Association of high plasma TNF-alpha levels and TNF-alpha/IL-10 ratios with TNF2 allele in severe P. falciparum malaria patients in Sri Lanka.

Plasma levels of pro- and anti-inflammatory cytokines of Plasmodium falciparum-infected patients with severe malaria (SM; n = 62) and uncomplicated malaria (UM; n = 69) from Sri Lanka were assessed. SM patients had significantly higher levels of TNF-alpha (P < 0·01), IL-6 (P < 0·01), and IL-10 (P < 0·05) compared to the UM patients. Plasma IL-2 levels of these patients were undetectable. TNF-alpha levels of a third group of patients with uncomplicated P. falciparum malaria, who were recruited during their fever episodes (UMF; n = 14) were significantly higher than those of the UM patients (P < 0·001) and comparable to SM patients. Plasma IFN-gamma levels of SM patients were higher compared to UM patients, but was not statistically significant. Body temperature in both SM and UMF groups were significantly higher compared to UM group, whereas percentages of parasitemia in all three groups were comparable. Analysis of plasma TNF-alpha levels and the ratio of TNF-alpha/IL-10 in UM (n = 34) and SM (n = 34) patients carrying TNF1 and TNF2 allelic types showed that SM patients carrying TNF2 had significantly higher TNF-alpha levels as well as TNF-alpha/IL-10 ratio compared to UM patients carrying TNF1, UM patients carrying TNF2 and SM patients carrying TNF1 (P < 0·05). These results suggest that the high circulating TNF-alpha levels and the inadequate IL-10 response in the SM patients carrying TNF2 allele could have contributed to the development of severe falciparum malarial disease.

ABO-blood-group types and protection against severe, Plasmodium falciparum malaria.

Although the ABO blood group of the human host has been reported to influence malarial infection, there have been few clinical observations on this effect. A hospital-based, comparative study was therefore performed to investigate the relationship between blood-group type and severe disease i nPlasmodium falciparum malaria. Overall, 243 cases of malaria (163 uncomplicated and 80 severe) and 65 patients with severe, non-malarial infections were studied. In terms of ABO-blood-group composition, the patients with severe malaria were significantly different from the patients with the uncomplicated disease (P<0.001) and also from a population control described previously (P<0.0001). The patients with uncomplicated malaria or severe but non-malarial disease were, however, similar to the population control. The cases of severe malaria were significantly less likely to be of blood group O (P=0.0003), and significantly more likely to be of group AB (P<0.0001), than the patients with nonsevere malaria. It appears that individuals who are of blood-group O are relatively resistant to the severe disease caused by P. falciparum infection.

Cognitive performance at school entry of children living in malaria-endemic areas of Sri Lanka.

In a cross-sectional study, carried out in January 1997 at the beginning of the school year, the impact of repeated attacks of malarial infection on the cognitive performance of children at school entry in moderate malaria-endemic areas of Sri Lanka was investigated. The cognitive performance of 325 schoolchildren in grade 1 (mostly aged 5-6 years) in 2 districts of Sri Lanka which are endemic for malaria (Anuradhapura and Moneragala) was assessed by an entry performance test developed by the National Institute of Education, Sri Lanka. The indices assessed included writing, language and mathematical skills. There was no difference in any of the cognitive performance indices between children from Anuradhapura and Moneragala districts. The scores of most of the indices decreased as the number of malaria infections experienced by a child increased and the ability to identify letters was significantly impaired by the number of malaria infections a child had experienced after controlling for socio-economic and nutritional status. These findings suggests that repeated attacks of malaria in children can have an adverse impact on their development.

The impact of repeated malaria attacks on the school performance of children.

The impact of repeated malarial infections on the school performance of children was investigated in 571 school children 6-14 years of age in a malaria-endemic area in southern Sri Lanka where both Plasmodium falciparum and P. vivax infections are prevalent. Malaria infections confirmed by microscopy were monitored over a six-year period. School performance was assessed by two specially designed, school grade-specific, test papers for Sinhala language and mathematics. The scores for Sinhala language and mathematics for each school term test for the year 1997 were obtained. Malarial infections were a major predictor of children's performance in language and mathematics after controlling for parent's education, monthly family income, and house type. The education of the father predicted language scores but not mathematics scores. A child who experienced more than five attacks of malaria scored approximately 15% less than a child who experienced less than three attacks of malaria. The data suggest that repeated attacks of malaria have an adverse impact on the school performance of children.

Quantifying genetic and nongenetic contributions to malarial infection in a Sri Lankan population.

Explaining the causes of variation in the severity of malarial disease remains a major challenge in the treatment and control of malaria. Many factors are known to contribute to this variation, including parasite genetics, host genetics, acquired immunity, and exposure levels. However, the relative importance of each of these to the overall burden of malarial disease in human populations has not been assessed. Here, we have partitioned variation in the incidence of malarial infection and the clinical intensity of malarial disease in a rural population in Sri Lanka into its component causes by pedigree analysis of longitudinal data. We found that human genetics, housing, and predisposing systematic effects (e. g., sex, age, occupation, history of infections, village) each explained approximately 15% of the variation in the frequency of malarial infection. For clinical intensity of illness, 20% of the variation was explained by repeatable differences between patients, about half of which was attributable to host genetics. The other half was attributable to semipermanent differences among patients, most of which could be explained by known predisposing factors. Three percent of variation in clinical intensity was explained by housing, and an additional 7% was explained by current influences relating to infection status (e.g., parasitemia, parasite species). Genetic control of Plasmodium falciparum infections appeared to modulate the frequency and intensity of infections, whereas genetic control of Plasmodium vivax infections appeared to confer absolute susceptibility or refractoriness but not intensity of disease. Overall, the data show consistent, repeatable differences among hosts in their susceptibility to clinical disease, about half of which are attributable to host genes.

Spatial targeting of interventions against malaria.

Malaria transmission is strongly associated with location. This association has two main features. First, the disease is focused around specific mosquito breeding sites and can normally be transmitted only within certain distances from them: in Africa these are typically between a few hundred metres and a kilometre and rarely exceed 2-3 kilometres. Second, there is a marked clustering of persons with malaria parasites and clinical symptoms at particular sites, usually households. In localities of low endemicity the level of malaria risk or case incidence may vary widely between households because the specific characteristics of houses and their locations affect contact between humans and vectors. Where endemicity is high, differences in human/vector contact rates between different households may have less effect on malaria case incidences. This is because superinfection and exposure-acquired immunity blur the proportional relationship between inoculation rates and case incidences. Accurate information on the distribution of malaria on the ground permits interventions to be targeted towards the foci of transmission and the locations and households of high malaria risk within them. Such targeting greatly increases the effectiveness of control measures. On the other hand, the inadvertent exclusion of these locations causes potentially effective control measures to fail. The computerized mapping and management of location data in geographical information systems should greatly assist the targeting of interventions against malaria at the focal and household levels, leading to improved effectiveness and cost-effectiveness of control.

TNFalpha*2 marks high risk of severe disease during Plasmodium falciparum malaria and other infections in Sri Lankans.

We have investigated the association between alleles of the genes for tumour necrosis factor-alpha (TNF-alpha) and TNF-beta and severity of disease during malarial (Plasmodium falciparum) and other infections in the Sri Lankan population. Patients were categorized as having either (i) uncomplicated malaria, (ii) severe and complicated malaria, or (iii) severe and complicated infection in which a diagnosis of malaria had been excluded. For all the patients, as well as for a group of matched healthy controls, TNF-alpha and TNF-beta allelic types were identified using the polymerase chain reaction (PCR) and allele-specific oligonucleotide probes and restriction enzyme digestion. The odds in favour of carrying the TNFalpha*2 allele, mainly of the heterozygous genotype (TNFalpha*1,*2), were two to three times greater among individuals with severe disease, of either malarial or other infectious origin, relative to healthy controls or to those with uncomplicated malarial infections. No significant risk was associated with either of the alleles of TNF-beta.

Malaria risk factors in an endemic region of Sri Lanka, and the impact and cost implications of risk factor-based interventions.

In an 18-month study of malaria in a population of 1,875 residents in 423 houses in an endemic area in southern Sri Lanka, the risk of malaria was found to be 2.5-fold higher in residents of poorly constructed houses than in those living in houses of good construction type. In residents of poorly constructed houses but not in others, the risk was even greater when the house was located near a source of water that could act as a potential breeding place for malaria vector mosquitoes (P = 0.0001). Based on previous findings that confirmed that house construction type was itself a risk determinant, and not merely a marker of other behavioral factors, we have estimated the potential impact of two feasible interventions to reduce the risk of malaria: 1) the imposition of a buffer zone of 200 meters around bodies of water from which houses of poor construction were excluded, which was estimated to lead to a 21% reduction of the malaria incidence in the overall population and a 43% reduction in the relocated community; and 2) the conversion of houses of poor construction type located in the buffer zone to those of a good construction type, which was estimated to lead to a 36% reduction in the incidence rates in the whole population and a 76% reduction in the residents of houses whose construction type was improved. Taking into consideration the cost to the Government of malaria prevention, we estimated the worth of a Government's investment in improving house construction type. The investment in housing was estimated to be offset in 7.2 years by savings to the Government on malaria costs alone, and beyond this period, to bring a return on the Government's investment by way of savings to the malaria control program.

Demonstration of anti-disease immunity to Plasmodium vivax malaria in Sri Lanka using a quantitative method to assess clinical disease.

Clinical immunity to malaria was studied by quantifying the intensity of symptoms as well as by measurement of several hematologic indicators of pathology (the erythrocyte sedimentation rate [ESR], serum bilirubin, reticulocyte count, plasma tumor necrosis factor-alpha [TNF-alpha], and blood glucose levels) in 39 Plasmodium vivax malaria patients exposed to endemic malaria in southern Sri Lanka, and for comparison in 43 nonimmune patients who were residents of nonmalarious regions of the country. The intensity of 11 symptoms was scored numerically in all patients using a questionnaire. This clinical score was validated by introducing internal controls to the questionnaire, and by correlating it with the underlying pathology. Both the intensity of clinical disease as well as the degree of underlying pathology were found to be significantly lower in endemic area patients (mean clinical score = 8.8, median ESR = 8 mm) compared with the nonendemic area patients (mean clinical score = 19.0, median ESR 31.5 mm). Endemic area patients also had lower parasite densities (mean = 0.06%) than those from the nonendemic area (0.12%) (P < 0.05). However, at any parasite density, both clinical disease and pathology were significantly less in the endemic area patients (P < 0.001, for both clinical score and ESR), indicating that the clinical immunity seen in the endemic area patients was a true tolerance of parasites. Although plasma TNF-alpha levels were elevated in both groups of patients, they were significantly higher in the nonendemic area patients than in patients from the endemic area (P < 0.01). Furthermore, at comparable levels of plasma TNF-alpha, nonendemic area patients had both a higher intensity of clinical disease and an underlying pathology than those from the endemic area, suggesting that if TNF-alpha is indeed a mediator of clinical disease, the endemic area patients may be tolerant to its effects. Hypoglycemia was not observed in any of these P. vivax patients despite some with high levels of plasma TNF-alpha.

Malaria mortality rates in South Asia and in Africa: implications for malaria control.

Malaria mortality in human populations varies greatly under different circumstances. The intense malaria transmission conditions found in many parts of tropical Africa, the much lower malaria inoculation rates currently sustained in areas of southern Asia, and the epidemic outbreaks of malaria occasionally seen on both continents, present highly contrasting patterns of malaria-related mortality. Here Harsha Alles, Kamini Mendis and Richard Carter examine malaria-related mortality under different circumstances and discuss implications for the management of malaria in these settings. They emphasize the power of rapid case treatment to save lives at risk under virtually all circumstances of malaria transmission.

Optimizing the malaria data recording system through a study of case detection and treatment in Sri Lanka.

The potential of using malaria incidence data routinely collected from endemic regions for disease control and research has increased with the availability of advanced computer-based technologies, but will depend on the quality of the data itself. We report here an investigation into the relevance of malaria statistics provided by the routine data collection system in Moneragala, a rural malaria-endemic region in Sri Lanka. All patients (n = 321) treated for malaria in 2 clusters of health care centres (HCCs) of both the private and the public sector in the administrative regions of Moneragala and Buttala Divisional Secretariat (D.S.). Divisions were studied in December 1995/ January 1996. The catchment area of these HCCs included a population resident in 53 Grama Niladhari (GN) areas, the smallest administrative units of the country. Almost equal numbers of malaria patients were detected and treated at Government and private health care institutions, and in 70% of them treatment was based on a diagnosis confirmed by microscopy. The routine data recording system, however, included only statistics from the Government sector, and only of patients whose diagnosis was microscopically confirmed. In compiling data, the origin of a case of malaria is attributed to the D.S. Division in which the institution (at which the patient was treated) was located, rather than the area in which the patient was resident, which was inaccurate because 90% of malaria patients sought health care at institutions located closest to their residence, thus crossing administrative boundaries. It also led to a loss of resolution of spatial data because patients' addresses recorded at the Government HCCs to the village-level are replaced in the statistics by the D.S. Division, which is a coarse spatial unit. Modifications to the system for malaria case recording needed to correct these anomalies are defined here. If implemented, these could result in major improvements to the quality of data, a valuable resource for the future of malaria control. The paper reiterates the call for the use of a standard spatial unit within a country to facilitate exchange of data among health and other sectors for the control of tropical diseases.

The ParaSight-F dipstick test as a routine diagnostic tool for malaria in Sri Lanka.

Blood from 1053 persons who presented for treatment at outpatient clinics of government health institutions in Sri Lanka, and 250 who took part in a blood survey for malaria, was examined by thick blood film microscopy under routine field conditions, and by the ParaSight-F dipstick method. All the samples were also examined microscopically under laboratory conditions when 4 times the number of microscope fields were examined. Compared with this reference standard, the sensitivity and specificity of the ParaSight-F test were 90.2% and 99.1%, and those of microscopy in the field were 92.4% and 98.4% respectively, there being no statistically significant difference between the 2 methods. The ParaSight-F test reading correlated significantly and positively with the intensity of clinical disease of patients but not with their peripheral parasitaemia, indicating that it may be a more accurate measure of the true parasite load than microscopy, which detects only parasites which are in the peripheral blood and not those which are sequestered in deep organs. The ParaSight-F test, however, failed to detect Plasmodium falciparum infections with only gametocytes in the blood (19.6% of the infected blood samples in this study). The time taken for a patient to revert to negativity by the ParaSight-F test was also significantly longer, up to 14 d. This would make the test unsuitable for checking the response to antimalarial treatment within 14 d. In an endemic area it would therefore fail to detect drug resistant populations of parasites.

Features of recrudescent chloroquine-resistant Plasmodium falciparum infections confer a survival advantage on parasites and have implications for disease control.

This paper reports on the features of recrudescent infections of chloroquine-resistant Plasmodium falciparum (CQRPf) malaria from a study in vivo of patients from a malaria endemic (n = 527) and non-endemic (n = 129) region of Sri Lanka where the incidence of RI resistance was 30% and 55%, respectively. In both groups of patients, the recrudescent infections which emerged after treatment of the primary infection with chloroquine (CQ) and primaquine had significantly lower peripheral parasitaemia (0.036% and 0.108% in endemic and non-endemic patients, respectively) compared to their primary infections (mean parasitaemia 0.13% and 0.49%; P = 0.021 and 0.002, respectively). The recrudescences of CQ resistant infections also gave rise to clinical disease of markedly reduced severity (average clinical scores of 10.1 and 8.2) compared to their primary infections (average clinical scores of 12.4 and 12.3; P = 0.003 and 0.001, respectively, in endemic and non-endemic patients). CQ resistant recrudescent infections therefore had a lower probability of being diagnosed and treated. In endemic patients, a higher proportion of CQRPf infections (57%) had gametocytaemia compared to the chloroquine sensitive ones (29%) (P = 0.014, chi 2 = 5.96) and were significantly more infective to mosquitoes (P = 0.047). these findings imply that, in areas where CQ resistance is prevalent, the continued use of the drug may confer a survival and propagation advantage on resistant parasites and favour the rapid expansion of their reservoir. In support of this, we also present epidemiological evidence showing that, in endemic areas, the proportion of P. falciparum patients carrying gametocytes has increased significantly since the emergence of chloroquine resistance. These findings are relevant to the management of drug resistance and malaria control in countries where P.falciparum is only partially resistant to CQ.

Cloning and characterisation of a gene from Plasmodium vivax and P. knowlesi: homology with valine-tRNA synthetase.

We have previously described a lambdagt11 clone detected by immune screening with a monoclonal antibody (mAb) A12. This mAb is capable of completely blocking Plasmodium vivax transmission in the mosquito vector. An epitope recognised by A12 was mapped to six amino acids (aa) within the translated sequence of this clone. Here, we describe the complete sequence of the gene within which we mapped this epitope. Surprisingly, the translated sequence of the full-length open reading frame shows homology with that of valine-tRNA synthetases (Val-tRS) from other organisms. DNA cross-hybridisation with several of these species was observed by Southern blot. In addition, the corresponding gene has been obtained from the closely related simian malaria parasite, P. knowlesi. The two aa sequences show 66% identity and yet are very divergent from other Val-tRS sequences, apart from conserved blocks related to functional activity. Multiple sequence alignments reflect this dichotomy, as do predicted differences in antigenicity.

A malaria parasite toxin associated with Plasmodium vivax paroxysms.

We have previously demonstrated a correlation between clinical paroxysms in Plasmodium vivax malarial infections and the appearance in patients' plasma of factors that kill blood stage parasites (gametocytes). This activity was, as previously shown, dependent on the presence in paroxysm plasma of tumour necrosis factor-alpha (TNF-alpha), which acts in conjunction with other 'complementary' factors. Here we have identified a parasite component which is essential for this activity and functions as a 'complementary' factor together with TNF, and a third component of unknown origin. The P. vivax parasite component present in paroxysm plasma can be substituted with a blood-stage schizont extract of either P. vivax or P. falciparum. This was demonstrated by restoring the parasite-killing activity to post-paroxysm plasma (from which it was absent) with the addition of the extracts together with TNF. The active materials in these extracts, however, are different from the natural components in P. vivax paroxysm plasma, i.e. while the schizont extracts are immunologically cross-reactive between species, the activity of the natural P. vivax toxin(s) in patients' plasma is neutralized only by the homologous antisera. Plasmodium falciparum infections have neither distinct paroxysms nor parasite-killing activity in plasma. The pronounced paroxysms of P. vivax infections may thus be due in part of a species-specific toxin(s).

Clinical disease and pathogenesis in malaria.

This is the report of a meeting held in Ahungalla, Sri Lanka, 16-19 January 1994, under the sponsorship of the Rockefeller Foundation, Health Sciences Division. The meeting was initiated jointly by the Rockefeller Foundation and the TDR Special Programme of the World Health Organization in order to bring together scientists with a wide spectrum of experience relating to malarial disease and pathogenesis. The objective was to generate interdisciplinary discussion ranging from the clinical pictures of malarial infections and their impact in different parts of the world, to current investigations on mechanisms of pathogenesis and clinical immunity and the genetic determinants in human and parasite populations affecting the nature of the disease.