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Three methods for the quantitation of parasitaemia in malaria were compared with the standard method for ascertaining the accuracy in patients, Plasmodium berghei infected mice and P. knowlesi infected Rhesus monkeys. Technique I, where parasitaemia was calculated from the number of PRBCs in 10,000 RBCs in thin blood film and the total RBC count of the host, was used as the standard. Technique II, where parasitaemia was calculated based on the number of PRBCs per WBC and average total WBC count (8000/microliter), was least accurate. Technique IV, where parasitaemia was calculated from the number of PRBCs per oil immersion field (OIF) of microscope and the estimated amount of blood in one OIF of a thick smear, was most accurate when parasitaemia was low as in malaria patients and experimental animals with < 1 per cent parasitaemia. In mice with moderate parasitaemia (5-10%) and in falciparum malaria cases (with 3-7% parasitaemia) also technique IV was most accurate. In both animal models showing high (15-25%) and in monkeys with moderate parasitaemia, technique III based on the number of PRBCs per WBC and actual total WBC count, was the most accurate. Thus, technique IV being simpler and cost effective, with standardization of the amount of blood used in making a thick smear, may be used routinely for quantitation of parasitaemia.

The morbidity associated with malaria plays a key role in the staggering of the social and economic development of human race. The investigations on the cellular, biochemical and molecular organisation of the malarial parasite are important to understand the host parasite interactions in a better way. The parasite induces several biochemical and biophysical alterations in the host red cells. It is well recognized that cation homeostasis is vital to basic aspects of cell functions. Though the pathogenesis of anaemia associated with Plasmodium falciparum infection is multifactorial, the complex mechanisms involving the role of oxidant stress and calcium imbalance of infected red cells plays an important role.

It is known that the calcium channel blocker (CCB), nifedipine, can inhibit phagocyte oxidative burst in Plasmodium berghei-infected mice. The extent of immunopathological changes as seen by the course of infection and membrane lipid peroxidation in nifedipine-treated mice was examined in comparison with untreated mice at different parasite loads. The glutathione antioxidant system was also studied in these animals to assess its capacity to neutralize reactive oxygen species (ROS) in infected erythrocytes. The survival period of nifedipine-treated, infected mice decreased significantly. It was observed that the accumulation of reduced glutathione was greater and the decrease in glutathione peroxidase activity was less marked in drug-treated animals, suggesting better protection of the parasites against oxidative injury. The accumulation of the lipid peroxidation product, malonyldialdehyde was significantly lower in nifedipine-treated animals at all parasitemia levels studied, indicating decreased ROS generation and parasite damage. These observations reveal the shortcomings of using CCB to reverse the chloroquine resistance in malaria as this would minimize oxidative damage of parasitized red cells and phagocyte-mediated parasite killing.

The status and kinetics of monocyte activation during acute P. knowlesi infection was investigated by latex-induced, luminol-dependent chemiluminescence (CL) response. The contribution of various reactive oxygen species (ROS) to CL response was estimated before infection and at peak parasitaemia (day 7 post infection) by using scavengers of ROS (benzoate, catalase and superoxide dismutase). The chemiluminescence index (CLI) was not found to be significantly different from controls on day 2 postinfection, but was significantly higher on days 5 and 7 postinfection. Hydroxyl radical (OH.) production was considerably elevated, whereas superoxide anion (O2-.) and hydrogen peroxide (H2O2) production dropped following infection. These changes in generation of ROS are discussed in relation to the progression of parasitaemia to high levels, immunopathology and immunosuppression during acute P. knowlesi infection.

The effect of nifedipine (a calcium channel blocker) on the course of P. berghei infection was examined. It was observed that mice receiving a daily dose of 0.015 mg/kg of nifedipine had significantly shorter prepatent, patent and survival periods as compared to untreated P. berghei-infected animals (p < 0.001). This shows that the calcium channel blockers, in addition to possessing the property of reversing drug resistance during combined therapy with chloroquine, may also alter the pathophysiology of malaria infection. The decreased resistance of the host to the invading parasite suggests that the effect of CCB on the host-parasite interaction in human malaria needs to be investigated further before CCB can be used in combination with chloroquine for the treatment of chloroquine-resistant malaria or for chemoprophylaxis.

The macrophages and neutrophils from nifedipine-treated mice, both Plasmodium berghei-infected and uninfected, showed suppressed capacity to generate oxygen free radicals as compared with untreated controls. Nifedipine treatment did not affect resting state free calcium levels in these cells. But the rise in intracellular calcium levels of macrophages and neutrophils following P. berghei infection was significantly less (P < 0.05) in nifedipine-treated mice as compared with untreated groups at various parasitaemia levels. Probably this reflects a more potent effect of nifedipine on these cells in the depolarized state. Similarly, the rise in intracellular calcium levels of these cells following formyl-Met-Leu-Phe (fMLP) stimulation was also significantly less in nifedipine-treated groups than in untreated controls at different parasitaemia levels. A positive correlation between this fMLP-stimulated rise in calcium levels and the chemiluminescence response of macrophages and neutrophils was observed in nifedipine-treated and untreated groups at various parasitaemia levels. Thus the respiratory-burst responses of these cells during P. berghei infection depend on the calcium homeostasis in the cells. The disturbances of the calcium-regulating mechanisms by nifedipine treatment resulted in subnormal phagocytic cell responses which lead to more severe and rapidly fatal P. berghei infection in these animals.

The oxidative metabolism of peritoneal macrophages (PM) and neutrophils from nifedipine (calcium channel blocker)-treated, Plasmodium berghei (NK 65)-infected and normal infected Swiss Albino mice was studied. A significant fall in oxidative metabolism as evidenced by decreased chemiluminescence (CL) response (P less than 0.001) was recorded both in PM and neutrophils from nifedipine-treated mice compared to the control animals. When the oxidative metabolism of these phagocytes was studied after infection of the host, higher CL response was recorded from both PM and neutrophils isolated during the early course of infection (0-1 and 5-10% parasitaemia) when compared to uninfected mice (P less than 0.001). A similar pattern was observed in the case of nifedipine-treated and infected mice even though the CL response was much lower. The increasing parasite load not only resulted in subnormal CL response but also prolonged the time required for the phagocytes to exhibit peak oxidative activity both in normal infected and CCB-treated infected mice, but the time taken to show peak CL response was shortened following drug administration compared to controls. These observations revealed the profound in vivo effect of CCB on the functioning of phagocytic leucocytes and thereby questions the use of CCB in combination with chloroquine for reversal of drug resistance.

Very little information is available as regards the methods to be advocated to prevent transfusion malaria, especially in endemic countries. Most of the malaria non-endemic countries follow the rule of donor deferral for 3 years after malaria infection. This criterion cannot be followed in endemic areas since the majority of the population is continuously exposed to this infection. Therefore, there was a long felt need for a suitable screening procedure for blood donors to make the transfusion therapy safe. A total of 6,435 blood donors and 3,621 patients who received blood from these donors were studied by blood smears examination and malarial antigen detection by specific monoclonal antibody. Smear examination by Giemsa and acridine orange staining methods showed poor results (0.06% and 0.1% positivity respectively), probably due to low concentration of parasites. However, antigen detection by monoclonal antibody confirmed specific diagnosis in majority of these subjects. Blood smear examination failed to reveal malaria infection in 92.3% of antigen positive blood donors. It is, therefore, recommended that antigen detection by monoclonal antibody should be adopted as a routine screening procedure by the blood transfusion services in malaria endemic countries like India.

Transfusion-associated malaria is one of the dreaded threats to the safety of transfusion services in this malaria endemic world. The main drawback in preventing this menace is that routine blood donor screening techniques are not very satisfactory. In this study, blood donors were screened for antimalarial antibody by micro ELISA and Indirect Fluorescence Antibody (IFA) test and malarial antigen by monoclonal antibody (MAB) technique. A total of 19.37% and 12.39% blood donors showed significantly high antibody by ELISA and IFA test respectively, and 0.35% donors showed the presence of antigen by the MAB technique. Solitary dependence on malarial antibody detection as a screening test might have led to rejection of 19.02% blood donors without any apparent infection. So antigen detection in blood donors with the help of the MAB technique seems to be more sensitive and a practically feasible screening test to prevent transfusion malaria.

Transfusion-associated malaria is often severe or even fatal, because diagnosis is frequently delayed and it complicates an already serious underlying disorder. Detection of infected donors is difficult in endemic areas due to the lack of a suitable donor screening test. Blood smear staining techniques show poor results due to the low parasite concentration in many infected persons, and the antibody detection test is not helpful due to the universal presence of antibody in healthy donors in these areas. For comparative evaluation of various screening tests, 9131 blood smears from voluntary donors and a group of patients were screened by Giemsa staining. Ten (0.10%) subjects showed parasitaemia, whereas Acridine Orange fluorescence staining showed 13 (0.14%) parasitaemia in almost the same number of smears screened on the same samples. Significantly high levels of malarial antibody were detected in 12.6% and 19.86% of subjects by indirect fluorescent antibody and enzyme-linked immunoassay tests, respectively. Malarial antigen detection by monoclonal antibody showed positive results in 9.48% of subjects, demonstrating excellent results and showing direct evidence of infection. We recommend that this should be adopted as a screening technique by transfusion services in endemic areas in order to prevent post transfusion malaria.

Generation of reactive oxygen radicals by peripheral blood monocytes was measured by luminol-dependent chemiluminescence in 23 P. vivax- and 7 P. falciparum-infected patients. The chemiluminescence index (CLI) was not found to be significantly higher in P. vivax-infected cases than in healthy controls. But in patients with P. falciparum infection, the CLI was significantly higher compared to controls as well as to P. vivax-infected patients. In two severe and complicated P. falciparum-infected cases, CLI was found to be higher than in mild cases. As immunosuppression is more marked in falciparum malaria than in vivax cases, the role of oxygen radical generation in immunopathology and causation of immunosuppression in falciparum malaria needs further investigation.

The oxygen free radical generation (as determined by cytochrome c reduction) and respiratory burst enzyme activities were measured in the peripheral blood monocytes before infecting normal animals, during primary Plasmodium knowlesi infection, after treatment of primary infection with chloroquine, after administration of various subcurative doses following reinfection and after establishment of chronicity. For all the parameters of the oxidative response, no significant difference was observed during primary infection and after curing of primary infection. However, the response was significantly increased in the reinfected monkeys given subcurative therapy; this was followed by a sharp decline after the 4th subcurative dose, after which the response continued to be low until the attainment of chronicity. The results of the present study exclude the possibility of the increase in oxidative metabolism being the effect of the drug (i.e. chloroquine), since no significant difference was observed during primary infection and after its cure. The sustained low grade parasitaemia in the chronic stage, in spite of the sharp decrease in the oxidative response, suggests that some nonoxidant parasitic killing mechanism might be playing a role in the regulation of parasitaemia.

A total of 125 beta-thalassaemia patients receiving repeated blood transfusions were screened by Giemsa stain, Acridine-orange stain and antigen detection for evidence of malaria infection on each visit. A total of 8 (6.4%) of the patients developed post-transfusion malaria (PMT) as confirmed by tracing the infected blood donors. A high incidence of PTM in thalassaemia patients appears to be due to the use of fresh blood and the high frequency of blood transfusions required by these patients. Antigen detection using monoclonal antibody was found to be more sensitive for diagnosis of PTM and for screening suspected donors than the conventional blood smear examination methods and is therefore recommended for routine blood donor screening to rule out malaria infection.

In the present study, the percentage of lymphocyte subpopulations and their relation to T cell subsets was estimated during acute and chronic Plasmodium berghei malaria. During acute infection, a decreased percentage of T lymphocytes was observed, whereas no change was observed during chronic infection. However, no change in the percentage of B cells was observed in both types of infection. The T cell population was characterized by an increased percentage of T suppressor cytotoxic (CD8+) cells and a decreased percentage of T helper cells (CD4+) during acute infection. This resulted in an inversion of the CD8+/CD4+ cell ratio. During chronic infection no change in the CD8+ cell percentage was observed; however some increase in CD4+ cell percentage was observed in later stages. Thus failure of the immune system to overcome acute infection may be due to an increase in T suppressor cells, and maintenance of parasites at subpatent levels during chronic infection may involve the normal CD8+/CD4+ cell ratio with some increase in CD4+ cells.

Marked suppression of IgM, IgG and IgA plaque forming cells was observed in mice immunized with sheep erythocytes (T-dependent antigen) during acute Plasmodium berghei infection whereas during chronic infection mild immunosuppression was observed. When mice were immunized with polyvinylpyrrolidone (T-independent antigen), suppression in the number of plaque forming cells was observed at higher parasitaemias only during acute infection whereas during chronic infection the number remained within normal limits. Adoptive transfer studies suggested that the immunosuppression was mainly mediated by macrophages and their products. The suppressor role of macrophages and their products during acute malarial infection is highlighted.

A comparative study of specific and non-specific immunosuppression has been carried out in acute and chronic Plasmodium berghei infected mice in an in vivo system. In our previous studies, immunosuppression during acute P. berghei infection was attributed to T lymphocytes when we studied modulation of blastogenic response of lymphocytes in an in vitro system. In the present study, delayed type hypersensitivity (DTH) was evident from the homing of radiolabelled bone marrow cells into the delayed lesions of antigen challenged foot pads during chronic infection. This response was suppressed during acute infection especially in early stages. A greater concentration of bone marrow cells in the liver and spleen occurred during chronic infection in comparison with acute infection. When radiolabelled bone marrow cells from infected mice were injected into the normal mice previously given malaria antigen in foot pads, no significant change in homing pattern in liver, spleen or foot pads was observed. Contact sensitivity to picryl chloride was suppressed during acute infection, but was intact during chronic infection. Since these responses are mediated by T lymphocytes, significance of these responses is discussed.

A study of lymphocyte subpopulation and leucocyte migration inhibition index (LMI) in 78 pregnant and 22 nonpregnant women with malarial infection (P. vivax 60% and P. falciparum 40%) was done in a hospital in Northern India. Eighty pregnant and 20 nonpregnant women without any infection were also included in the study as controls. T-cell percentage in controls decreased to its lowest in first trimester and gradually rose to nonpregnant levels by the end of puerperium. With malarial infection, there was further drop in T-cell percentage, more so in third trimester. LMI in controls corresponded with T-cell percentage and was depressed in pregnant patients. Enhancement in LMI with malarial infection was depressed in pregnant patients maximally in the third trimester. Parity did not have any influence on these cellular immune parameters. Thus, cellular immunity status in malarial patients was found to be depressed during pregnancy especially in the third trimester.

Generation of reactive oxygen species (ROS) by blood monocytes and Kupffer cells of normal and Plasmodium berghei infected mice treated at different levels of parasitaemia with chloroquine, were studied. Cells isolated at lower level of parasitaemia (less than 2%) produced ROS within the range of normal animals, whereas ROS production by the cells isolated from the animals at higher level of parasitaemia (greater than 20%), was significantly higher even without stimulation with latex particles. The ROS generation capacity of both normal and infected animals was less after the chloroquine treatment. This inhibitory effect of chloroquine may be beneficial in protecting the host from the adverse effect of reactive oxygen species by controlling their overproduction.

The immunopharmacokinetics of chloroquine were studied in Plasmodium knowlesi-infected and uninfected rhesus monkeys. In control monkeys, chloroquine depressed the percentage of lymphocyte subpopulation and their proliferative response after the third dose of drug administration. The effect was maximum during day 6 to 10 after initiation of treatment. During this period the plasma level of chloroquine was high. The drug was slowly excreted and recovery in immune functions was noticed by the 20th day of treatment. On the other hand, the drug caused an irreversible effect on the proliferative response of normal lymphocytes following in vitro exposure of cells. Interestingly, while the drug depressed the immune functions of normal animals, it reversed the immunosuppression caused by malarial parasites in infected animals. The probable mechanism of action of the drug has been discussed.

The role of lymphocytotoxic antibodies in elimination of lymphocytes and characterization of these antibodies during Plasmodium berghei infection was examined in BALB/c mice. When assayed at 15 degrees, an increase in lymphocytotoxic levels above base line values was detected in acute infection when parasitaemia exceeded 10%. There was an increase in lymphocytotoxins with increase in parasitaemia to a plateau beyond 25% parasitaemia. When assayed at 37 degrees, lymphocytotoxic activity during acute infection was lower than at 15 degrees but the increase detected was significant. During chronic infection there was a lower increase in the activity detected at 15 degrees and no significant increase in activity assayed at 37 degrees (P greater than 0.05). In addition, peripheral blood lymphocyte counts also dropped during acute infection while there was little change in lymphocyte numbers in the chronic infection group. The lymphocytotoxic antibodies present in sera of infected mice were predominantly of IgM class during acute infection and IgM or IgG class during chronic infection. The relevance of these observations to plasmodium infection is discussed.

A total of 1689 peripheral blood smears and serum samples were collected from healthy subjects from four villages of U.T. Chandigarh during the pre-monsoon season (February to May 1987) while 1809 such samples were collected during the post-monsoon season (October 1987-January 1988). None of the peripheral blood smears examined by Giemsa and acridine-orange stains showed malarial parasite. Out of 1689 serum samples tested by indirect haemagglutination (IHA) test during pre-monsoon season, 81 per cent showed malarial antibody titres of less than 1:8 and only 19 per cent showed titres of 1:8 or above. In contrast, out of 1809 serum samples tested during post-monsoon season, 58.3 per cent showed antibody titres of less than 1:8 while 41.6 per cent samples showed titres of 1:8 and above. Total number of malaria cases from these villages from June 1987 to January 1988 was also low (total 65 cases) as compared to corresponding period of previous year (total cases 191). Serological findings independent of positive cases of malaria suggest that though, no proved clinical cases of malaria were observed in the population surveyed, malaria transmission had certainly taken place as evidenced by higher antibody titres observed during the post-monsoon season compared to pre-monsoon season.

A single oral dose of 20 mg/kg body weight of chloroquine was administered to normal and P. knowlesi infected Rhesus monkeys. The plasma chloroquine half-life was significantly increased (p less than 0.05) in infected monkeys as compared to the normal monkeys. The clearance and volume of distribution were significantly decreased in the infected monkeys. The decreased elimination half-life of the drug in infected monkeys could be due to hepatic involvement during malaria infection.

The effect of chloroquine on the phagocytic function of monocytes was studied in normal and Plasmodium knowlesi-infected rhesus monkeys during a course of therapy. In normal animals, chloroquine inhibited phagocytic activity after three doses. The effect of the drug and concentration was greatest after six to eight days. Recovery in function of monocytes was noticed after the drug had been excreted. In P. knowlesi-infected monkeys, the degree of inhibition of phagocytosis depends on the level of parasitaemia. Chloroquine treatment reversed the trend of inhibition in the animals that received the drug when parasitaemia was less than 30%.

To investigate the involvement of immune complexes in the pathogenesis of cerebral malaria, a comparative study between Plasmodium berghei infected Swiss albino mice and BALB/c mice was conducted. Rise in parasitemia was found to be same with differences in manifestation of disease and mortality, level of immune complexes and histopathology. Increased parasitized red cells with swelling of endothelial cells in the brain capillaries along with increased levels of circulating immune complexes were observed in the BALB/c mice.

The superoxide anion generation profile of peripheral blood monocytes of rhesus monkeys was investigated during the different stages of an acute Plasmodium knowlesi infection. An initial increase in superoxide anion was followed by a significant decline (P less than 0.001), paralleled by a drop in NADPH oxidase activity; there was no alteration in the activity of the hexose monophosphate shunt enzymes. This lowered activity of the NADPH oxidase, with the resulting decreased O2 generation, might be responsible for the failure of the animals to control the parasitaemia; as a result they succumbed to the infection.

The role of macrophages in immunosuppression which develops during Plasmodium berghei infection in mice has been studied. The transfer of activated macrophages (or their extracts) from the infected mice with low parasitaemia (less than 10%) to uninfected mice enhanced their humoral response to sheep red blood cells (SRBC) as indicated by the number of splenic IgM and IgG plaque-forming cells (PFC). In contrast, when macrophages obtained during higher parasitaemia (16%) were used, significant inhibition was observed. These findings indicate that during Plasmodium berghei infection in mice macrophages may play a dual role.

A prospective hospital-based study was conducted from June 1984 to November 1985 in northern India to study the morbidity pattern of malaria in pregnant women. 78 pregnant and 22 non-pregnant women with malarial infection (60% Plasmodium vivax and 40% P. falciparum) were included in the study. The results were analysed according to period of gestation, parity and type of infection. Severity of clinical illness was significantly higher in pregnant patients for both P. vivax and P. falciparum infections. Some striking differences were found in our study when compared with those from African countries. Multigravidas had a slightly higher morbidity than primigravidas, though the difference was not statistically significant. Even though only a small proportion (1.4%) of our pregnant patients were affected by malaria, cerebral malaria, maternal deaths, intrauterine foetal deaths and pre-term labours were much more common.

The role of Plasmodium falciparum and activated blood motiocytes in bringing about erythrocyte membrane lipid peroxidation and in altering the enzyme activity associated with Ca2+ and K+ efflux was studied. An attempt was made to investigate the role of parasite and monocyte-mediated reactive oxygen species (ROS) in inhibiting Ca2+-Mg2+ ATPase and Na+-K+ ATPase in order to find out the cause of reported high intra-erythrocytic calcium and depleted potassium levels in parasitized erythrocytes (PRBC). The PRBC showed enhanced lipid peroxidation as indicated by increased malonyldialdehyde (MDA) formation which coincided with the maturity of the parasite. This was further enhanced following exposure of PRBC to activated blood monocytes. The Ca2+-Mg2+ ATPase activity was decreased as the parasite matured and was further hampered significantly in mature parasiteinfected red cells exposed to activated blood monocytes. There was a good negative correlation between MDA formation and Ca2+-efflux from red blood cells suggesting the negative influence of ROS on Ca2+-efflux. The Na+-K2+ ATPase activity did not reveal any significant change, both during parasite maturation as well as upon exposure to ROS from activated monocytes. We therefore suggest that inhibition of Ca2+-efflux and the resulting increased cytosolic Ca2+ in PRBC might have a role in structural and functional abnormalities of red blood cell, thus enhancing the red cell loss during P. falciparum infection. [ABSTRACT FROM AUTHOR]

The role of lymphocytotoxic antibodies in elimination of lymphocytes and characterization of these antibodies during Plasmodium berghei infection was examined in BALB/c mice. When assayed at 15°, an increase in lymphocytotoxic levels above base line values was detected in acute infection when parasitaemia exceeded 10%. There was an increase in lymphocytotoxins with increase in parasitaemia to a plateau beyond 25% parasitaemia. When assayed at 37°, lymphocytotoxic activity during acute infection was tower than at 15° but the increase detected was significant. During chronic infection there was a lower increase in the activity detected at 15° and no significant increase in activity assayed at 370 (P >0.05). In addition, peripheral blood lymphocyte counts also dropped during acute infection while there was little change in lymphocyte numbers in the chronic infection group. The lymphocytotoxic antibodies present in sera of infected mice were predominantly of IgM class during acute infection and IgM or IgG class during chronic infection. The relevance of these observations to plasmodium infection is discussed. [ABSTRACT FROM AUTHOR]

Background & objectives: Severe anaemia in Plasmodium falciparum (Pf) associated malaria is a leading cause of death despite low levels of parasitaemia. In an effort to understand the pathogenesis of anaemia we studied expression level of RBC complement regulatory proteins, CR1 (CD35), CD55 and CD59 with haemoglobin status in a group of malaria cases from Assam, Goa and Chennai, and in healthy controls. Methods: Flowcytometry was used to study expression of CRI, CD55 and CD59 in 50 Pf cases and 30 normal healthy volunteers. Giemsa stained thick and thin blood films were used for microscopic detection and identification of malarial parasites and parasite count. Results: No correlation was found between degree of expression of RBC surface receptors CR1, CD55 and CD59 with haemoglobin level. However, expression of CD55 was less in malaria cases than in healthy controls. Interpretation & conclusions: The present findings indicate that malaria infection changes the expression profile of complement regulatory protein CD55 irrespective of severity status of anaemia. Further studies are needed to explore the pathophysiology of anaemia in malaria cases in Assam where expression of RBC complement receptors appears to be low even in normal healthy population. [ABSTRACT FROM AUTHOR]

Background & objectives: The increase in Plasmodium falciparum infections which are associated with severe and complicated malaria and drug resistance has made control of malaria a difficult task. Extensive genetic polymorphism in P. falciparum has been reported from several parts of the world which affects the efficacy of sub-unit vaccines. The knowledge of genotypes of the parasite in a geographical region is therefore, important for effective management and control. The aim of the present study was to investigate the usefulness of random amplified polymorphic DNA (RAPD)-PCR technique for differentiation of P. falciparum isolates from patients presenting with severe (cerebral malaria) and mild malaria. Methods: Genetic polymorphism in 21 P. falciparum isolates obtained from patients found positive for P. falciparum by light microscopy was studied by RAPD-PCR analysis. Eleven RAPD primers were used for analysis of 21 P. falciparum isolates obtained from cerebral and non-cerebral malaria patients. Results: Of the 11 primers, only three (E-4, E-8, and R-8) produced useful polymorphic patterns. The cluster analysis based on UPGMA demonstrated that isolates causing cerebral malaria cluster separately from those causing uncomplicated malaria. However, the analysis of phylogenic tree showed that P. falciparum isolates causing non-cerebral and cerebral malaria clustered separately but showed relatedness. Interpretation & conclusions: The results of the present study showed that the RAPD-PCR was able to differentiate the isolates causing severe and mild malaria. The cluster analysis of the phylogenic tree suggested that the virulent strains evolved from less virulent strains as it clustered separately. RAPD technique may be useful in discriminating between the different isolates of the same species resulting in different clinical profiles. [ABSTRACT FROM AUTHOR]