors

Malaria transmission is a multifactorial phenomenon. Climate is a major limiting factor in the spatial and temporal distribution of malaria, but many non-climatic factors may alter or override the effect of climate. Thirty years of monthly malaria incidence data from KwaZulu-Natal province, South Africa, reveal strong medium and long-term trends, which were not present in the climate data. This paper explores various non-climatic factors that may have contributed towards the observed trends. The development of antimalarial drug resistance, available information on human immunodeficiency virus (HIV) prevalence, cross-border people movements, agricultural activities, emergence of insecticide resistance and the case reporting system are reviewed and their potential effect on malaria transmission examined. Single-variable linear regression analysis showed significant association between seasonal case totals (log-transformed) and the measured level of drug resistance (log-transformed) (r2=0.558, n=10, P=0.013) as well as relative measures of HIV infection since 1990 (r2=0.846, n=11, P=0.001). The other factors appear to have affected the level of malaria transmission at certain periods and to some degree. The importance of surveillance and inclusion of non-climatic variables in analysis of malaria data is demonstrated.