An immunohistochemical assay was developed combining an avidin-biotin-glucose oxidase complex procedure (ABC-GO) with light microscopy to detect specific antibody against Plasmodium falciparum. Thin blood films were prepared from culture material of P. falciparum and fixed with acetone. Antibody was detected by successive incubations with test serum, biotinylated goat antihuman antibody, avidin-biotin-glucose oxidase complex, and glucose oxidase substrate. In the presence of reactive serum, a blue precipitate formed on the parasites and could be visually observed with a 40x objective. Sera from patients with single infections for P. vivax or P. ovale were unreactive. No cross-reactivity was observed with sera from patients with rheumatoid arthritis, filariasis, amebiasis, schistosomiasis, dengue, scrub typhus, leptospirosis, or toxoplasmosis. The sensitivity of ABC-GO is comparable to that of the indirect fluorescent antibody test.
A visual, enzyme-linked immunosorbent assay using urease (ELISA-U) as the enzyme marker was adapted for rapid detection of antibody against Plasmodium falciparum. Flat-bottom, 96-well microtiter plates were coated with P. falciparum soluble antigen obtained by saponin and NP-40 treatment of parasite cultures. Antibody was detected by successive incubations with test sera, urease-conjugated rabbit-human antibody, and urease substrate. Reactive sera developed a definite and easily visualized purple color. Sera from patients with single infections of P. vivax or P. ovale were unreactive. No cross-reactivity was noted with sera from patients with rheumatoid arthritis, filariasis, amebiasis, schistosomiasis, dengue, scrub typhus, leptospirosis, or toxoplasmosis. The procedure can be performed at room temperature and completed within 1 hr. The sensitivity of the assay is comparable to that of the indirect fluorescent antibody test at all but the lowest dilutions tested.
The drug susceptibility of 70 isolates of Plasmodium falciparum to standard and experimental antimalarials was evaluated using a radioisotope microdilution method. All isolates were from forest fringe dwelling Orang Asli, the aborigines of Peninsular Malaysia. The geometric mean IC50 values were: chloroquine, 10 ng/ml; amodiaquine, 4.7 ng/ml; mefloquine, 2.8 ng/ml; quinine, 40.5 ng/ml; halofantrine, 1.5 ng/ml; enpiroline, 3 ng/ml; and pyrimethamine, 21 ng/ml. Four isolates exhibited decreased susceptibility to chloroquine (IC50 greater than 60 ng/ml), and one exhibited decreased susceptibility to quinine (IC50 = 161 ng/ml). Three isolates showed decreased susceptibility to mefloquine (IC50 = 10-11 ng/ml). The lack of drug pressure may account for the high prevalence of P. falciparum isolates susceptible to chloroquine.
Two hundred and seventy-five Orang Asli volunteers living in nine villages in the Pos Legap Valley of Perak State, peninsular Malaysia, participated in a prospective study designed to characterize the epidemiological, parasitological, and entomological characteristics of Plasmodium falciparum, P. vivax, and P. malariae malaria transmission. Prevalence rates for the three plasmodial species at initiation of the study ranged from 56% in the 0-4-year-old age group to 0% in individuals over the age of 40. Entomological surveys were conducted, enabling us to determine mosquito salivary gland-positive rates and entomological inoculation rates of 1.2 infectious mosquito bites per person per month for P. falciparum, 2.4 for P. vivax, and 0.3 for P. malariae. Cumulative incidence rates over the 16 weeks of the study, following radical cure of all volunteers, were 22.5% for P. falciparum, 12.7% for P. vivax, and 1.5% for P. malariae. The median baseline antibody titer against the immunodominant repetitive B cell epitope of P. falciparum or P. vivax circumsporozoite protein was significantly higher for volunteers who did not become parasitemic. Volunteers were selected for further study if they had evidence of being challenged with P. falciparum sporozoites during the study, based on a two-fold or greater increase in antibody titer against the immunodominant repetitive B cell epitope of the circumsporozoite protein. Resistance to infection was seen in six of 10 individuals who had high (greater than 25 OD units) baseline ELISA titers, compared with only three of 24 individuals who had low baseline ELISA titers (chi 2 P less than 0.02). A similar analysis for P. vivax did not show a significant correlation.
Monoclonal antibodies (MAbs) directed against different epitope regions on three sexual stage-specific gamete surface proteins of Plasmodium falciparum, Pfs 25, Pfs 230, and Pfs 48/45, were used to study the genetic diversity of these epitopes among fresh isolates of P. falciparum from Malaysia, using immunofluorescence microscopy (IFA). Among 45 Malaysian isolates, one epitope of Pfs 25, designated region I, showed evidence of variable reactivity with MAbs among different isolates; the Pfs 25 epitope, region II, was universally recognized by MAbs in all isolates. Two apparently distinct epitope regions of Pfs 230 were defined by MAbs, one of which was universally recognized by MAbs among the 45 isolates; the other was conserved in all but three isolates. The epitope regions of gamete-surface protein Pfs 48/45, designated regions I, IIa, IIb, IIc, III, and IV, were examined for reactivity by IFA in 33 isolates. Epitope regions I, IIb, III, and IV were conserved in all isolates; regions IIa and IIc existed in variant forms.