The optimal conditions for the determination of exposure to scrub typhus by the whole blood lymphocyte transformation assay was 7 days culture of 10% blood in RPMI 1640 medium supplemented with 10% human AB-negative serum and L-glutamine with 50-200 micrograms protein/ml of Karp, Kato, or Gilliam strain membrane antigen. A simple exponentially decaying linear model shows the decrease in lymphocyte viability, the ability of sensitized cells to be stimulated with PHA mitogen, and the corresponding decrease in stimulation by scrub typhus antigens with increasing time of preincubation on ice. The lower limit of stimulation index for the detection of scrub typhus by whole blood lymphocyte transformation assay was 4.0 with a type I error of 1%.
A modified version of the standard 2-site sporozoite enzyme-linked immunosorbent assay (ELISA) using 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate chromogen solution was adapted for rapid detection and identification of Plasmodium falciparum and P. vivax circumsporozoite (CS) proteins. The TMB-ELISA was evaluated using sporozoites from experimentally infected mosquitoes and laboratory colonized uninfected mosquitoes. Our data indicate comparable sensitivity levels between the TMB-ELISA and the standard ELISA, i.e., 50 P. falciparum or P. vivax sporozoites/50 microliters of test solution. Reactions inherent to the method were specific and background reactivity was minimal. The TMB-ELISA is rapid (1 hr), simple, uses a minimal amount of monoclonal antibodies, and is suitable for use in a wide range of laboratories.
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.
A seroepidemiological survey of 837 people and 383 febrile patients was performed in rural areas of Sabah. We determined that the rickettsial diseases scrub typhus and endemic typhus were uncommon causes of febrile illness, as was tick typhus, except in forest dwelling peoples. The rate of occurrence of SFGR specific antibody was 16.5% among 412 forest dwellers, indicating that tick typhus may be a frequent cause of illness in this population.
An indirect immunoperoxidase test was compared with an indirect fluorescent antibody test and the Weil-Felix OXK test for serodiagnosis of scrub typhus by measuring the rickettsial antigen specific activity of IgG, IgM, and whole globulin. Acute and convalescent sera from 50 Rickettsia tsutsugamushi isolate-positive scrub typhus patients and from 45 febrile patients diagnosed as having diseases other than scrub typhus were tested. The receiver operating characteristic for each test showed that the indirect immunoperoxidase and indirect fluorescent antibody tests were more sensitive and specific than the Weil-Felix test using convalescent and acute as well as paired sera. The indirect immunoperoxidase test showed no cross-reactivity when R. tsutsugamushi antigen was tested against sera collected from patients living outside the scrub typhus-endemic area with diseases other than scrub typhus. The indirect immunoperoxidase and indirect fluorescent antibody tests were comparable in measured response to R. tsutsugamushi, R. typhi, and TT-118 (spotted fever group) antigen. Thus the indirect immunoperoxidase test represents a sensitive, specific, reproducible, and practical semiquantitative test for rickettsial disease diagnosis.
A seroepidemiologic survey of Plasmodium vivax and Plasmodium falciparum transmission was conducted in 94 Orang Asli children and adults. The prevalence of malaria was 46% in this population, and infections due to P. vivax and P. falciparum occurred with equal frequency. Multi-species infection was common, particularly in children less than 10 years of age. Circumsporozoite (CS) antibodies to P. vivax were detected by ELISA, using the recombinant protein NS181V20, in sera from 53-95% of all subjects in this study. The specificity of reactivity to NS181V20 was confirmed by immunofluorescence using air-dried sporozoites. CS antibodies to P. falciparum were present in less than 50% of the population less than 30 years of age. These data support further testing of this protein as a candidate vivax vaccine.
Duffy phenotypes were determined for 314 Malaysian Orang Asli. The most common gene, Fya, was present in 313; there were no Duffy negative individuals. A previous study found evidence of Plasmodium vivax infection in 5 of 7 Orang Asli reported to be of the Duffy negative genotype. In this study, 5 of the 7 previously tested Orang Asli were retested in triplicate, and each of the 5 was found to be Duffy positive, having the Fya gene and a phenotype of Fy (a + b-).
Dexamethasone has recently been shown to block the production of cachectin (implicated in the pathogenesis of cerebral malaria) if administered prior to endotoxin induction of mouse macrophages. Using the hamster cheek pouch-cerebral malaria model, we tested the hypothesis that dexamethasone is effective as a therapeutic agent in severe malaria if given before some yet undefined trigger point in the disease. Infected hamsters were treated with dexamethasone (0.7 mg/kg) daily on days 7-12, 4-12, or 1-12 post-challenge. When treatment was started on day 1, whole body oxygen consumption (used as a measure of erythrocyte transport to sites of diffusion) on day 12 was greater than (P less than 0.05) that of infected control animals, though the degree of anemia was no different in treated and untreated groups. Furthermore, treatment produced a reduction in monocyte accumulation, capillary malfunction, and monocyte/red blood cell aggregate formation observable in the cheek pouch in vivo and a similar reduction in monocyte presence, capillary pathologic change, and multifocal hemorrhage in the brain on postmortem. These data suggest that mediator(s), whose production can be blocked by pretreatment with dexamethasone, are involved in the pathogenesis of disease leading to death of the Plasmodium berghei infected hamster.
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.
Scrub typhus is a major cause of febrile illness throughout the Asia-Pacific region. It is commonly undiagnosed, partly because of the lack of a simple, reliable diagnostic test which can be used in clinical laboratories. The indirect immunoperoxidase technique, configured into a test kit, was provided to technicians who were trained in its use. They used the kit during a 2 year field trial in their respective clinical hospital laboratories throughout Malaysia. In an evaluation using 1,722 consecutive sera tested in those laboratories, the kit was found to have a median sensitivity for IgG detection of 0.85 (range 0.33-0.95), a median specificity of 0.94 (range 0.88-1.00), reproducibility of 0.86, and efficiency of 0.92 when compared to the reference laboratory. In a proficiency survey in which 10 laboratories received 3 coded test samples, all but 2 laboratories had results within 1 dilution of the reference laboratory in quantitating specific IgG, whereas 7 laboratories were within 1 dilution in quantitating IgM. The shelf life of the kit was at least 1 year at 4 degrees C.