Comparison of the diagnosis of malaria by microscopy, immunochromatography and PCR in endemic areas of Venezuela.

Whole blood samples (N = 295) were obtained from different locations in Amazonas and Sucre States, in Venezuela. Malaria was diagnosed by microscopy, OptiMAL and polymerase chain reaction (PCR), with Plasmodium vivax, P. falciparum, and P. malariae being detected when possible. We identified 93 infections, 66 of which were caused by P. vivax, 26 by P. falciparum, and 1 was a mixed infection. No infection caused by P. malariae was detected. The sensitivity and specificity of each diagnostic method were high: 95.7 and 97.9% for microscopy, 87.0 and 97.9% for OptiMAL, and 98.0 and 100% for PCR, respectively. Most samples (72.2%) showed more than 5000 parasites/microL blood. The sensitivity of the diagnosis by microscopy and OptiMAL decreased with lower parasitemia. All samples showing disagreement among the methods were reevaluated, but the first result was used for the calculations. Parasites were detected in the 6 false-negative samples by microscopy after the second examination. The mixed infection was only detected by PCR, while the other methods diagnosed it as P. falciparum (microscopy) or P. vivax (OptiMAL) infection. Most of the false results obtained with the OptiMAL strip were related to the P. falciparum-specific band, including 3 species misdiagnoses, which could be related to the test itself or to genetic variation of the Venezuelan strains. The use of the microscopic method for malaria detection is recommended for its low cost but is very difficult to implement in large scale, population-based studies; thus, we report here more efficient methods suitable for this purpose.

Comparison of the diagnosis of malaria by microscopy, immunochromatography and PCR in endemic areas of Venezuela

Whole blood samples (N = 295) were obtained from different locations in Amazonas and Sucre States, in Venezuela. Malaria was diagnosed by microscopy, OptiMAL™ and polymerase chain reaction (PCR), with Plasmodium vivax, P. falciparum, and P. malariae being detected when possible. We identified 93 infections, 66 of which were caused by P. vivax, 26 by P. falciparum, and 1 was a mixed infection. No infection caused by P. malariae was detected. The sensitivity and specificity of each diagnostic method were high: 95.7 and 97.9 percent for microscopy, 87.0 and 97.9 percent for OptiMAL, and 98.0 and 100 percent for PCR, respectively. Most samples (72.2 percent) showed more than 5000 parasites/æL blood. The sensitivity of the diagnosis by microscopy and OptiMAL decreased with lower parasitemia. All samples showing disagreement among the methods were reevaluated, but the first result was used for the calculations. Parasites were detected in the 6 false-negative samples by microscopy after the second examination. The mixed infection was only detected by PCR, while the other methods diagnosed it as P. falciparum (microscopy) or P. vivax (OptiMAL) infection. Most of the false results obtained with the OptiMAL strip were related to the P. falciparum-specific band, including 3 species misdiagnoses, which could be related to the test itself or to genetic variation of the Venezuelan strains. The use of the microscopic method for malaria detection is recommended for its low cost but is very difficult to implement in large scale, population-based studies; thus, we report here more efficient methods suitable for this purpose.

Detection of specific antibodies to Plasmodium falciparum in blood bank donors from malaria-endemic and non-endemic areas of Venezuela.

Malaria antibody detection is valuable in providing retrospective confirmation of an attack of malaria. Blood bank screening is another area were malaria serology is potentially useful. In the present study, we tested the presence of antibodies to Plasmodium falciparum in sera from blood bank donors of non-endemic and malaria-endemic areas of Venezuela. Sera from 1,000 blood donors were tested by an indirect immunofluorescent antibody (IFA) assay and an IgG-ELISA for the presence of malaria antibodies using a synchronized in vitro-cultured Venezuelan isolate of P. falciparum as the antigen source. A selected group of positive and negative sera (n = 100) was also tested by a dot-IgG-ELISA. Positive results (reciprocal titer > or = 40) were found in 0.8% and 3.8% of blood donors when tested by the IFA assay and in 0.8% and 2% (optical density > or = 0.2) when tested by the IgG-ELISA in Caracas (non-endemic area) and Bolivar City (endemic area), respectively. The presence of anti-malarial antibodies in some sera from non-endemic areas such as Caracas reflects the increased potential risk of post-transfusional malaria in those areas due to the mobility of the blood donors. The data obtained indicate the need to implement new blood donor policy in blood banks in developing areas. Our results also indicate that the IFA assay is the most reliable test to use in malaria serodiagnosis.

Mapping of specific and promiscuous HLA-DR-restricted T-cell epitopes on the Plasmodium falciparum 27-kilodalton sexual stage-specific antigen.

We have characterized HLA-DR-restricted T-cell epitopes on the 27-kDa protein (Pfg27), a sexual stage-specific antigen, of the human malaria parasite Plasmodium falciparum in subjects with a history of malaria. Pfg27, expressed early in the sexual stages, is recognized by monoclonal antibodies capable of reducing the infectivity of gametocytes in mosquitoes. By using 16 Pfg27-specific CD4(+)-T-cell clones derived from three donors, seven different T-cell epitopes were identified. Among them, P11 (amino acids 191 to 210 of the Pfg27 sequence, IDVVDSYIIKPIPALPVTPD) was found to contain a previously described binding motif for multiple HLA-DR allotypes. Indeed, P11 was found to be promiscuous in that it could be recognized by T cells in the context of at least five different HLA-DR molecules. The cytokine profile of the clones was mixed. Seven of nine T-cell clones exhibited a Th0-like cytokine profile, producing high levels of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) upon stimulation with specific peptides and mitogens. The other two clones had a Th1-like cytokine profile with high expression of IFN-gamma and no IL-4. Identification of a promiscuous epitope in Pfg27 could play a significant role in the design of a subunit vaccine for suppressing malaria transmission.

Detection of human antibodies against Plasmodium falciparum antigens in blood meals of anopheline mosquitoes.

Human IgG antibodies against Plasmodium falciparum asexual stages, gametocytes and sporozoites were detected by indirect fluorescent antibody (IFA) techniques in the blood meals of Anopheles gambiae s.l. from a malaria-endemic area of western Kenya. Field-collected mosquitoes, which had been stored dry for over 2 years, were screened first for human IgG by ELISA. In 141 blood meal samples from human-fed mosquitoes, the prevalence of stage-specific antibodies was 87.9% for asexual-stage parasites, 78.0% for gametocytes, and 87.9% for sporozoites. There were no differences in the prevalence of stage-specific antibodies for mosquitoes collected from 2 sites, before and after the long rainy season of 1988. The detection of specific human antibodies in mosquito blood meals by IFA, or by more efficient methods, may provide alternative approaches for large-scale, epidemiologic studies of malaria and other vector-borne diseases.

RESA-IFA assay in Plasmodium falciparum malaria, observations on relationship between serum antibody titers, immunity, and antigenic diversity.

RESA-IFA assays were conducted using 63 adult sera from 7 different malarious areas against 7 different strains of Plasmodium falciparum, and 28 children's sera against 3 different parasite strains. Generally, where immunity to malaria was high, there was little or no antigenic diversity among the different strains examined. However, where sera were collected from semi-immunes, or from children, some variation in the RESA-IFA endpoint titers was discernible. Correlation between antibody titers determined by RESA-IFA and in vitro parasite invasion inhibition was not complete. Sera having high RESA-IFA titers were predictably inhibitory; however, many sera having low RESA-IFA titers were as inhibitory as sera having high titers, indicating that antibodies with specificities different from the RESA may be as important, or more important, to clinical immunity to blood-stage infections.

Human repetitive and unique sequences coexist in a large circulating DNA species found in cryoprecipitates from SLE patients.

Cryoprecipitates from systemic lupus erythematosus (SLE) patients with high levels of anti DNA antibodies show a sharply migrating large circulating DNA species of about 17-20 kb (M. Rieber et al., Clin. exp. Immunol. (1986) 66, 61). We have now used Southern blot analysis of circulating DNA from different individuals to analyse the relative cross-hybridization of circulating DNA from different individuals, as well as their homology with genomic DNA from different species. Molecular hybridization showed significant homology of the various circulating DNA examined, only with human genomic DNA, but limited cross-reactivity among circulating DNA from different individuals. This suggests that the circulating DNA is composed of sequences repeated in human genomic DNA and by specific sequences unique to circulating DNA from some individuals. Our data suggests the possibility of using probes derived from the specific sequences now reported in the circulating DNA, in gene typing and in the analysis of susceptibility to disease.

Novel DNA-protein complex and a large DNA in SLE cryoprecipitates.

Agarose gel electrophoresis of cryoprecipitates from systemic lupus erythematosus (SLE) patients revealed the presence of a slowly migrating DNAse I-sensitive DNA species at the top of the gel. Upon deproteinization, electrophoretic migration was modified favouring the migration of a 17.5 kb DNA fragment. Mixing experiments adding human serum or plasma to a lambda phage DNA digest revealed a DNA-protein interaction shown by an accumulation of high mol. wt polynucleotide at the top of the gels, and a slowed migration of the DNA bands. No comparable effect was observed when serum albumin was added to the lambda DNA digest. Dot hybridization analysis showed preferential reactivity of the 17.5 kb DNA to human DNA, implying its human origin. Our data suggests that most of this high molecular weight DNA exists as a DNA-2 protein complex. Our mixing experiments also suggest the occurrence of an excess of free DNA antibodies. We propose that the DNA-protein association may play a role in the stabilization and immunogenicity of the nucleoprotein complex.

Respuesta inmunologica en pacientes con uveitis. / Immunological response in patients with uveitis

Se estudio la respuesta inmunologica del compartimiento humoral en el suero sanguineo y humor acuoso en tres grupos de pacientes: Grupo I: pacientes con uveitis; Grupo II con enfermedad sistemica autoinmune sin uveitis, y Grupo III: control.Los resultados senalan alteraciones locales en este sentido encontrandose elevacion de la IgG e IgA en los pacientes del Grupo I asi como la presencia del factor B y C3, los cuales se reportan en forma original; los complejos inmunologicos fueron detectados por dos metodos, siendo significativos los resultados en el Grupo I

Solid phase C1q microassay for the rapid detection of circulating immune complexes.

A C1q solid phase microassay was designed for the rapid detection of circulating immune complexes. Its level of sensitivity is comparable to that of the Raji cell and greater than the C1q binding assay; furthermore, it is faster and low in cost. These conditions make it more practical and applicable in the clinical setting.

Physiological aspects of circulating immune complexes in the normal population.

Circulating immune complexes (CIC) have been investigated in 100 normal subjects; the RIA-Raji and the C1q-BA conventional methods, as well as a new solid phase microassay utilizing purified C1q and the systematic search of cryoprecipitates were employed. CIC serum levels did not differ in regards to sex; in relation to age, values for C1q-BA were identical in subjects from 0 to 60 years and also in those beyond age 60; the differences encountered by RIA-Raji or by the C1q-SP microassay in these two main groups were not statistically significant. Cryoprecipitates were present in 100% of the 68 examined subjects. Immunoglobulins (G, A and M), anti-nucleic acid (DNA and Poly A) and CIC (by the three methods) were present in the cryoprecipitates while lymphocytotoxins, rheumatoid factor and C3 were undetectable; protein content of the cryoprecipitates increased significantly with age, reaching a normal superior limit of 0.52 mg/ml beyond age 30. These findings further support the role played by CIC in normal immune response and may help in the understanding of the physiopathology of clinical conditions associated with immune complexes.

Immunology of human gastric cancer: a preliminary report.

The immunological spectrum in fifteen patients with gastric cancer is presented. Patients were divided in three groups. Those with nonadvanced cancer, those with advanced but resectable lesions and those with advanced but nonresectable tumors. Preoperatively, elevated levels of circulating immune complexes (CIC) associated with hyporesponsiveness to phytohemagglutinin (PHA) and in mixed lymphocyte culture (MLC) as well as a positive leukocyte inhibitory serum factor (LIF-S) were found in nearly half of the patients. Inhibitory or enhancing autologous serum factors were detected. Postoperatively, immunologic parameters return to normal in patients with nonadvanced cancer, while in advanced cancer, antibody and cell-mediated immune response remained altered, with some changes associated with chemotherapy. These findings are probably related with the presence or absence of tumor and offer a distinct approach in evaluating the immunologic response of a tumor-bearing patient.

Immunopathological aspects of Plasmodium berghei infection in five strains of mice. I. Immune complexes and other serological features during the infection.

The development of circulating immune complexes was studied in mice of the BALB/c, A/J, OF1, CBA and C57B1 strains infected with P. berghei. Complexes were evaluated in relation to levels of parasitaemia, soluble antigen, specific antibody and C3. Susceptibility to infection was greatest in BALB/c, A/J and OF/a mice. The maximum parasitaemia was 30% in CBA and 70% in all other strains. Levels of soluble antigen paralleled those of parasitaemia. Specific antibody was detected in all strains, but the titre continued to rise throughout the infection only in CBA mice. Circulating immune complexes occurred in mice of all strains from day 6; the level fell after day 9 in C57B1 whereas it was maintained in CBA mice. The development of immune complexes was associated with marked depression of C3 levels in all except CBA mice, in which a transient reduction was followed by recovery. Partial characterization of the complexes showed that IgM-containing complexes appeared earliest and reached highest levels in BALB/c mice while in CBA mice, IgM complexes were found in lesser amounts and the level fell in late infection. IgG complexes rose throughout infection in CBA and fell in later stages in BALB/c and C57B1 mice. In nude BALB/c mice, immune complexes were usually not detectable and only low levels of antibody of IgM class were produced. Differences in mortality pattern could not be related to any single serological factor.

Immunopathological aspects of Plasmodium berghei infection in five strains of mice. II. Immunopathology of cerebral and other tissue lesions during the infection.

Histological changes during the course of P. berghei infection were investigated in A/J, BALB/c, OF1, CBA and C57B1 mice. The findings were studied in relation to serological aspects (Contreras et al., 1980). High mortality and acute deaths occurred in A/J, BALB/c and OF1 mice and marked cerebral lesions were found in these strains from day 15, including congestion of meningeal and cerebral veins and capillaries, blocking of these vessels by heavily parasitized RBC, cerebral oedema and haemorrhages. Such lesions were minimal in CBA and C57B1 mice, and absent in mice examined 21 and 24 days after infection. Small deposits of IgG and traces of C3 were detected by immunofluorescence in the choroid plexus of most mice from day 9. Renal lesions included congestion, plugging of veins and capillaries, low-grade mononuclear infiltration and mesangial thickening; these changes were most marked in CBA, C57B1 and A/J mice. Glomerular deposits of IgM were present in all strains in the first week of infection. IgG and C3 were detected in the second week, but only traces were found in CBA mice. The livers showed congestion, accumulation of pigment in swollen Kupffer cells and mononuclear portal infiltration; these were most pronounced in A/J mice. In the spleen, there was a great increase in the reticuloendothelial cell population, white pulp proliferation, congestion and accumulation of pigment and plasma cell reaction; the pattern of white pulp expansion varied in the different strains. The results suggest that cerebral lesions play a significant role in the aetiology of acute deaths in this malaria model.

Circulating and tissue-bound immune complex formation in murine malaria.

Immune complex formation during Plasmodium berghei infection of OF1 mice was investigated. Circulating immune complexes (CIC) were detected by the Clg-binding assay and the conglutinin-binding solid-phase assay in lethal or drug-limited infections. CIC appeared on day 9 of infection, peaked on day 11, and disappeared only after complete cure of the infection. Analysis of the immune complexes detected by the Clq-binding assay revealed the following characteristics: sedimentation coefficients of 13S to 21S, resistance to DNAse, and selective removal by filtration through protein A bound to Sepharose. Glomerular deposits of IgM preceded the appearance of CIC, whereas deposits of IgG and C3 were concomitant with the appearance of CIC. Tissue-bound immunoglobulins were also found in the choroid plexus. The appearance of anti-malarial antibodies and malarial antigens in the serum was closely associated with a depression of C3 levels and the presence of CIC. Drug treatment was followed by normalization of C3 levels, and clearance of both CIC and malarial antigens.

Improved detection of immune complexes in human and mouse serum using a microassay adaptation of the C1Q binding test.

A microassay adaptation of the [125I]C1q binding test for the detection of circulating immune complexes is described. This technique is more rapid to perform, requires smaller volumes of serum and reagents, and surprisingly, increases the sensitivity of the assay when compared to the previously reported C1q binding method.