Computer-assisted multiple categorization of absorbance values in ELISA through pictorial emulation of 96-well plates.

A simple algorithm is proposed by which multiple categorization of absorbance values from ELISA plates is performed under a microcomputer control. The printed output is a pictorial emulation of a 96-well plate with the color intensities represented for each reaction. Although the method is presented as a colorimeter computer interfaced system, a provision for manual entry of absorbance values via keyboard is also included. Simulation is based solely on the magnitude of absorbance values. Therefore, it is possible to utilize any enzyme/substrate combination within the range of filters of the colorimeter. We have tested the present system for titration of anti-malarial antibodies in human serum and for the screening of mouse hybridoma culture supernatants.

Diagnosis of toxoplasmosis by electron microscopic fine-structural analysis.

The analysis of fine structures by electron microscopic examination of ultrathin tissue sections permitted a diagnosis of toxoplasmosis in a fatal human case that would have gone undiagnosed by conventional methods. Examination of histologically prepared sections revealed organisms that were morphologically nondiagnostic. Fine-structural analysis showed the organisms to be 3 x 2 micrometer in size, to contain few micronemes, to contain as many as nine rhoptries, to possess an apical conoid, and to multiply by endodyogeny. The features corresponded to those observed in fine-structural analyses of Toxoplasma gondii grown in animals by Scholtyseck and Mehlhorn. Fine-structural analysis can be a valuable adjunct in the diagnosis of toxoplasmosis.

Toxoplasma gondii isolated from amniotic fluid.

A pregnant woman contracted toxoplasmosis from exposure to oocysts shed by cats. She underwent amniocentesis for a therapeutic abortion, and Toxoplasma gondii was isolated from the amniotic fluid and placenta. This method may be useful in determining whether the fetus is infected in cases of toxoplasmosis acquired during pregnancy.

Indirect immunofluorescence test for human Babesia microti infection: antigenic specificity.

An indirect immunofluorescence (IIF) test was performed with human sera to detect cross-reactivity of Babesia microti antibodies with other species of Babesia parasites, with other blood and tissue parasites, and with various tick-borne organisms. Antisera to B. microti cross reacted with other Babesia species, but at lower dilutions than with the homologous antigens, and occurred most often during the acute phase of the disease. Cross-reactions with antibodies to malaria, Colorado tick fever, and a variety of other parasitic diseases were uncommon. However, acute and convalescent phase sera from 4 patients with suspected or confirmed Rocky Mountain spotted fever showed a rise in titer to B. microti antigen. In addition, 6 of 185 serum samples from children on an Indian reservation in North Carolina had IIF titers of greater than or equal to 1:256, suggesting a possible focus of B. microti infections in humans.

Comparison of the complement fixation, indirect immunofluorescence, and indirect hemagglutination tests for malaria.

The complement fixation (CF), indirect immunofluorescence (IIF), and indirect hemagglutination (IHA)tests for malaria were compared by using sera from U.S. citizens with either natural infections or heroin-associated, needle-induced infections. In natural Plasmodium vivax infections, the CF, IIF, and IHA tests apparently detect malarial antibodies equally efficiently for the first 2 months after the onset of symptoms, but the titers obtained by CF and IIF rapidly decline within a year, while the IHA titers remain elevated. In the sera from heroin addicts who developed needle-induced P. vivax infections, sensitivities of all three tests were decreased: the IIF and IHA tests each detected 83%, but the CF test detected only 57.1%. False-positive reactions with this group were very high for the CF (76.6%) and IHA (15.9%) tests, but only 2% for IIF.

Evaluation of the indirect immunofluorescence and complement fixation tests for the serodiagnosis of schistosomiasis.

Sera from patients with a variety of infections were used to compare the sensitivity and specificity of the complement fixation (CF) and the indirect immunofluorescence (IIF) tests for schistosomiasis. Adult antigens were used in both tests. The sensitivities of the IIF and CF tests were 95% and 69%, respectively; the specificities were 98% and 100%, respectively. There was no statistical difference between the number of positive reactors among those individuals with no detectable helminth infection and those infected with helminths other than schistosomes. Infected people native to endemic areas had lower reactivity in both tests than did infected U.S. citizens who resided in endemic areas for only a few years. We concluded that the IIF test with adult antigen was more sensitive than and as specific as the CF test; therefore, the IIF test is the procedure of choice for routine diagnostic serology of schistosomiasis.

A case of asymptomatic babesiosis in Georgia.

An asymptomatic infection with Babesia sp. was diagnosed in an epidemiologic investigation of transfusion-acquired malaria. This is the seventh human piroplasmosis infection that has been reported in the literature.

An oocyst-transmitted outbreak of toxoplasmosis: patterns of immunoglobulin G and M over one year.

Sera from 32 patients who became ill after jungle combat training were tested for antibodies to Toxoplasma gondii using the indirect immunofluorescence test. Swift rises of both IgG and IgM antibodies occurred within 2 weeks of infection. Reduction in IgM titers, due to competitive suppression by IgG antibody, occurred in most but not all cases. Suppression of IgM reaction by IgG antibody could be prevented by adsorption of serum with Staphylococcus aureus containing protein A. Antibody of the IgM class could be detected at greater than or equal 1:256 level in many sera at 6-month and 1-year intervals after exposure. In groups with exposures such as were experienced in this study, the presence of IgM antibody titers in single serum specimens cannot be used to indicate recent exposure. Both IgG and IgM antibody may rise together to high levels very rapidly after infection; IgM did not precede IgG antibody in our 32 subjects.

Parasitized erythrocyte membrane antigens of Plasmodium brasilianum: relationships with the ring-infected erythrocyte surface antigen of Plasmodium falciparum.

An antigen, designated here as the parasitized erythrocyte membrane antigen (PEMA), is present in the erythrocyte membrane surrounding all intraerythrocytic stages of Plasmodium brasilianum. An antibody specific for PEMA appeared in 21 (50%) of 42 antisera from Saimiri sciureus monkeys naturally infected with P. brasilianum. Of these 42 sera, nine (21.4%) contained antibody to the ring-infected erythrocyte membrane antigen (RESA); of these nine sera, six did not react with PEMA. Sera of humans infected with P. malariae reacted with PEMA and RESA in a similar pattern; i.e., of 83 antisera, 71 (85.5%) reacted with PEMA and 30 (36%) reacted with RESA. Only one of these latter 30 sera were not reactive with PEMA. Of 167 sera from humans infected with P. falciparum but not P. malariae, 133 (79.6%) reacted with RESA; of these, 43 (25.7% of the total) reacted with PEMA but not RESA. Although PEMA was demonstrated with P. brasilianum and RESA with P. falciparum, neither PEMA or RESA could be demonstrated with P. malariae. Interactions of PEMA and RESA and the corresponding antibodies offer a method whereby the two morphologically similar quartan species, P. malariae and P. brasilianum, can be readily distinguished from each other and may furnish clues to genetic separation of the two and the mechanisms of interaction of quartan malaria and P. falciparum where they are coendemic.

Development and persistence of antibody in persons infected with Babesia microti.

An indirect immunofluorescent test was used to determine antibody titers to Babesia microti in serum samples collected from 16 patients between 1 week and 1 year after the onset of illness. Titers rose to greater than or equal to 1:1,024 during the first few weeks of illness and then fell gradually to 1:16 to 1:256 over the next 5-6 months.

Evaluation of the micro enzyme-linked immunosorbent assay for antibodies to Trypanosoma cruzi.

A micro enzyme-linked immunosorbent assay (ELISA) for antibodies to Trypanosoma cruzi was evaluated and the results obtained by ELISA were compared with those obtained by the complement fixation test (CF) and indirect fluorescent antibody test (IFA). Fifty sera collected from residents of the southeastern United States all had reciprocal ELISA titers less than or equal to 320. Similarly, serum samples from 17 patients with T. cruzi infection proven by xenodiagnosis had reciprocal ELISA titers of greater than or equal to 1,280. Specimens from 302 El Salvador Army recruits were tested by ELISA, IFA, and CF. Excellent correlation was observed between results obtained by the three serologic tests; 62.9% of the samples were negative by each of the three tests and 24.5% were positive by all. Overall, 29.5% of the sera were positive for antibodies to T. cruzi by ELISA, 29.5% by IFA, and 31.5% by CF. The data suggest that the micro ELISA is a promising serologic test for measuring antibodies to T. cruzi in individuals and in populations.

Babesia microti infection in man: evaluation of an indirect immunofluorescent antibody test.

An indirect immunofluorescent antibody test was used to detect antibody to Babesia microti in human sera. Nine patients from Nantucket Island, Massachusetts infected with B. microti had serum titers greater than or equal to 1,024. Of 84 control sera from New York City residents, 246 sera from patients with possible exposure to ticks, and 36 sera from patients with suspected or confirmed tick bites, none was reactive at titers of 1:16 or above. The within-test reproducibility was within one fourfold dilution in 95% of trials. Test-to-test reproducibility was within one fourfold dilution in 33% of trials and within two fourfold dilutions in 100% of trials. Although cross-reactions among infected patients' sera and antigens of B. argentina, B. equi, B. bigemina, Plasmodium vivax, P. falciparum, and P. brasilianum were common, titers were highest to the homologous antigen.

Imported malaria in Montagnard refugees settling in North Carolina: implications for prevention and control.

In the winter of 1992, some 402 Southeast Asian refugees were resettled in North Carolina. They received very limited medical screening before immigration and many arrived in the United States with significant health problems, including several tropical infectious diseases. These refugees had lived for many years in remote areas along the Vietnam-Cambodia border, where there is intense transmission of malaria, including Plasmodium falciparum resistant to most antimalarial drugs available in the United States. Of 322 refugees screened after arrival in North Carolina, 187 (58%) were infected: 33% with P. falciparum, 23.5% with P. vivax, 23.5% with P. malariae, and 2.1% with P. ovale. Most infected persons were asymptomatic and infections with multiple species were common. Because of the documented high infection prevalence and the probable presence of many subpatent infections, all nonpregnant refugees were treated with halofantrine; those with P. vivax or P. ovale infections were given primaquine as well. This group accounted for the largest cluster of malaria cases reported in the United States in the last 50 years. Their rapid relocation, with minimal medical screening prior to arrival, resulted in a significant burden to the refugees and to the health-care system. Coordination between immigration agencies, the public health community, and medical workers in communities where the refugees are settled is critical for U.S.-based management of imported tropical diseases.

Immunogenicity of the Plasmodium falciparum asexual blood-stage synthetic peptide vaccine SPf66.

The immunogenicity of the cocktail vaccine SPf66 against Plasmodium falciparum was investigated in rabbits, monkeys, and human volunteers. This polymerized peptide vaccine incorporates a portion of each of the 35-kD, 55-kD, and 83-kD blood-stage proteins, linked by an amino acid sequence reproducing one repeat from the circumsporozoite protein of P. falciparum. Results from this study show that vaccination with this vaccine molecule elicited high titers of antibodies to SPf66 and its constituents, but these antibodies did not correlate with regular or sensitive indirect fluorescent antibody (IFA) titers to blood-stage malaria parasites. However, rabbits injected with individual peptides produced antibodies with low affinity to indefinite parasite structures by immunoelectron microscopy, and rabbits injected with SPf66 had high antibody titers against the peptide (NANP)40. Consequently, these anti-SPf66 serum samples recognized P. falciparum sporozoites in the IFA. Such reactivity was not observed in monkeys or human volunteers vaccinated with SPf66. In addition, SPf66 components were recognized by antibodies induced by natural infection in humans and by laboratory-monitored infections in Aotus monkeys. These results suggest that this vaccine candidate merits further developmental work to better define immune response elicited by the copolymer to the parasite.

Infectious disease patterns in the Waorani, an isolated Amerindian population.

The Waorani Indians of eastern Ecuador provide a unique opportunity for studying exposure of an isolated human population to various infectious disease agents. Using serologic tests to determine antibody prevalence, skin test data, and stool examination for parasites, we have been able to construct a profile of infectious diseases which are endemic, and others which have been introduced into the Waorani population. These findings are compared with similar data reported from elsewhere in the Amazon. Serologic studies demonstrating the presence of antibody to measles and poliovirus type 3 after vaccination indicate that the Waorani respond normally to viral challenge with these agents. The question of genetic inability among aboriginal Amerindians to respond to viral agents is discussed. Finally, general recommendations are made regarding the future health care of the Waorani.

Protective immunity induced in squirrel monkeys with recombinant apical membrane antigen-1 of Plasmodium fragile.

Saimiri sciureus boliviensis monkeys were immunized with the Plasmodium fragile form of the merozoite apical membrane antigen-1 produced using the baculovirus expression system and combined with Montanide ISA 720 adjuvant. Following three immunizations, monkeys were challenged with 10,000 P. fragile trophozoite parasites. Antibody titers determined by fluorescence microscopy indicated an enhanced response following the second immunization. Four of five control animals had parasite counts > 5% 18-26 days following challenge. Four of five immunized monkeys had reduced levels of maximum parasitemia or delays in accumulated parasite counts, suggestive of protection. Rechallenge of the animals with P. falciparum resulted in three of four adjuvant control animals developing patent parasitemia whereas none of five immunized animals were infected, suggesting some level of heterologous protection.

Assessment of a synthetic DNA probe for Plasmodium falciparum in African blood specimens.

Synthetic DNA oligomers homologous to 21-base long repetitive sequences of Plasmodium falciparum DNA were labeled with 32P using T4 kinase, and were hybridized with purified DNA and with processed blood samples from Africa. The sequence PFR1, its antiparallel oligomer PFR1R, and PFR1 covalently attached to biotin hybridized similarly to P. falciparum DNA. One-microliter aliquots of blood from Zaire spotted on prewet nylon filters and hybridized with PFR1 gave detectable autoradiogram signals from samples with parasitemias as low as 1,000 parasites/mm3. Blood lysis and protein digestion followed by alkylation allowed dot-blot processing of larger aliquots of blood. After hybridization with PFR 1 and autoradiography, 26 samples were scored positive visually, compared with 34 scored positive by microscopy. The effective sensitivity for processed 10-microliter samples was about 500 parasites/mm3. Signals from hybridized probes were quantitated by liquid scintillation counting and densitometry, and were proportional to the amounts of purified P. falciparum DNA applied to the filter. Autoradiogram signals also were roughly proportional (correlation coefficient, r = 0.77) to the number of parasites/mm3 of blood from field samples as determined by microscopic examination.

Serum immunoglobulin levels and malaria antibodies in Burkitt's lymphoma.

Data are presented to support a relationship between malaria infection and Burkitt's lymphoma in African children. IgG, IgM and IgA levels were measured in sera from Burkitt's lymphoma patients and from sex- and age-matched, nearest-neighbour controls. All three classes of immunoglobulins were present in significantly lower amounts in the sera from Burkitt's lymphoma patients than in the sera from controls. The mechanism of this apparent B-cell suppression is not yet clear. Malaria-specific IgG and IgM antibody titres were determined in the indirect immunofluorescence test. No significant difference in the IgG malaria-specific antibodies was detected between the two groups of sera. Malaria antibody levels measured using IgM specific conjugates were significantly lower in the sera from Burkitt's lymphoma patients in reactions with Plasmodium falciparum antigen. No significant difference was observed when P. malariae was used. Confirmation of this finding would serve as a positive link between Burkitt's lymphoma and P. falciparum infection.

Malaria and pregnancy in Cameroonian women. Naturally acquired antibody responses to asexual blood-stage antigens and the circumsporozoite protein of Plasmodium falciparum.

Antibody responses to Plasmodium falciparum antigens in women during pregnancy were investigated in Mfou, a rural community in Cameroon. The study consisted of cross-sectional analyses involving 225 pregnant women and 75 non-pregnant controls. Blood samples were collected from each woman to determine serological reactivity to intraerythrocytic malarial antigens, ring-infected erythrocyte surface antigen (RESA) and circumsporozoite (CS) repeat peptide (NANP)5 by the indirect fluorescent antibody assay, modified immunofluorescent antibody assay, and enzyme-linked immunosorbent assay, respectively. Reactivity to intraerythrocytic asexual blood-stage antigens and to the CS repeat region was similar in both pregnant and non-pregnant women, and no correlation with parasitaemia was found. In contrast, anti-RESA antibody levels were significantly lower in pregnant than in non-pregnant women (P = 0.02) and in primigravidae than in multigravidae (P = 0.002), and were inversely correlated with parasitaemia (r = -0.36; P < 0.01). These data suggest that the increased susceptibility to malarial infection in pregnant women may be explained in part by their lower reactivity to RESA.

Study of coinciding foci of malaria and leptospirosis in the Peruvian Amazon area.

A hyperendemic malaria focus, found in 1973 in a secluded valley in South-eastern Peru, was restudied in 1975. Tests for antibodies to Plasmodium and Leptospira were performed on blood serum and blood slides collected at three locations on the Rio Ene and confluent streams and at two locations in the neighbouring highlands. The hyperendemic focus of P. vivax-P. malariae found at Mission Cutivirini in 1973 was confirmed in this study. Another hyperendemic focus of predominantly P. vivax was found at the village of Saoreni. Lesser amounts of malaria were found at other locations. Serology indicated past or present contact with Leptospira of from 50 to 75% of individuals at all locations. The two hyperendemic malaria foci therefore were embedded in a much larger hyperendemic focus of leptospirosis. The value of the indirect immunofluorescence test for malarial antibodies as a sero-epidemiological tool was emphasized by this study.