Diagnosis of Plasmodium falciparum infection in man: detection of parasite antigens by ELISA

An ELISA method has been developed for the diagnosis of Plasmodium falciparum infection in man. Parasites from in vitro cultures of P. falciparum were used as source of antigen for the solid phase and the source of specific antibody was immune Gambian sera; binding of antibody in antigen-coated wells was registered by means of alkaline phosphatase-conjugated anti-human IgG. Parasites were detected on the basis of inhibition of antibody-binding. The test was applied to the detection of parasites in human red blood cells (RBC) from in vitro cultures of P. falciparum and in RBC from infected Gambians; RBC from 100 Geneva blood donors served as normal, uninfected controls. In titration experiments, the degree of antibody-binding inhibition correlated with the number of parasites in the test RBC. Parasites were detected at a level of 8 parasites/10⁶ RBC. Samples of RBC were tested from 126 Gambians with microscopically proven infection; significant antibody-binding inhibition was found in 86% of these cases, where parasitaemia ranged from 10 to 125 000/μl of blood. The presence of high-titre antibody in the test preparations was found to reduce the sensitivity of parasite detection in infected RBC from in vitro cultures mixed with equal volumes of different antibody-containing sera. The sensitivity was restored in most cases by recovering the RBC by centrifugation before testing. In a preliminary experiment, there was no significant difference in antibody-binding inhibition using fresh infected RBC and RBC dried on filter-paper and recovered by elution, although there was greater variation in the latter samples.     

Pf332-C231-reactive antibodies affect growth and development of intra-erythrocytic Plasmodium falciparum parasites

The Plasmodium falciparum antigen 332 (Pf332), is a megadalton parasite protein expressed at the surface of infected red cells during later stages of the parasite's developmental cycle. Antibodies to different parts of this antigen have been shown to inhibit parasite growth and adherence to host cells with or without ancillary cells. However, the mechanisms involved in these inhibitions remain largely unknown. We further analysed the activities of specific antibodies with regard to their specific mechanisms of action. For these analyses, affinity purified human antibodies against epitopes in the C-terminal fragment of Pf332 (Pf332-C231) were employed. All purified antibodies recognized Pf332-C231 both by immunofluorescence and ELISA. IgG was the main antibody isotype detected, although all sera investigated had varying proportions of IgG and IgM content. All the antibodies showed a capacity to inhibit parasite growth in P. falciparum cultures to different extents, mainly by acting on the more mature parasite stages. Morphological analysis revealed the antibody effects to be characterized by the presence of a high proportion of abnormal schizonts (15-30%) and pyknotic parasites. There was also an apparent antibody effect on the red cell integrity, as many developing parasites (up to 10% of trophozoites and schizonts) were extracellular. In some cases, the infected red cells appeared to be disintegrating/fading, staining paler than surrounding infected and uninfected cells. Antigen reversal of inhibition confirmed that these inhibitions were antigen specific. Furthermore, the growth of parasites after 22-42 h exposure to antibodies was investigated. Following the removal of antibody pressure, a decreased growth rate of these parasites was seen compared to that of control parasites. The present study confirms the potential of Pf332 as a target antigen for parasite neutralizing antibodies, and further indicates that epitopes within the C231 region of Pf332 should constitute important tools in the dissection of the role of Pf332 in the biology of the malaria parasite, as well as in the design of a malaria vaccine.     

Altered red cell membrane fluidity during schizogonic development of malarial parasites (Plasmodium falciparum and P. lophurae)

The plasma membranes of human or duckling erythrocytes infected with malarial parasites (Plasmodium falciparum and P. lophurae respectively) were stained by the fluorescent dye merocyanine 540 in the presence of serum. Unparasitized erythrocytes from infected ducklings or from in vitro cultures remained unstained in the presence of serum. Because merocyanine 540 has a greater affinity for fluid phased or disordered lipid bilayers the results suggest that upon infection of the red blood cell the erythrocyte plasma membrane becomes disordered or is increased in its fluidity. Such alterations of the host erythrocyte are probably due to parasite-induced modifications in the underlying spectrin network (required for lipid leaflet asymmetry) as well as changes in erythrocyte membrane lipid composition.     

Thick-smear Plasmodium falciparum antigen from in vitro cultures for the indirect fluorescent antibody test

An important prerequisite for serological studies is the availability of specific antigens with which a high, consistently reproducible degree of test sensitivity can be obtained. In the present study, thick films of P. falciparum antigen were made from in vitro cultures in order to evaluate this antigen in terms of the sensitivity and reproducibility of the indirect fluorescent antibody test, as compared with an antigen prepared from washed infected erythrocytes of Aotus trivirgatus monkeys. The test was performed in 132 serum samples from 4 different sources. The results showed that the sensitivity and reproducibility of the two antigens compared well. No binding of donors'' antibody to antigen was noticed within the 24-hour period of in vitroculture.     

Development of an in vitro microtest for determining the susceptibility of Plasmodium falciparum to sulfadoxine-pyrimethamine: laboratory investigations and field studies in Port-au-Prince, Haiti

An in vitro microtest for assessing the susceptibility of Plasmodium falciparum to sulfadoxine—pyrimethamine (S—P) was developed following WHO guidelines. Paraaminobenzoic acid and folic acid were depleted in the culture medium used, the test wells were predosed with sulfadoxine and pyrimethamine at a constant ratio of 80:1, and the parasites were incubated for 48 hours. Optimum parasite multiplication was obtained with a 2% erythrocyte suspension in medium supplemented with 12% serum. During in vitro studies with laboratory-adapted isolates, response patterns were obtained which distinguished 3 isolates with documented in vivo sensitivity to S—P from 2 isolates with documented in vivo resistance to S—P. In addition, among the three S—P-sensitive isolates, one isolate that was pyrimethamine-resistant in vitro had a higher S—P inhibitory endpoint than 2 isolates that were pyrimethamine-sensitive in vitro. The S—P microtest was further evaluated in combined in vivo and in vitro studies in Port-au-Prince, Haiti. Twenty-six patients infected with P. falciparum were treated with standard doses of S—P, resulting in prompt clearance of parasitaemia, with no recurrence in the 24 patients who completed a 28-day follow-up period. Parallel in vitro tests with pyrimethamine alone showed 3 pyrimethamine-resistant isolates out of 22 successful tests on the patients'' blood samples. In 23 successful S—P tests, the known in vivo S—P-sensitive parasites were inhibited at S—P concentrations that were generally lower for in vitro pyrimethamine-sensitive isolates than for in vitro pyrimethamine-resistant ones.     

The fine structure of Plasmodium falciparum and its host erythrocytes in natural malarial infections in man

Electron micrographs of ultra-thin sections of erythrocytes taken from two Liberian children ill with Plasmodium falciparum malaria show that the appliqué forms of this parasite are clearly within the host cell. The general fine structure of the parasites resembles that of other mammalian malaria parasites, as does the mode of ingestion of host cell material by pinocytosis. Granules of haemozoin were usually found in small vesicles pinched off from the large food vacuole. Scattered through the cytoplasm of infected red cells were narrow clear clefts bounded on each side by two unit membranes. These probably represent the Maurer''s clefts seen in light microscopy. The surface of infected erythrocytes was notably distorted, a phenomenon which may have a bearing on the stickiness of the infected red cells in human falciparum malaria and the segregation of these cells in the capillaries. Many uninfected erythrocytes showed a multiple alveolar, blister-like abnormality of a portion of the cell membrane; this was not seen in otherwise comparable blood from a case of P. ovale infection.     

Use of the in vitro microtechnique for the assessment of drug sensitivity of Plasmodium falciparum in Sennar, Sudan

In 1978, studies on the chloroquine sensitivity of Plasmodium falciparum were carried out in the district of Sennar, Sudan. The results of the in vivo tests showed parasites resistant at the RI level only, but the mean clearance time of trophozoites from the blood was higher than for strains found in many other areas of tropical Africa. The in vitro tests, using the microtechnique, indicated a lower sensitivity to chloroquine in the local P. falciparum isolates than in those of most other African countries. However, similar results have been reported from Ethiopia. The chloroquine sensitivity of P. falciparum from Sennar is close to the critical level of resistance. The in vitro microtechnique was also used to test for the sensitivity to Dabequin, 4-aminobenzo-quinoline, and was generally found to be a suitable and reproducible method, with a greater potential than the standard macro method. At parasite densities of over 100 000 asexual parasites per microlitre of blood the effect of a given concentration of chloroquine was related to the parasite density owing to the selective uptake of the compound by the parasitized cells.     

Specificity of the indirect haemagglutination test with Plasmodium falciparum test cells. Comparison of indirect haemagglutination and fluorescent antibody tests on African sera

The specificity of the indirect haemagglutination (IHA) test with Plasmodium falciparum placental antigen-sensitized test cells was examined with sera from healthy blood donors and from patients with diseases other than malaria. Only 1 nonspecific antibody reaction was seen in more than 700 tests. A comparison of IHA titres and indirect fluorescent antibody (IFA) titres with IgG and IgM conjugates on 503 sera from inhabitants of Mto Wa Mbu in Tanzania showed in successive age groups an increasing number of seropositive reactors with both tests. The increase in the proportion of positive reactions and in the mean titre levels started earlier but was more gradual in the IFA test than in the IHA test. Parasite carriers had higher antibody levels than people without an apparent parasitaemia. Parasite carriers in the younger age groups especially were more frequently seronegative in the IHA test than in the IFA test with anti-IgG conjugates. The reactivity of the IHA test with P. falciparum (Palo Alto/Aotus) antigen was higher than that with P. falciparum placental antigen and was thus closer to that of the IFA test with IgG conjugates.     

Cultivation techniques for the erythrocytic stages of malaria parasites

The study of the biochemistry, physiology, and immunology of plasmodia has been restricted by the difficulty of maintaining the parasites in isolation from the host. Some success has been achieved in cultivating them in vitro, using tissue cultures and chick embryo techniques to study exoerythrocytic states and the sporogonic cycle, but no completely successful method has been found for studying the asexual and sexual stages of plasmodia in circulating red blood cells. The relative slowness with which techniques for continuous in vitro cultivation have been developed is the result of inadequate knowledge of the biochemistry of the parasites and of the blood and its constituents. However, radioactive labelling techniques applied to P. knowlesi cultures are beginning to yield data of fundamental importance. Existing methods for the short-term in vitro cultivation of plasmodia are potentially very useful for analysing malarial antigens, for developing vaccines, and for screening and studying antimalarial drugs. Investigations of the physicochemical requirements for the in vitro preservation of red blood cells are required, and more emphasis should be given to the study of plasmodia with longer cycles. Differences between the metabolism of plasmodia in vivo and in vitro should be studied and the growth factors in normal plasma identified. Studies of the membrane of the parasites and of the red blood cells, of the immune response, and of extracellular methods for the cultivation of plasmodia should be extended.     

Chloroquine sensitivity of Plasmodium falciparum in Ibadan,Nigeria: II. Correlation of in vitrowith in vivo sensitivity

Plasmodium falciparum malaria was treated in 82 children with 25 mg/kg chloroquine orally over three days. They were observed for 28 days during which blood films were examined periodically for malaria parasites. Asexual forms of P. falciparum, present in the blood films of all the patients before commencing treatment, disappeared rapidly and by the third day no parasites were seen in blood films from any of them. Among the patients observed for more than three days, blood films remained negative throughout the observation period. In vitro tests of sensitivity of blood samples from 10 patients showed chloroquine concentrations of 0·5 to 0·8 nmol/ml to inhibit completely maturation from ring forms to schizonts.This suggests that P. falciparum in the Ibadan area is probably still fully sensitive to chloroquine.     

Plasmodium falciparum sensitivity to chloroquine in the Buyo Region, Ivory Coast

As part of a project, supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, for achieving a reduction of mortality due to Plasmodium falciparum by means of a primary health care programme, in vivo and in vitro sensitivity testing of P. falciparum to chloroquine was carried out in the Buyo Region of the Ivory Coast. Blood samples from a total of 595 children aged 2-13 years from 5 villages were screened microscopically and 32 of these children were selected for in vivo testing and 36 for in vitro testing. All 32 in vivo test patients were treated with 25 mg chloroquine base per kg, given in divided doses over 3 days, and all of them completed the 7-day observation period. Daily blood slides were taken and these were negative for asexual parasites on days 3 to 7. The mean parasite clearance time was 1.8 days. The 36 in vitro tests produced satisfactory results in 19 isolates. Complete inhibition was achieved in all 19 at 5.7 pmol chloroquine base per well (1.14 μmol/l of blood); 7 of these isolates showed complete inhibition at 1 pmol, 15 at 2 pmol and 17 at 4 pmol.     

Antibodies to Pf155, a major antigen of Plasmodium falciparum: seroepidemiological studies in Haiti

The presence of malaria parasites and the serological antibody responses against whole Plasmodium falciparum and the Pf155 antigen were studied in the population of a small rural locality in Haiti in December 1985. Only 7 (1.5%) of the individuals were found to be infected with P. falciparum, the only species observed. Antibodies to P. falciparum were detected in an ELISA in 38.2% of the sera, the positivity rates being age-related. Anti-Pf155 antibodies were detected in 12.5% and 13.6% of individuals by two different techniques used. The anti-Pf155 positivity rates increased only after 25 years of age. No trends were detected for a clear-cut protective value of Pf155 antibodies against clinical malaria and further longitudinally conducted field surveys are needed to satisfactorily assess the potential protective effect of Pf155 antibodies.     

Fansidar resistant falciparum malaria acquired in South East Asia

A case of Plasmodium falciparum malaria resistant to Fansidar (sulphadoxine plus pyrimethamine) at a level corresponding to R III and resistant to chloroquine is reported. The infection was most certainly acquired in Malaysia, but diagnosed and treated in a non-malarious area.Normal resorption and elimination rates of the Fansidar components excludes cure failure due to abnormal drug fate in the host.P. falciparum parasites from the patient have been maintained in in vitro cultures.The patient was permanently cured with mefloquine.     

Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype

Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives.     

Indirect ELISA test for malaria in blood donors

To determine the potential risk of transfusion malaria at the Hospital Militar Central in Bogota, Colombia, sera from 3114 blood donors were tested for malaria antibodies by the indirect ELISA technique. Positive results were found in 8·6 per thousand of the serum samples using P. falciparum antigen containing more than 60% mature forms as substrate. Three cases of transfusion-induced malaria were confirmed during the study. The first patient developed a P. vivax infection one week after the administration of one unit of infected blood. The other two patients received a red blood cell concentrate and a platelet preparation, respectively, derived from a single donor and developed a P. falciparum infection eight days after transfusion. The application of the ELISA technique would be of use in attempts to control transfusion-induced malaria.     

Plasmodium falciparum: assay of antigens and antibodies by means of a solid phase radioimmunoassay with radioiodinated staphylococcal protein A

Human red blood cells (RBC) infected in vitro with Plasmodium falciparum were employed to prepare several types of antigens (sonicated, infected RBC and purified, sonicated merozoites and schizonts). These antigens, as well as control preparations derived from non-infected RBC, were used to coat plastic tubes, which were subsequently tested for capacity to bind anti-P. falciparum antibodies. Binding was detected by means of radio-iodinated staphylococcus protein A. Sera from patients with recent disease or patients who had a history of P. falciparum infection gave strong binding, while sera of normal individuals had only a low binding activity. Some of the antibodies in the positive sera were directed against RBC, since they could bind to tubes coated with normal RBC antigens and could be removed by absorption with RBC. The specificity of the P. falciparum antibodies was confirmed by inhibition tests: preparations derived from infected blood but not from normal blood inhibited the binding activity of the positive sera, to antigen coated tubes.     

Serotypes of Plasmodium falciparum defined by immune serum inhibition of in vitro growth

In vitro growth inhibition assays were used to detect antigenic differences among geographically distinct strains of Plasmodium falciparum. Owl monkeys were immunized against the Camp and FCR-3/FMG strains of P. falciparum by infection, drug treatment, and rechallenge with homologous parasites. Camp-immune monkey serum was used to inhibit the in vitro growth of eight strains of P. falciparum. Inhibition was maximum for the homologous Camp strain (an average of 62% inhibition by 100 ml/litre Camp-immune serum). Four other strains were inhibited to a lesser degree, and three strains (FCR-3/FMG, FVO, and Smith) were not significantly inhibited by Camp-immune serum at concentrations as high as 400 ml/litre. FCR-3/FMG-immune serum at a concentration of 50 ml/litre caused significant inhibition of the FCR-3/FMG strain, but not the Camp strain. Thus Camp and FCR-3/FMG strains appear to bear distinct antigenic determinants recognized by the homologous, but not the heterologous, antiserum. Inhibition of in vitro growth by immune serum may be useful for serotyping P. falciparum and may have application in the selection of strains for inclusion in a malaria vaccine.     

Circumsporozoite antibodies and falciparum malaria incidence in children living in a malaria endemic area

In a case—control study we examined the association of Plasmodium falciparum circumsporozoite antibodies (anti-R32tet32) with subsequent P. falciparum infections. A study population of 140 children living in an endemic area was followed longitudinally for 25 weeks with weekly blood smears for malaria parasites and, once every two weeks, serum samples for circumsporozoite antibody determinations. From the malaria cases, antibody measurements occurring between two and six weeks prior to the onset of parasitaemia were utilized. For each case, two controls were selected. The results from 17 cases and 34 controls failed to show a statistically significant difference in antibody levels prior to the infection (P=0.07, one-tailed Student''s t-test). However, 8 of the 17 cases had antibody present, indicating a level that was not protective against patent infection.     

Myocardial Dysfunction: A Primary Cause of Death Due To Severe Malaria in A Plasmodium falciparum-Infected Humanized Mouse Model

Background

Our study aimed at substantiating the recent claim of myocardial complications in severe malaria by experimentally inducing severe Plasmodium falciparum infection in a humanized mouse model employed as human surrogate.

Methods

Twenty five humanized mice were inoculated with standard in vitro cultured P. falciparum and blood extracts collected from the inner cardiac muscles of infected mice that died were examined for the presence of the infectious cause of death. The therapeutic effect of quinine on 7 mice severely infected with P. falciparum was also evaluated.

Results

All the 25 humanized mice inoculated with the in vitro cultured P. falciparum revealed peripheral parasitemia with a total of 10 deaths recorded. Postmortem examination of the inner cardiac muscles of the dead mice also revealed massive sequestration of mature P. falciparum as well as significant infiltration of inflammatory cells such as lymphocytes and monocytes. Postmortem evaluation of the inner cardiac muscles of the P. falciparum-infected mice after quinine therapy showed significant decline in parasite density with no death of mice recorded.

Conclusions

Data obtained from our study significantly corroborated the findings of myocardial dysfunction as the primary cause of death in recent case reports of humans infected with P. falciparum.     

Diagnosis of Plasmodium falciparum infection by spot hybridization assay: specificity, sensitivity, and field applicability

The spot hybridization assay for the detection of Plasmodium falciparum reported here uses as probe a repetitive DNA sequence from this species and exhibits a high degree of species specificity. Isolates from African, Asian, and South American patients were positive in the assay and gametocytes could be detected at the same level of parasitaemia as asexual parasites. An RNA probe containing the same repetitive sequence as the DNA probe has a detection limit of 1 parasite per 10⁶ red blood cells. Comparison of the results of the assay with those obtained by microscopic examination of blood films indicated that the assay was more sensitive than microscopy if the blood films were examined for only 10 minutes; however, 40 minutes'' examination by microscopy was slightly more sensitive than the assay.