Efficacy of permethrin-impregnated bed nets on malaria control in a hyperendemic area in Irian Jaya, Indonesia III. Antibodies to circumsporozoite protein and ring-infected erythrocyte surface antigen.

A two years intervention study was carried out using permethrin impregnated bed nets in a hyperendemic area, in Irian Jaya, Indonesia. To assess the influence of this intervention on natural immunity, concurrent immunological studies to determine levels of antibodies to the circumsporozoite (CS) and ring-infected erythrocyte surface antigen (RESA) proteins were conducted. Prevalence and titers of immunoglobulins (Ig)G and IgG subclasses were periodically measured in 138 individuals (30 children under the age of ten and 108 villagers ten years old and older). In the younger group, seropositivity of total IgG against CS fluctuated according to the parasite infection rates; however, IgG seropositive reaction against RESA gradually increased. In the older age group, seropositivity of both kinds of antibodies was stable during the whole study period. Nevertheless, the geometric mean titers of total IgG against CS and RESA were significantly reduced in this latter group in individuals who contained these antibodies before and after intervention. The geometric mean titer of IgG3 subclass against RESA was decreased at a highly significant level (p = 0.0005), and that of IgG4 against the same antigen was also decreased although to a lesser extent (p = 0.02).

Efficacy of permethrin-impregnated bed nets on malaria control in a hyperendemic area in Irian Jaya, Indonesia: influence of seasonal rainfall fluctuations.

A malaria intervention study was carried out using permethrin impregnated bed nets in the south-central part of Irian Jaya with perennial transmission, from April 1993 to April 1995. Malariometric surveys were carried out periodically for parasite prevalence by species and for spleen rates. Prior to intervention, the percentage of Plasmodium falciparum infected inhabitants was significantly higher in Hiripau, where permethrin-impregnated bed nets were used during the study, than in the placebo-treated control village, Kaugapu. After two years of intervention the situation was reversed and figures higher in the control village (RR 0.19, 95% CI 0.10-0.36, p < 0.0001). Similarly, P. vivax infection rates, 12.4% in Hiripau vs 5.7% in Kaugapu in April 1993. were reversed in April 1995 (3.6% in Hiripau and 11.3% in Kaugapu, p < 0.001). In the treated village, pre-control hyperendemicity was reduced to a low mesoendemic level (spleen rate 12.5%) during two years of intervention, whereas the level was mesoendemic (spleen rate 35.2%) in the control village. Impregnated bed nets were found an effective intervention both in moderate (April 1993 through April 1994, 1,626 mm rainfall) and high (April 1994 through April 1995/1995, 3,321 mm) transmission seasons.

Efficacy of permethrin-impregnated bed nets on malaria control in a hyperendemic area in Irian Jaya, Indonesia: differentiation between two age groups.

A malaria intervention trial was conducted for two years to evaluate the efficacy of permethrin-impregnated bed nets in reducing malaria infection and splenomegaly in two different age groups, ie below and over age of ten, in a hyperendemic area in Irian Jaya, Indonesia. Permethrin-impregnated or placebo-treated bed nets were provided to a treated and a control village, respectively. Immediately after periods with moderate rainfall in the first year, treated bed nets decreased P. falciparum and P. vivax density in the blood of children <10 years (group 1) but did not reduce the percentage of infection with either species. Children >10 and adults (group 2) showed significant reduction only in P. falciparum infection rates and density, whereas P. vivax was not influenced. After an excessive rainfall season in the second year, the risk for P. falciparum infections in both age groups using treated nets was less than half of that in the control village. P. vivax infection rates were significantly lower in the treated village at the beginning of and after these heavy rainfalls. In the treated village, spleen enlargement was markedly reduced in the younger age group during the second year.

Malaria situation in several villages around Timika, south central Irian Jaya, Indonesia.

Malaria in Timika area, south central Irian Jaya, is a public health problem causing morbidity and mortality, particularly to the vulnerable age group. In August/September 1992 malariometric surveys were conducted simultaneously with sensitivity studies of Plasmodium falciparum to antimalarials, and bionomics of vectors in six villages around Timika (Mwapi, Kaugapu, Hiripau, Pomako, Mapurujaya, Kwamki Lama). The average overall spleen rate was 44.0%, the highest rate observed in Kwamki Lama (68.3%) and the lowest in Mapurujaya (13.7%). The average parasite rate in children aged 2-9 years was 60.6%. The highest rate was found in Mwapi (92.0%) and the lowest rate in Mapurujaya (4.8%). In the study area the dominant species was P. falciparum, (except in Kaugapu), followed by P. vivax. P. malariae and P. ovale were not observed. In vivo sensitivity studies done in 7 villages showed P. falciparum was resistant to chloroquine [51.3% S/R I (sensitive or 1st grade resistant), 43.6% R II and 5.1% R III] in Kwamki Lama, SP I and SP II (transmigrant settlements) and Timika health service center. In vitro sensitivity test in Kwamki Lama, SP I, SP II and Timika health service center showed 64.4% resistant to chloroquine, and remain sensitive to sulfadoxine-pyrimethamine, quinine and mefloquine. Vector studies revealed that Anophelese punctulatus and An. koliensis were the potential vectors as was confirmed by ELISA positive test with a sporozoite rate of 1.43% and 0.33% respectively. The vectors were indoor and outdoor resting.

Anthelminthic treatment raises plasma iron levels but does not decrease the acute-phase response in Jakarta school children.

The study was conducted to investigate the impact of intestinal helminthiasis and treatment on iron status and acute phase response (APR) among urban Indonesian primary school children, aged 8-11 years old. The prevalence of helminthiasis among these children was; Ascaris lumbricoides, 81.6%; Trichuris trichiura, 88.3%; and mixed infection of A. lumbricoides and T. trichiura, 70.0%. Of 120 children enrolled in the investigation, 59 received a single 400 mg dose of albendazole, and 61 received a placebo. Ten days following treatment, the prevalence of ascariasis and trichuriasis in the treatment group diminished to 0% and 27%, respectively, and in the placebo group to 63.9% and 68.9%. Plasma iron, hemoglobin, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), white blood cell (WBC), interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor (TNF) concentrations were determined prior to the intervention and 10 days after. Plasma iron concentrations and WBC count rose in the treatment group (p=< or =0.05) when compared to baseline status. Increases in hemoglobin concentrations observed in the treatment group 10 days post-treatment were not statistically significant. CRP, IL-1, IL-6 and TNF were found to be within normal limits for both groups both before and after treatment. ESR increased significantly in both treatment and placebo groups when compared the rates measured before treatment. These findings show that treatment with albendazole is associated not only with a decreased worm burden in school children, but also a rise in plasma iron.

Seroepidemiological studies of malaria in different endemic areas of Indonesia.

A total of 618 sera from inhabitants living in various endemic areas in Indonesia were examined for IgG against Plasmodium falciparum utilizing young trophozoites and mature schizonts as antigens by the method of ELISA and IFAT. In general, antibodies against trophozites (RESA) based on ELISA and antibodies against schizonts based on IFAT showed a correlation of malarial antibodies with the level of endemicity of the area examined. Anti-RESA antibody, detected either by ELISA or IFAT was more pronounced in the aparasitemic group compared to the parasitemic group. On the contrary, anti-schizont antibody measured by IFAT was more pronounced in the parasitemic group. Malarial antibody levels against the schizont-merozoite fraction of P. falciparum as assayed by ELISA appeared to develop more slowly compared to levels based on IFAT.

Antibody responses to human lymphatic filarial parasites.

The lymphatic filarial parasites, Brugia and Wuchereria, continue to present an immunological puzzle, particularly with respect to the development of natural resistance or damaging disease. We have approached this question by examining humoral responses to a few defined antigens of selected interest from these parasites, using sera from each category in the spectrum of filarial disease. Many antigens, such as the major adult surface protein of Mr 29,000 (29K), appear to be recognized at all stages of infection, but two components show interesting patterns of differential recognition. A triplet of proteins of Mr 65-75K associated with the microfilarial surface is preferentially bound by serum from patent microfilaraemic infections, whereas an unrelated 75K protein has been found to react only with antibody from amicrofilaraemic individuals. In general, however, the data obtained so far emphasize the importance of undertaking an antigenic analysis at the level of single epitopes. Such studies are now under way using recombinant proteins expressed in bacterial hosts.

Antigenic characterization of adult Wuchereria bancrofti filarial nematodes.

Adult Wuchereria bancrofti were recovered from infected Presbytis cristatus monkeys and radio-isotope labelled extrinsically with 125I and in vitro with [35S]methionine. 125I labelling of the surface of adult W. bancrofti permitted a comparison between the major surface antigens of this species and those from the related lymphatic filariae, Brugia malayi and B. pahangi. All species bear a prominent Mr 29,000 surface antigen but among the differences observed were the strongly labelled molecules with Mr 58,000 and 67,000 in W. bancrofti which are extremely faint in the Brugia species. The [35S]methionine label was effectively incorporated into somatic parasite proteins in vitro although it was not possible to identify any secreted proteins in this way. The antigenicity of these products was investigated using a variety of sera from homologous and heterologous infections and the immunoprecipitation patterns highlighted particular differences between somatic proteins of male and female worms. One secreted antigen was detected, however, by virtue of its phosphorylcholine epitopes, in the culture medium of mixed adult worms; medium from male W. bancrofti adults was negative although homogenates of either sex of adult W. bancrofti were strongly positive in the same system.

Identification of Brugia malayi in vectors with a species-specific DNA probe.

We evaluated the potential value of a cloned sequence of genomic DNA of Brugia malayi as a species-specific probe. Clone pBm 15 reacted with all stages of 8 different geographic isolates of B. malayi and cross-hybridized with microfilariae of B. timori. It did not hybridize with Wuchereria bancrofti or with B. pahangi, W. kalimantani, Dirofilaria repens, Breinlia booliati or Cardiofilaria species, animal filariids that can be sympatric with B. malayi. P32-labeled clone pBm 15 correctly identified mosquitoes infected even with 1 infective larva of B. malayi. This specific DNA probe should be an invaluable tool to monitor control programs of Brugian filariasis.

Secreted and surface antigens from larval stages of Wuchereria bancrofti, the major human lymphatic filarial parasite.

Antigenic proteins of microfilariae and infective larvae of Wuchereria bancrofti have been identified by intrinsic and extrinsic radiolabelling, and specific immunoprecipitation with sera from filarial patients. From 125I surface-labelling experiments, the most prominent antigen on both stages is of relative molecular mass (Mr) 17 000, while a molecule of similar size is both synthesized and released in vitro following labelling with [35S]methionine. A second similarity between the two stages is the production and secretion of a Mr 21 000 component, which is, however, not detected on the worm surfaces. A series of additional proteins from larval W. bancrofti are described from each parasite compartment (secreted, surface and somatic) and the antigenicity and specificity of these components explored with serum from patients with filariasis due to W. bancrofti or Brugia species, and with onchocerciasis. Among additional molecules released in vitro we have found a Mr 51 000 antigen from both stages, and also several proteins which are not recognised by antibody from human filarial patients.

Secreted and circulating antigens of the filarial parasite Brugia pahangi: analysis of in vitro released components and detection of parasite products in vivo.

A range of excretory-secretory (ES) antigens have been characterised following in vitro culture of adult Brugia pahangi filarial nematodes in serum-free medium. Analysis by radioiodination, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and immunoprecipitation of purified macromolecules with antibodies from human and experimental animal infections reveals both host and parasite components. Two host molecules appear by molecular weight and immunoprecipitation analysis to be immunoglobulin and serum albumin, presumed to be taken up from the jird host from which adult worms were recovered. A further prominent component, of 19 kDa, reacts with neither anti-host nor anti-filarial antibodies, and may represent a non-immunogenic parasite product. Three additional bands, although less intensely radiolabelled, did prove to be consistently antigenic, with apparent molecular weights of 15, 29 and 40 kDa. A further ES antigen, which does not take up radio-iodine or lend itself to electrophoretic analysis, has also been detected. This molecule reacts in a immunoradiometric assay in which monoclonal antibody directed against a repetitive epitope acts both to capture and indicate antigen presence. The same antibody, Bp-1, may also be employed to detect circulating antigen in the serum of animals experimentally infected with Brugia pahangi, and in the serum of patients with each of the three species of human lymphatic filariasis, Brugia malayi, Brugia timori and Wuchereria bancrofti.

Specificity of surface molecules of adult Brugia parasites: cross-reactivity with antibody from Wuchereria, Onchocerca and other human filarial infections.

The extent of structural and immunological similarity between surface antigens of three species of Brugia filarial parasites was studied by lodogen-mediated surface labelling of adult worms of B. malayi, B. pahangi and B. timori. The close homology and cross-reactivity between these antigens reported in previous surface labelling studies with Bolton-Hunter reagent, was verified in this system. The surface antigens of adult B. pahangi are also recognised by antibody from patients with Wuchereria bancrofti, the major human lymphatic filariae, and by antibody to Loa loa and Mansonella perstans. Further experiments have begun to establish the boundaries of these cross-reactions: antibodies to nonfilarial nematodes such as Trichinella, Necator and Strongyloides does not recognise the adult surface antigens; however, although most anti-Onchocerca sera show little or no reaction to the major (29 kDa) surface antigen, there is consistent reactivity to the secondary 20 kDa antigen, and extensive recognition of a minor antigen of 15 kDa.

Adverse reactions to a single dose of diethylcarbamazine in patients with Brugia malayi infection in Riau Province, West Indonesia.

A study on the adverse reactions, occurring after treating microfilaremic patients infected with B. malayi, revealed that all reacted to a single oral dose of DEC (5 mg/kg). The major reactions were fever, headache, anorexia, abdominal pain, muscle and joint pains, nausea and vomiting. There seemed to be no association between the time of fever onset and microfilarial density, but the number of cases observed was too small to make any firm conclusion. There was a tendency for more severe reactions to occur in patients with higher microfilaria counts. Local reactions, probably due to destruction of adult worms, were seen in 3 patients. The reactions were serious enough to necessitate the patients spending approximately 48 hours in bed.

Surface antigens of a filarial nematode: analysis of adult Brugia pahangi surface components and their use in monoclonal antibody production.

The surface antigens of adult worms of the filarial nematode Brugia pahangi have been investigated further by surface radioiodination and detergent solubilisation techniques. In addition to yielding new information on the distribution of antigenic components of this stage, detergent-solubilised molecules were used in both radiometric and enzyme-linked assays for human and mouse antibody. These assays were subsequently used in screening for monoclonal antibodies from hybrid cells derived from animals infected with living parasites and boosted with detergent-extracted antigen. Three monoclonal antibody-producing cell lines were isolated, with differing antigenic specificities: Bp-1, which binds a non-iodinatable antigen with high ELISA activity; Bp-2, which reacts with a determinant found on but not unique to the major surface Iodogen-labelled 29 kDa antigen; and Bp-3, which is specific for a minor antigen of 20 kDa revealed by Iodogen labelling.