A DNA hybridization assay for use in drug sensitivity tests in vitro for Plasmodium falciparum under field conditions.

A deoxyribonucleic acid (DNA) hybridization technique was applied to in vitro drug sensitivity testing of P. falciparum using a synthetic 21-mer oligonucleotide coupled to alkaline phosphatase (PFR1-AP) to monitor development of parasite stages in culture. The density of the coloured spot clearly distinguished schizonts from ring forms. This assay system was applied in the field on Hainan Island, China. Blood samples obtained from patients were cultivated in the presence of antimalarial drugs and the minimum drug concentration required to inhibit development of parasites was determined by the DNA hybridization assay and by microscopical observation of Giemsa-stained blood smears. The 2 methods yielded identical results, indicating that the DNA hybridization assay can be used for in vitro drug sensitivity testing under field conditions.     

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.     

Chloroquine sensitivity of Plasmodium falciparum in Ibadan,Nigeria

Thirty-five children with Plasmodium falciparum malaria were treated with 25 mg/kg chloroquine over three days and observed for seven days during which blood films were examined daily for malaria parasites.Asexual forms of P. falciparum which were present in the blood films of all the patients before commencing treatment disappeared rapidly from the blood so that by the third day no parasites were seen in the blood film. The blood films remained negative for the rest of the seven-day observation period.Plasma chloroquine determination in eight of the patients showed high blood levels during the first three days.The results do not confirm the suspicion of chloroquine-resistant P. falciparum in the area studied although RI level of resistance by WHO criteria was not excluded.     

Prolonged Plasmodium falciparum infection in immigrants, Paris

Few immigrant travelers have Plasmodium falciparum infections >2 months after leaving malaria-endemic areas. We conducted a case-control study to identify factors associated with prolonged P. falciparum infection in immigrant travelers. Results suggest that P. falciparum infection should be systematically suspected, even months after travel, especially in pregnant women and first-arrival immigrants.     

Assessment of chloroquine sensitivity of Plasmodium falciparum in Choluteca, Honduras

During an outbreak of urban malaria in Choluteca, Honduras, the response of local isolates of Plasmodium falciparum to chloroquine was assessed. The 7-day WHO alternative standard field test was used together with three in vitro tests: the Rieckmann macro- and micromethods and a new 48-hour test which underwent its first field trial in this study. No chloroquine resistance was found in in vivo tests in 10 patients or in the in vitro tests on blood samples from 6 patients.     

In vitro drug sensitivity of Plasmodium falciparum in Acre, Brazil

In Acre, the westernmost state of Brazil in the Amazon region, the sensitivity of Plasmodium falciparum to chloroquine, amodiaquine, mefloquine, quinine and sulfadoxine/pyrimethamine was determined in vitro by the Rieckmann microtechnique. The study was performed between January and June 1987; the in vitro parasite responses to all antimalarial drugs were determined according to the recommendations of WHO. Of 83 isolates of P. falciparum, all were sensitive to mefloquine and of 87 isolates of P. falciparum, 84 (97%) were sensitive to quinine. The EC50 for mefloquine was 0.27 mumol/l and for quinine 4.60 mumol/l. In contrast, 65 of 89 (73%) and 70 of 83 (84%) isolates were resistant to amodiaquine and chloroquine, respectively; 11 isolates even grew at 6.4 mumol chloroquine/l. The EC50 for amodiaquine was 0.34 mumol/l and for chloroquine 0.73 mumol/l. Sulfadoxine/pyrimethamine resistance was seen in 23 of 25 (92%) cases. These data clearly indicate that in the western part of the Amazon region the 4-aminoquinolines, as well as sulfadoxine/pyrimethamine, can no longer be recommended for the treatment of P. falciparum infections.     

A qPCR-based multiplex assay for the detection of Wuchereria bancrofti, Plasmodium falciparum and Plasmodium vivax DNA

The purpose of this study was to develop real-time multiplex quantitative PCR (qPCR) assays for the simultaneous detection of Wuchereria bancrofti (Wb), Plasmodium falciparum (Pf) and P. vivax (Pv) in mosquitoes. We optimized the assays with purified DNA samples and then used these assays to test DNA samples isolated from Anopheles punctulatus mosquitoes collected in villages in Papua New Guinea where these infections are co-endemic. Singleplex assays detected Wb, Pf and Pv DNA in 32%, 19% and 15% of the mosquito pools, respectively, either alone or together with other parasites. Multiplex assay results agreed with singleplex results in most cases. Overall parasite DNA rates in mosquitoes, estimated by PoolScreen 2 software, for Wb, Pf and Pv were 4.9%, 2.7% and 2.1%, respectively. Parasite DNA rates were consistently higher in blood-fed mosquitoes than in host-seeking mosquitoes. Our results show that multiplex qPCR can be used to detect and estimate prevalence rates for multiple parasite species in arthropod vectors. We believe that multiplex molecular xenodiagnosis has great potential as a tool for non-invasively assessing the distribution and prevalence of vector-borne pathogens such as W. bancrofti and Plasmodium spp. in human populations and for assessing the impact of interventions aimed at controlling or eliminating these diseases.     

The sensitivity of Plasmodium falciparum to chloroquine and amodiaquine in Ibadan, Nigeria

An extended in vivo test of the sensitivity of Plasmodium falciparum to antimalarial drugs in Nigerian children showed no evidence of resistance to chloroquine and amodiaquine. However, the results of a small number of in vitro tests suggest a decreased sensitivity of the parasite to chloroquine when compared with the results of earlier studies in the same locality.     

Diagnosis of Plasmodium falciparum infection using a solid-phase radioimmunoassay for the detection of malaria antigens

A serodiagnostic test has been developed for the detection of Plasmodium falciparum in infected blood. Using parasite antigens and infected red blood cells from in vitro cultures of P. falciparum and malaria antibody from high-titre Gambian sera, parasites were detected in a solid-phase radioimmunoassay (RIA) that measured antibody-binding inhibition. Lysed RBC were incubated with labelled IgG purified from immune sera and were then placed in antigen-coated microtubes and incubated. The degree of inhibition of antibody binding in the tubes correlated with the level of parasitaemia in the test RBC and, using dilutions of RBC from in vitro cultures of P. falciparum, the test detected infection at a level of 8 parasites/10⁶ red blood cells. The test was applied to RBC from 100 healthy European blood donors and to samples of RBC from 500 Gambians from the up-country villages of Keneba and Manduar. More samples were positive by RIA than by microscopy and there was a highly significant degree of correlation between the RIA and microscopy results.     

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.     

Susceptibility of Anopheles culicifacies species A and B to Plasmodium vivax and Plasmodium falciparum as determined by immunoradiometric assay

We have used a two-site immunoradiometric assay and species-specific antisporozoite monoclonal antibodies to determine the relative roles that sibling species A and B of the Anopheles culicifacies complex play in malaria transmission in western Uttar Pradesh, India. The results unequivocally establish species A as the primary vector of both Plasmodium vivax and P. falciparum in this area. Our results indicate active transmission of P. vivax from May to October and of P. falciparum from August to December. The identification of species A as the primary malaria vector in northern India will now allow suitable malaria control strategies to be designed.     

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.     

Anaemia caused by asymptomatic Plasmodium falciparum infection in semi-immune African schoolchildren

A cohort of 250 Ghanaian schoolchildren aged 5-15 years was followed clinically and parasitologically for 4 months in 1997/98 in order to study the effect of asymptomatic Plasmodium falciparum infections on haematological indices and bone-marrow responses. Of the 250 children 65 met the predefined study criteria. Thus, 14 children were parasite-free throughout (group 1), 44 had P. falciparum in all blood samples collected but no symptoms of malaria (group 2), and 7 had 1 malaria attack during the study period (group 3). At the end of the study the mean haemoglobin (Hb) level in group 1 was 123 g/L, significantly higher than the value of 114 g/L in groups 2 and 3 (P < 0.02, adjusted for age and splenomegaly). The low Hb in group 2 was associated with subnormal plasma iron. Low Hb was associated with elevated erythropoietin (EPO) levels, and there was a positive correlation between EPO and reticulocyte counts. However, the reticulocyte response to EPO was more pronounced in uninfected than in infected children, suggesting a partial interference with erythropoiesis in asymptomatic infections. Children with asymptomatic infections had significantly higher plasma levels of tumour necrosis factor than uninfected children (geometric means 50 ng/L and 27 ng/L, respectively, P < 0.001) and this cytokine may contribute to bone-marrow suppression and disturbed iron metabolism. We suggest that asymptomatic malaria leads to a homeostatic imbalance in which erythrocyte loss due to parasite replication is only partially compensated for by increased erythropoiesis. The consequences of the reduced Hb levels on the development and cognitive abilities of children with asymptomatic infections, and the risk of precipitation of iron deficiency, deserve further study and should be considered in malaria control programmes that aim at reducing morbidity rather than transmission.     

Pyrimethamine sensitivity in Plasmodium falciparum: determination in vitro by a modified 48-hour test

Four strains of Plasmodium falciparum recently isolated in culture were assessed in vitro for their response to pyrimethamine. A simple modified 48-hour test was used, which showed two strains to be sensitive to the drug in vitro, while the other two were resistant at a very high level. In the two strains for which relevant clinical information was available the in vivo response to pyrimethamine was corroborated by the in vitro findings. This modified 48-hour test is thus useful for determining patterns of drug sensitivity in laboratory-adapted strains, and would be a valuable asset if found to be equally applicable under field conditions.