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A simple diagnostic strip test for Plasmodium falciparum malaria (ParaSight F test, Becton Dickinson Advanced Diagnostics) detects a water soluble antigen, histidine rich protein 2, which is produced by blood [...]

A rapid, simple diagnostic polymerase chain reaction (PCR) method for the diagnosis of dengue fever was developed using a pair of consensus oligonucleotide primers and validated with laboratory-derived strains of dengue serotypes 1–4 and other common flaviviruses. A cluster of 13 patients with clinical dengue fever admitted to a single infectious diseases unit over a period of 3 months allowed evaluation of this technology. The PCR was positive in all 11 acute dengue cases and negative in 2 convalescent cases and 10 febrile patients recently returned from the tropics in whom an alternative diagnosis was established. In some of the acute cases, viraemia was detected before the development of a dignostic antibody response (indirect Immunoglobulin (Ig) G enzyme-linked immunosorbent assay (elisa) and capture IgM elisa). In patients from whom sequential sera were taken, defervescence and recovery from thrombocytopenia coincided with the disappearance of dengue ribonucleic acid from the blood. Nucleotide sequencing of the PCR products was undertaken in 2 cases (from India and Guyana) and the results showed a close match with previously reported serotype 2 sequencies, suggesting a potential for use of this region of the genome in epidemiological studies.

The speed and stage specificity of antimalarial drug action on the metabolic activities of cultured Plasmodium falciparumwere studied for chloroquine (CQ), quinine (QN), artemisinin (AR), and sodium artelinate (SA). CQ had the most rapid onset of action on [3H]hypoxanthine and [3H]isoleucine uptake, reaching 50% of its maximum effect in 1.8 hr compared with 3.5–7.4 hr for the other three drugs. In contrast there was a lag time of 1–4 hr before AR and SA had a measurable inhibitory effect, although after this delay antimalarial action was very rapid. Parasite glycolysis was relatively drug resistant; the inhibition of lactate production was <60% of that for [3H]hypoxanthine and [3H]isoleucine uptake. The susceptibility of P. falciparumchanged markedly as the parasite matured. Maximum drug effects occurred at the late ring and early trophozoite stage, which corresponds to the time at which the most rapid increases in synthetic and glycolytic activities occur. Mature schizonts and young rings were relatively unaffected by the antimalarial drugs. Young rings were particularly resistant to QN. Schizonts multiplied successfully in the presence of relatively high concentrations of all four drugs. The two artemisinin compounds had the broadest time window of action and may be particularly suitable for the treatment of severe malaria.

The effect of well-characterized monoclonal antibodies to red cell surface molecules on the invasion of human red cells by the malarial parasites Plasmodium falciparum and Plasmodium knowlesi was examined. Antibodies to glycophorin A (GP alpha) inhibit invasion for both parasite species, and this is highly correlated with the degree to which they decrease red cell membrane deformability as measured by ektacytometry. This effect on rigidity and invasion was also seen with monovalent Fab fragments. The closer the antibody binding site was to the membrane bilayer, the greater was its effect on inducing membrane rigidity and decreasing parasite invasion. Antibodies to the Wright determinant in particular were the most inhibitory. This differential effect of the various antibodies was not correlated with their binding affinities or the number of sites bound per cell. Antibodies to surface molecules other than GP alpha were without effect. A novel mechanism is described whereby monoclonal antibodies and their Fab fragments directed at determinants on the external surface of red cells might act to inhibit invasion by malarial parasites by altering membrane material properties.

We find Wr(a+b-) erythrocytes of donor M. Fr., which appear to carry a rare glycophorin A variant, to be fully susceptible to invasion by nine isolates of Plasmodium falciparum. Thus we fail to confirm the previous publication on the refractoriness of these erythrocytes. In addition the serum of donor M. Fr., which is known to contain anti-Wrb directed against an epitope located on glycophorin A in close proximity to the erythrocyte membrane, was not found to inhibit P. falciparum invasion of blood group 0 Rh- red blood cells.

In an attempt to determine the mechanism whereby thalassemia in its milder forms may protect against malaria, we have examined the expression of neoantigen at the surface of Plasmodium falciparum-parasitized thalassemic red cells. Neoantigen expression was estimated by measurement of antibody bound after incubation in serum from adults living in a malaria-endemic area, using a quantitative radiometric antiglobulin assay. We found that P. falciparum-parasitized alpha- and beta-thalassemic red cells bind greater levels of antibody from endemic serum than controls: mean binding ratios (+/- SE), respectively, for alpha- and beta-thalassemia compared with controls were 1.69 +/- 0.12 and 1.23 +/- 0.06 on a cell for cell basis, and 1.97 +/- 0.11 and 1.47 +/- 0.08 after a correction for surface area differences. Binding of antibody increased exponentially during parasite maturation. In addition, we found a small but significant degree of binding of naturally occurring antibody to parasitized red cells, the extent of which was also greater in thalassemia. The apparent protective effect of thalassemia against malaria may be related to enhanced immune recognition and hence clearance of parasitized erythrocytes.

6 out of 7 patients with severe neutropenia associated with the use of amodiaquine for malaria prophylaxis amodiaquine (400 mg weekly) plus proguanil (200 mg daily); 1 of these patients had also taken co-trimoxazole and another had taken sulphaguanidine. The 7th patient had taken amodiaquine alone, but at a higher dose. A retrospective analysis suggests that the frequency of severe neutropenia complicating amodiaquine taken prophylactically may be as high as 1 in 2000.

Glycophorin both in solution and inserted into liposomes blocks invasion of erythrocytes by the malaria parasite Plasmodium falciparum. Furthermore, one sugar, N-acetyl-D-glucosamine (GlcNAc), completely blocks invasion of the erythrocyte by this parasite. GlcNAc coupled to bovine serum albumin to prevent the sugar entering infected erythrocytes was at least 100,000 times more effective than GlcNAc alone. Bovine serum albumin coupled to lactose or bovine serum albumin alone had no effect on invasion. These results suggest that the binding of P. falciparum to erythrocytes is lectin-like and is determined by carbohydrates on glycophorin.

The diagnosis of infection caused by Mycobacterium tuberculosis is of increased public health concern following increases in the number of cases in developed countries and major increases in developing countries associated with the spread of human immunodeficiency virus (HIV) infection. The specificity of purified protein derivative skin testing for the detection of infection is compromised by exposure to environmental mycobacteria. Examination of sputum detects the most infectious patients, but not those with extrapulmonary disease. The 38-kDa antigen of M. tuberculosis contains two M. tuberculosis-specific B-cell epitopes. We overexpressed the gene for this antigen in Escherichia coli and evaluated the recombinant product in in vitro assays of T-cell function and as a target for the antibody response in humans. The sensitivity and specificity of the antigen as a skin test reagent were also assessed in outbred guinea pigs. We found that 69% of healthy sensitized humans recognize the antigen in vitro, as manifested by both cell proliferation and the production of gamma interferon. Untreated patients initially have a lower frequency of response (38%); this recovers to 72% during therapy. A total of 292 patients (20 with HIV coinfection) and 58 controls were examined for production of antibody to the 38-kDa antigen by using a commercially available kit. The sensitivity of the test in comparison with that of culture was 72.6%, and the specificity was 94.9%. The antigen was also tested for its ability to induce skin reactions in outbred guinea pigs sensitized by various mycobacterial species. The antigen provoked significant skin reactions in M. tuberculosis-, M. bovis BCG-, and M. intracellulare-sensitized animals. The significance of these findings and the usefulness of this antigen in immunodiagnosis are discussed.

The topography and specificity of B- and T-cell stimulatory epitopes from the 19-kDa protein of Mycobacterium tuberculosis were investigated by using overlapping synthetic peptides. Murine antisera identified two cryptic epitopes (residues 11 to 30 and 61 to 80) and one species-specific immunodominant epitope (residues 140 to 159). Immunoglobulins G1 and G2a antibody isotypes varied for the respective peptide immunogens but without relationship to the T-cell cytokine profiles which were characterized by high gamma interferon and low interleukin 5 levels. Antisera to recombinant M. tuberculosis 19-kDa protein (rGST-19) cross-reacted with homologous proteins of similar size from organisms of the Mycobacterium avium-intracellulare complex. Two-dimensional gel electrophoresis revealed differences in the number, relative mobility, and charge of isoforms of the 19-kDa protein, possibly reflecting posttranslational modifications. The immunodominant T-cell epitope from the M. tuberculosis 19-kDa protein (residues 61 to 80) and the corresponding peptide sequence from Mycobacterium avium subsp. intracellulare (residues 64 to 83), differing at five residues, were both recognized in a genetically permissive manner. Peptides 61-80 and 64-83 stimulated cross-reactive responses in BALB/c (H-2d) mice, while in the C57BL/10 (H-2b) strain, responses to peptide 61-80 were species specific. In purified protein derivative-positive healthy individuals, the M. avium subsp. intracellulare peptide stimulated stronger responses than did the M. tuberculosis peptide, whereas patients with active tuberculosis had enhanced in vitro T-cell responses to both peptides.

Severe anaemia is an important cause of morbidity and mortality in African children, but the causes, particularly falciparum malaria, are difficult to determine. We assessed the contribution of falciparum malaria to anaemia in Kenyan children by clinical examination and measurement of parasitaemia and haemoglobin (Hb) concentration in 559 children in the community and in 2412 children admitted to Kilifi district hospital during a 2‐year period. We also attempted to characterize severe malarial anaemia by examining the causes and pathophysiology of anaemia in 101 children admitted with Hb concentration 50 g/l during a 1‐year period. Plasmodium falciparuminfection was associated with reduced Hb concentration in children in the community and in those admitted to hospital irrespective of diagnosis. Falciparum malaria was the primary cause in 46 cases (46%) of severe anaemia admitted to hospital. There was no difference in the frequency of haemolysis or dyserythropoiesis in the children with malarial anaemia and those with anaemia from other causes, such as iron deficiency or sickle cell disease. The mortality rate in the children with severe malarial anaemia was 8.6% compared with 3.6% in children with severe anaemia due to other causes. Falciparum malaria does not present with a characteristic clinical or haematological picture, but is a major cause of the morbidity and mortality in children with severe anaemia who live on the Kenyan coast, a malaria endemic area.

RELATIVE protection against Plasmodium falciparum malaria afforded to heterozygous carriers of the sickle-cell gene is now the accepted mechanism for the high frequency of the gene in areas where malaria is (or was) common1–3. However, the cellular mechanisms whereby Hb S protects the red cell against the malarial parasite are still not fully understood. We have compared the rates of invasion and growth of P. falciparum in normal red cells and in those of individuals with the sickling disorders, in both aerobic conditions and conditions of reduced oxygen tension. As a result of these studies, we suggest a possible mechanism for the protection of sickle-cell heterozygotes against P. falciparum malaria.

It has been suggested that one of the main factors which determines host susceptibility to different malarial parasites is the interaction of their invasive forms, merozoites, with specific receptors on the red cell membrane1–3. Thus the Duffy blood group determinants may be involved in the entry of Plasmodium vivax but not of Plasmodium falciparum into human red cells. When analysing red cells deficient in various blood group antigens, Miller et al. noted3that two samples of En(a−) cells showed a reduction of invasion by P. falciparum4. These cells lack both the major transmembrane sialoglycoprotein, glycophorin A (or MN glycoprotein), and the independently segregating Wrightb(Wrb) antigen and also show increased glycosylation of band 3, the major membrane-penetrating glycoprotein. Despite this, En(a−) individuals are haematologically normal5. We have now confirmed that En(a−) cells obtained fresh from three En(a−) individuals are indeed relatively resistant to invasion by P. falciparum although they are able to support parasite development. Our results also indicate that these cells may be a useful model in the search for a putative receptor for P. falciparum.

HIGH gene frequencies for the sickling disorders and β thalassaemia may be due to the relative protection against Plasmodium falciparum malaria which has been afforded to heterozygous carriers1. In sickling disorders the properties of the abnormal haemoglobin may be responsible for this phenomenon2–4but the red cells of adult β thalassaemia heterozygotes contain reduced amounts of normal haemoglobin5and are indistinguishable from normal red cells with respect to the rates of invasion and growth of P. falciparum (G.P., D.J.W. and R.J.M., in preparation). Hence it is not clear how the β thalassaemia polymorphism has been maintained. There is a possible explanation, however. At birth red cells contain mainly foetal haemoglobin (Hb F); adult haemoglobin (Hb A) replaces Hb F during the first year of life. The rate of decline of Hb F production during this period is retarded in infants heterozygous for β thalassaemia as compared with normal infants6. If the presence of Hb F were to protect red cells against P. falciparum, β thalassaemia carriers aged 6–18 months might be at an advantage when passive immunity to P. falciparum is waning but active immunity is not fully established and mortality from malaria is high7. To test this hypothesis we have compared the rates of invasion and growth of P. falciparum in red cells containing Hb A with those containing Hb F using a modification of the in vitro culture system8–10.

Attempts to control human malaria by immunological means could be compromised by antigenic variability within and between different strains of malarial parasites1. A useful alternative approach might be to block parasite antigens which are important in the mechanisms of invasion of red cells. As the major human parasite Plasmodium falciparum is highly specific for human red cells, isolation of the proteins involved in the recognition of red cells by this parasite might be of particular value. Recent studies suggest that the major red cell sialoglycoproteins (SGPs), glycophorins A, B and possibly C, may carry the sites recognized by the parasite2–4. Furthermore, because certain carbohydrates present on SGPs such as N-acetylglucosamine are able to block invasion by the parasite5, they may be involved in the initial interaction between parasite and red cell. We have now identified parasite proteins which bind to SGP or N-acetylglucosamine on Sepharose 4B columns. Three proteins, of molecular weights (MWs) 140,000 (140K), 70K and 35K, seem to be specifically bound by N-acetylglucosamine.

The rationale for exchange transfusion (ET) in severe Plasmodium falciparum malaria is to lower the parasite burden rapidly, to replenish unparasitized cells and to correct severe anaemia. In addition, parasite antigens or ‘toxins’ may be removed. Despite reports of successful ET in the treatment of malaria since 1974, it remains controversial and its role and technique have been poorly defined. We present a mathematical model of ET which relates volume of exchange to reduction in parasitaemia and change in haemoglobin concentration. This model fits published and unpublished clinical data well, and should facilitate standardization of ET in future.

Disease progression, support required, prognostic indicators and survival in hospital and after discharge were studied in 53 patients with community acquired infections who required admission to the intensive care unit from the Department of Infectious Diseases between January, 1985 and August, 1991. The Median age was 37 years and over two-thirds of patients were previously fully fit; 38 per cent of patients required intensive care within 6 hours of admission with rapidly progressive disease. Both APACHE II scores and number of organ systems failed were good indicators of prognosis: mortality was 85 per cent in those with three or more organ failures and 86 per cent in those with APACHE II scores of ≥25. No deaths were observed in those with scores of <10. Some organ failures (cardiovascular, renal and respiratory) occurred more frequently in those who died. Mechanical ventilation was required by 60 per cent of patients, inotropic drugs by 42 per cent and renal dialysis by 17 per cent. Mortality in hospital was 25 per cent with no further deaths at 6 months to 2 years. As a group these patients were young and previously fit. Deterioration was rapid, necessitating admission to the intensive care unit at short notice. Our results highlight the importance of having intensive care facilities in close proximity to infectious disease units. In contrast to many other groups of patients admitted to the intensive care unit, the overall outlook for patients with community acquired life-threatening infections is good, and long-term monitoring suggests that these patients remain well once recovery has occurred.

Human hemoglobin variants are known to protect the host against severe malaria due to P. falciparum. A new study demonstrates that such genetic variation may also be associated with increased transmission of this pathogen from the human host to the Anopheles vector.