The biotin-streptavidin system in a two-site ELISA for the detection of plasmodial sporozoite antigen in mosquitoes.

A two-site ELISA has been designed for the detection of sporozoite antigen in mosquitoes. Biotin-labelled monoclonal antibodies against sporozoites and a streptavidin-biotin-peroxidase complex were used to visualize the antigen. Evaluation of the sensitivity and specificity of the procedure was carried out and background levels of reactivity on the basis of negative mosquitoes were calculated. The test has been deliberately kept as simple as possible for use in the tropics and was designed using Anopheles stephensi infected with in vitro cultivated Plasmodium falciparum gametocytes. A minimum of about 100-350 sporozoites could be detected in mature salivary gland infections; in addition sporozoite antigen was detected in mosquitoes several days before the entry of sporozoites into the salivary glands. No reaction was demonstrable either with bloodstage or ookinete antigens of P. falciparum, or with mosquitoes carrying sporozoites of other plasmodial species. The number of sporozoites in positive mosquitoes and the generating capacity of a single oocyst could be assessed by the use of a calibration curve based on dilution data of a known sporozoite suspension. It was found that a single oocyst can produce about 10,000 sporozoite equivalents.

Cultivation of fertile Plasmodium falciparum gametocytes in semi-automated systems. 1. Static cultures.

A semi-automated cultivation apparatus for the in vitro culture of Plasmodium falciparum gametocytes is described. This apparatus has been designed to produce large numbers of fertile sexual stages for use in the development of a gamete vaccine or for the infection of suitable mosquitoes. These mosquitoes in turn may be used for the development of a possible sporozoite vaccine. Loss of red cells during medium change has been eliminated and the addition of warmed fresh medium simplified compared to similar systems described previously. Material harvested from this apparatus has been used for infecting mosquitoes. Up to 98% of Anopheles stephensi were infected with a mean oocyst count of 24 per positive gut (range one to 109). The importance of satisfactory presentation of gametocytes for mosquito infection is stressed. The possible presence of substances in normal human sera which inhibits exflagellation to a variable degree and reduces mosquito infectivity is also discussed.

The production of mature gametocytes of Plasmodium falciparum in continuous cultures of different isolates infective to mosquitoes.

In vitro gametocytogenesis of Plasmodium falciparum was observed in all 22 isolates established in this laboratory. Gametocytes were produced in variable numbers--up to 3% of red cells--for a limited period of time after which this stage was seen only very sporadically. Complete maturation of microgametocytes in vitro was obtained in all 14 of the isolates that were tested for exflagellation. Up to 88.2% of membrane-fed Anopheles stephensi were infected from material produced in culture. It was also possible to infect A. gambiae and A. freeborni. Addition of fresh red cells and serum to culture material promoted infectivity of gametocytes. Gametocyte infectivity declined rapidly with time in the membrane feeders held at 38 degrees C.

Chloroquine sensitivity of isolates of Plasmodium falciparum adapted to in vitro culture.

Thirteen isolates of Plasmodium falciparum obtained from cases of malaria imported into the Netherlands and established in culture were tested for their sensitivity to chloroquine. Reproducibility of the test results depended on the exposure of a standardized number of parasites in culture to the drug. The maximum activity of chloroquine was obtained when medium with the drug was added to parasite cultures twice at 24 hour intervals. The result of drug action over a period of 48 hours was estimated best when parasites were counted 72 hours after the commencement of the test. Sensitivity to chloroquine could not provide a basis for the characterisation of strains.

Parasitologic and serologic observations of infection with Pneumocystis in humans.

More than 500 specimens of lung tissue were examined for Pneumocystis. Of the 38 infections detected, most were in immunodeficient patients. Samples of serum from approximately 600 healthy normal subjects and 117 children with acute lymphatic leukemia were examined by an indirect fluorescent antibody test. The age-related data from the normal children suggested that nearly 100% of children are infected with Pneumocystis during the first two years of life. Groups of patients with leukemia who had symptoms of pneumocystis pneumonia had significantly higher titers of IgG antibody than groups of patients with leukemia who did not have clinical symptoms and normal subjects. Nevertheless, the diagnostic value of the indirect fluorescent antibody test is limited, but serologic follow-up study can be useful. Groups of children with leukemia had lower mean titers of IgM antibody regardless of their clinical condition.

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.

Comparison of indirect haemagglutination and immunofluorescence tests for malaria antibody in Aotus monkeys infected with Plasmodium falciparum. Application of indirect haemagglutination tests for malaria.

Longitudinal observations were made on 6 Aotus monkeys repeatedly infected with Plasmodium falciparum (West African and Palo Alto strains). Immunofluorescence titres were higher than indirect haemagglutination titres in the primary phase of the infections. Both serological tests were consistently positive later in the infections. Precipitins were often not detectable even during episodes of parasitaemia.

Application of the indirect haemagglutination test for malaria: reproducibility with Plasmodium falciparum-sensitized cells.

The reproducibility of the IHA test was assessed. Two batches of glutaraldehyde-fixed sheep cells were used and the cells were sensitized with different batches of P. falciparum antigen obtained from 4 owl monkeys. At the individual level the IHA test appears to have a reproducibility of approximately 90%.

Studies on various aspects of the indirect haemagglutination test for malaria.

This paper gives the results of studies on various technical aspects of the indirect haemagglutination (IHA) test for malaria, on the similarity of the results obtained in the IHA test and in the indirect fluorescent antibody test, on the use of various plasmodial extracts as sensitizing antigens in the IHA test, and on the influence of heterophile antibodies on the titres obtained in the IHA test. Some longitudinal observations on induced malaria infections of man and monkey showed that the infection can induce the production of heterophile antibodies: their appearance, however, remains unpredictable. In some infections agglutinins against host erythrocyte components are also produced. Absorption of sera with tanned sheep cells sensitized with noninfected host red blood cell antigens is advocated as a control on the IHA titre for specific agglutinins.