Comparison between microscopic examination, ELISA and quantitative buffy coat analysis in the diagnosis of falciparum malaria in an endemic population.

Monoclonal antibody-based ELISA and QBC (quantitative buffy coat analysis) were tested in two endemic areas with low and high incidence of malaria in Kanchanaburi Province, West Thailand with annual parasite incidence in 1992 of 119 and 5 per 1,000 population, respectively. The numbers of individuals positive by thick blood film examination (TBF) for P. falciparum with or without P. vivax, and P. vivax only were 82 and 69, respectively. The detection limit of ELISA was 10 parasites/10(6) red blood cells (RBC) (0.001% parasitemia). Of 1,095 individuals involved in the study at the beginning of the study, ELISA showed sensitivity, specificity, positive predictive value and negative predictive value of 78.1%, 94.9%, 72% and 98.1%, respectively. Nine of 18 (50%) TBF-positive but ELISA-positive individuals had parasitemia of less than 10 parasites/10(6) RBC. High and low incidence areas did not affect the validity of our result. Regression analysis showed good correlation between log parasitemia and ELISA percent OD increase (Y = 0 + 64.9*logX, r = 0.65), and agreement between TBF and ELISA results was 95.9%. In a fortnightly follow-up, in 82 TBF-positive individuals, both ELISA and TBF positive rates correlatively declined with agreement of 96.3%. With samples taken on the first day of the study, the TBF and QBC results were also correlated with agreement of 95.8% for P. falciparum, 95.6% for P. vivax. During 8 week follow-up involving altogether 191 samples, agreement between TBF and QBC results were 87.4% for P. falciparum. QBC detected more cases with P. falciparum infections but detected smaller number of cases with P. vivax infections.

Western blot analysis of antigens specifically recognized by natural immune responses of patients with Japanese encephalitis infections.

Specific recognition of antigenic proteins of Japanese encephalitis virus (JEV) by JE patients was investigated by using non-reducing and reducing Western immunoblot analysis. Under non-reducing conditions, the profile of JEV proteins recognized comprised E (52 kDa), NS1 (45 and 41 kDa), NS3 (66.2 kDa) and NS5 (103 and 97.4 kDa). When recognition patterns of sera from JE and dengue patients were compared, only slight differences between JE and dengue sera were found (under non-reducing conditions), involving only the 66.2 kDa protein: to this protein, JE sera exhibited greater reactivity, but not in greater frequency, than did dengue sera. In contrast, cerebrospinal fluid (CSF) from JE patients showed more differences from JE sera: CSF antibody lacked recognition of the 41 kDa protein and had lower frequencies, as well as less reactivities to several other proteins. These results suggested that restricted populations of lymphocytes were localized in the central nervous system of JE patients. The effect of reducing agent (2 beta-mercaptoethanol) on the recognition patterns of those groups of sera was also analysed: the reducing agent affected all the proteins mentioned above, however, the effects were not uniform. It is proposed that JE and dengue sera may recognize different epitopes on some or all of these proteins. Such differences cannot be detected by Western immunoblot analysis, but it would be feasible to test this hypothesis using epitope mapping with synthetic peptides in a multi-pin ELISA. Analysis in this fine detail is essential for designing improved JE vaccines.

Mapping of functional epitopes of Japanese encephalitis virus using monoclonal antibodies.

Epitopes involved in the important functions, hemagglutination (HA) and neutralization (NT), were mapped on Japanese encephalitis (JE) virus proteins by using monoclonal antibodies (MAbs). Fourteen MAbs raised against Nakayama-Yoken strain of JE virus characterized by hemagglutination inhibition (HI) and plaque reduction neutralization test (PRNT) were used to map the epitopes on the JE proteins by Western blot analysis in which non-reducing conditions were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). With these MAbs, at least 8 functional epitopes were demonstrated comprising (i) epitopes recognized by 5 MAbs which gave strong HI but weak NT activities and were mapped on the envelope (E) 53 kDa protein; (ii) epitopes recognized by 2 MAbs which showed weak HI but strong NT activities and were mapped also on the E protein; (iii) epitopes recognized by 2 MAbs which possessed weak HI but no NT activities and were mapped on the E protein; (iv) an epitope recognized by 1 MAb which gave weak NT and no HI activities and was mapped on the nonstructural protein 5 (NS5); (v) an epitope recognized by 1 MAb which showed activities similar to (i) but was mapped on both E and NS5; (vi) an epitope recognized by 1 MAb which had high activities to both HI and NT and was mapped on E and NS5; (vii and viii) epitopes recognized by 1 MAb which also gave low HI but high NT, and strong HI as well as strong NT activities respectively, but their location could not be demonstrated by SDS-PAGE under non-reducing condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoprecipitating antibodies against blood stage antigens of Plasmodium falciparum in cerebral and non-cerebral malaria patients.

Immunoprecipitating antibodies were determined in paired sera of 31 patients with cerebral malaria (CM), of whom 14 had complicated cerebral malaria (CCM) and 17 had uncomplicated cerebral malaria (UCCM), 15 single specimens of patients with acute uncomplicated (AM) malaria taken on the day of admission and 8 healthy controls. All but one patient were admitted within the first three days of the onset of fever. More than 20 precipitating bands were observed, of which the predominating molecules were the Mr greater than 200, 180, 157, 135, 130, 115, 103, 96, 91, 73, 71, 61, 49, 45, 43, 41 and 14.3 Kd. In general, there were no significant differences in the positive rates among the AM, CCM and UCCM patients except for the pf135 Kd molecule which was more frequently reactive in UCCM patients than the AM and CCM patients. If immunological naiveness in term of protective immunity is the feature in CM patients, the immunoprecipitation test used is inadequate to demonstrate the fundamental differences in immune responses between CM and AM patients.

Cross-sectional seroepidemiological survey of malaria in endemic areas with different activities of malaria control.

A single cross-sectional seroepidemiological survey of malaria antibody was conducted in 1982 in Klang District, Rayong Province in three villages under different phases of malaria control activity to determine whether a single survey could be used to delineate malaria endemicity in Thailand and to compare the usefulness of ELISA and the indirect haemagglutination test (IHA) in the assessment of malaria endemicity. Village 11 was a control area with high infection rate with an annual slide positive rate of 16.3% in 1981. Village 6 was also a control area but was in the late attack phase in which residual insecticide spraying has been ceased since 1976. Village 7 was a consolidation area. Finger-tipped blood was collected from 189, 191 and 132 individuals from villages 11, 6 and 7 respectively, and the plasma tested for anti-P. falciparum antibody with ELISA and IHA. With ELISA, it was shown that the seropositive rate in population of village 11 (84.6%) was significantly higher than those of other two villages (48.9% in village 6 and 28.8% in village 7). After age stratification, it was shown that the differences were observed in every age group except in the greater than or equal to 45 year age group of village 6. With IHA, a significantly higher seropositive rates in population of village 11 was evident when they were compared with the corresponding age groups of 6-14, 15-29 and 30-44 years in village 7, and the age group of less than or equal to 5 year in village 6.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum alpha-1 antichymotrypsin is a possible growth inhibitor of Plasmodium falciparum.

Serum protease inhibitors were determined in paired sera from 7 patients with cerebral malaria and 2 patients with acute malaria showing high and low growth inhibition activity in the initial and follow-up sera respectively. Alpha-1 antichymotrypsin and alpha-1 antitrypsin but not alpha-2 macroglobulin showed direct correlation with the growth inhibition activity. When alpha-1 antitrypsin was deliberately added to the malarial culture no growth inhibition occurred indicating that the alpha-1 antichymotrypsin was the most likely factor responsible for inhibition of growth of malarial parasites in vitro.

Multiple strains of Plasmodium falciparum are necessary for the growth inhibition assay.

Sixty-seven serum samples from individuals living in a malaria endemic area and sixty sera from healthy blood donors living in Bangkok (non-malarious area) were tested for growth inhibition activity against 3 strains (SO, SN and G-112) of Plasmodium falciparum. Forty-eight percent of the sera from the endemic area were positive when all 3 strains were tested. Among the positive sera, positive rates of 90.6% were observed for the SO and SN strain combination, 87.5% for the SO and G-112 combination, and 50% for the SN and G-112 combination. It is therefore recommended that multiple parasite strains should be tested in the growth inhibition assay. If facilities are limited, a minimum of two strains should be used, one of which is the SO strain or its equivalent.