Determination of immunoreactivity ofPlasmodium falciparum antigens, serum dilutions and biomaterials.

Immunoreactivity properties of serum dilutions andPlasmodium falciparum malaria antigens were measured and compared by ELISA technique using different ELISA plates to evaluate the role of antigens and serum dilutions for optimum binding. Also effort has been made to see the effect of reaction surface and material i.e. ELISA plates for binding capacity. Serological properties were estimated by ELISA methods for detection of malaria and determination of immunological characteristics. Three Pf antigens (PfAg) i.e. ring infected erythrocyte surface antigen: AR-1 (RESA), histidine-rich protein 2 antigen (HRP-2) and glycophospholipid antigen (grown and developed Pf antigen from PSJ-M strain): GPL1 have been used for serological testing of human blood samples by Enzyme Link Immunosorbant Assay (ELISA). 1∶100, 1∶1000 and 1∶10000 dilutions of Pf positive and negative serum (50 samples in each group) and 1∶1000 dilution of Pf antigens were used to measure immunoreactive properties by ELISA method. Result of PfAg-serum immunoreactivity study showed that GPL1 has the highest degree of immuno binding reactivity compared to other Pf antigens. HRP-2 and RESA antigens showed no significant difference to each other. Study also found that Costar and Fastec ELISA plates have a better Ag-Ab binding capability compared to immulon and Falcon plates at all dilutions of serum. Serum dilution of 1∶100 showed best binding and reactivity with Pf antigens followed by 1∶1000 and 1∶10000 showed lowest reactivity.

Design and development of an immunosensor for the detection of malaria in field conditions.

Eradication of malaria in Southeast Asian countries is still a distant goal, due to the absence of a simple, rapid and inexpensive diagnostic technique. Here, an immunosensor for the photometric detection of malaria, the malaria-detecting immunosensor (MDI), is developed to detect Plasmodium falciparum malarial antibodies in human blood. The method uses the principle of laser light-scattering by latex bead agglutinates in media monitored by a light-detecting device. Agglutination is induced by mixing antigen-coated latex beads with serum antibodies. Immunoreactions are measured in terms of the Tyndall effect in the transmitted beam detected by photodiodes. MDI sensitivity and specificity are compared with the results of enzyme-linked immunoabsorbent assay and laser light-scattering immunoassay techniques, which show that it is a good and sensitive monitoring device.

Testing of newly developed glycophospholipid antigen for the detection of P. falciparum malaria by laser light immunoassay in endemic and non-endemic areas.

A glycophospholipid (GPL) antigen isolated from Plasmodium falciparum culture supernatant has been tested for its antigenicity. Detection of malaria positive known blood samples and unknown field samples from endemic and non-endemic areas were compared. In this study laser light scattering immunoassay (LIA) was used for the detection of P. falciparum malaria. Test results of control (malaria negative samples from Surat) were compared with known positive samples and unknown malaria positive field samples. A positive correlation has been observed (97%) in falciparum positive samples from laboratory and unknown samples from endemic area (Haldwani) by LIA method using GPL antigen. From the results of the study it was found that GPL antigen has a better antigenic property and can detect almost all the cases of Pf malaria by LIA method.

Sensitivity and specificity of isolated antigen fromPlasmodium falciparum culture supernatant.

Immunological sensitivity and specificity properties of isolated Plasmodium falciparum (GPL) antigen from culture supernatant have been measured and compared with malarial antigens and non malarial filtered paper blood sera for potency and efficacy. Latex bead coded GPL, Pf and RESA antigens immunoreaction properties of human filter paper blood samples (FPB) were studied by laser light scattering immunoassay (LIA) and Enzyme linked immunoabsorbent assay (ELISA) techniques. Results of GP. antigen sensitivity study by LIA method showed a very high malaria antibody binding response (MABR) i.e. 6% compared with 78% with RESA and 88% Pf antigens. Malaria detection by ELISA method also found similar results. Specificity study of GPL antigen for different non malarial filter paper blood sera (NMFS) showed no immunoreaction however Pf and RESA antigen showed few positive immunological responses. These results suggest that sensitivity and specificity properties of isolated GPL antigen is better than other antigens.

Study of malaria in a village of lower Myanmar.

Malaria endemicity in lower Myanmar has been studied to identify the causes for the prevalence of malaria in Yeasitkan village of lower Myanmar. Vector mosquitoes were collected by mosquito net in cattlesheds and in human dwellings (indoor and outdoor) by biting and catching procedure for the identification of species, insecticide susceptibility test and sporozoites detection. Larvae of mosquitoes were also collected in and around the village for vector identification and for breeding sources. Malaria infection in humans was examined by blood examination and blood antibody detection by ELISA method. Results showed that malaria infection was 43.2% in children under 10 years of age and An. dirus and An. minimus were found as main vectors. Total parasite positive rate was found to be 41.28% and in this 78.87% were P. falciparum infections and remaining 18.31% were of P. vivax. Spleen positive rate has been found very high in children between 2 and 9 years (52.94%). Study indicates that villages near to dam areas are more prone to malaria infection.

Effect of serum dilution in diagnosis of malaria in community.

Recently, it has been shown that immunological methods can be used for the diagnosis of malaria other than sero-epidemiology. A study has been done to investigate optimum binding capacity of antigen-antibody (Ag-Ab) at different serum dilutions. For validating antigen-antibody (Ag-Ab) reaction at 1:100, 1:1000 and 1:1000 serum dilutions, have been tested in two different laboratories to establish validation of the ELISA method. Inter laboratory test on synthetic peptide (RI) ELISA was found comparable and meaningful for assessing malaria transmission in defined locality at 1:100 dilution. Results also showed that 1:1000 serum dilution can be useful for diagnostic purpose.

Well-breeding Anopheles dirus and their role in malaria transmission in Myanmar.

Mosquitos were collected with human and animal baits from March 1996 to January 1998 in four villages located along the Yadana gas pipe line in Yepyu township, Dawae district, Tanintharyi Division, southern Myanmar. A total of 23 anopheline species were collected. Anopheles dirus were abundant in pre-monsoon (May/June) an post-monsoon (October) months. All An. dirus caught both humans and cattle were assayed with specific, sporozoite enzyme-linked immunosorbent assays (ELISAs). A total of 5/250 (2%) caught with human bait was found positive with Plasmodium vivax from Eindayaza, Ohnbinkwin and Thaechaung during rainy and cool-dry months. Larval surveys also showed An. dirus larvae/pupae were caught from domestic wells (6 to 46% found positive). Clinical surveys indicated that transmission is hyperendemic and occur all year round in all four villages.

Hyperendemic malaria in a forested, hilly Myanmar village.

A 1-year longitudinal study of hyperendemic malaria was carried out at Tha-bye-wa village, Oktwin township, situated in the forested Bago mountain range in south-central Myanmar. Mosquito infectivity was assayed using specific, sporozoite enzyme-linked immunosorbent assays. Anopheles dirus was the predominant vector in the postmonsoon season (October); during the cool-dry season (January), both An. dirus and Anopheles minimus were vectors. Members of the Anopheles culicifacies complex were caught in the hot-dry season (April) but none was infective. The entomological inoculation rate was estimated to be at least 13.7 infective bites/person/year. Infective An. dirus were caught feeding on cattle as well as on humans. Three of the 4 positive An. dirus and both positive An. minimus were caught biting humans indoors in the second quarter of the night when most people were sleeping. This suggests that use of insecticide-impregnated bednets in this area could interrupt transmission.

The effect of tetanus toxoid in leprosy patients.

Since cases of lepra reaction following smallpox vaccination and BCG vaccination had been reported the effect of tetanus immunisation on leprosy patients (whether it may provoke a lepra reaction or not) was studied. Three doses of purified tetanus toxoid (one ml initially, one ml after six weeks and one ml after six months) were given to 357 leprosy patients and 60 patients living in the same environ were followed as controls. The antibody response following immunisation was followed in six lepromatous leprosy patients using toxin antitoxin neutralisation test at the Lf/1000 level in mice and in three of them the antibody titre of leprosy patients rose to satisfactory level. The number of lepra reactions in these patients was monitored for nine months (two months before vaccination, during the six months period of vaccination and one month after the last dose of vaccine). There was no significant rise in the number of patients with reaction following the vaccination.

Active immunization against tetanus III. Results of a five year study.

This is the first time in Burma that tetanus toxoids (purified and adsorbed) have been tested in over 250 non-immune adult volunteers and studied for a period of nearly five years. The safety and efficacy of these toxoids have been assessed by immunological, statistical and clinical methods. Both toxoids were found to be safe. The adsorbed toxoid was far superior to the fluid toxoid as an immunizing agent and optimum immunization regimens are proposed and presented.

Pre-exposure prophylaxis of humans against rabies: I. Studies on personnel at the Burma Pharmaceutical Industry.

This is the first time in Burma where personnel at risk against rabies have been pre-immunized and the effectiveness of such a procedure has been studied for nearly two years. The first batch of lyophilized, Semple-type, beta propiolactone inactivated anti rabies vaccine produced by the Burma Pharmaceutical Industry (B.P.I.) was used to immunize 55 B.P.I. workers previously unexposed to rabies and with no history of rabies vaccination. Three doses of 0.25 ml of the vaccine were given intradermally at one week intervals. Booster doses were given on the 98th, 392nd and 592nd day after the first dose. Blood samples were taken and serum neutralization tests were performed at varying time intervals after basic immunization and booster doses. Satisfactory antibody responses were obtained. The course of immunological response is presented and discussed.