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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.

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.

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.

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]

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]

Background & objective: Merozoite surface protein-I of Plasmodium vivax (Pvmsp-1) is a strong vaccine candidate against asexual blood stages. Extensive polymorphism in msp-1 gene has been reported in P. vivax isolates from different geographical regions which is necessary before a field trial of any malaria vaccine based on msp-1 is undertaken. There are only a few reports available on polymorphism in msp-1 gene in Indian field isolates of P. vivax. The aim of the present study was therefore to investigate the polymorphism in Pvmsp-1 gene in 25 isolates of P. vivax collected from malaria patients from regions of north and northwest India. Methods: Parasite DNA was extracted from whole blood samples collected in citrated anticoagulant. The polymorphic region-5, the most variable region of the Pvmsp-1 gene was amplified by PCR. The PCR products were further analyzed by restriction fragment length polymorphism (RFLP) using Mva-1 restriction enzyme. The DNA fragments obtained on PCR and RFLP were analyzed by agarose gel electrophoresis. Results: On the basis of PCR, significant size polymorphism was seen and 4 allelic types were observed amongst the 25 isolates. Further analysis by RFLP discriminated these 4 allelic types into 9 sub-allelic types indicating that PCR-RFLP can be a good tool to study polymorphism in msp-1 gene of Plasmodium. Interpretation & conclusion: Marked genetic polymorphism was observed in rasp-1 gene among the isolates of P. vivax. These observations stress the need to study larger numbers of isolates from different regions of India. The findings could have important implications on the vaccine development strategies for P. vivax. [ABSTRACT FROM AUTHOR]