Human cryptosporidiosis: detection of specific antibodies in the serum by an indirect immunofluorescence

Cryptosporidium sp., a coccidian parasite usually found in the faeces of cattle, has been recently implicated as an agent of human intestinal disease, mainly in immunocompromised patients. In the study realized, by an indirect immunofluorescence technique, specific immunoglobulins (IgG and IgM) have been demonstrated in human serum against Cryptosporidium oocysts. Purified oocysts were used as antigens in the indirect immunofluorecence assay. After analyzing this test in sera from selected groups of patients, the frequency of both specific IgG and IgM of immunocompetent children who were excreting oocysts in their faeces was 62% and in children with negative excretion of oocysts was 20% and 40%, respectively. In adults infected with the human immunodeficiency virus (HIV) and who were excreting Cryptosporidium in their stools, the frequency was 57% for IgG but only 2% for IgM. Twenty three percent of immunocompromised adults with not determined excretion of oocysts in their stools had anti-Cryptosporidium IgG in their sera. Children infected with human immunodeficiency virus had no IgM and only 14% had IgG detectable in their sera. The indirect immunoflorescence assay, when used with other parasitological techniques appears to be useful for retrospective population studies and for diagnosis of acute infection. The humoral immune response of HIV positive patients to this protozoan agent needs clarification.

Food frequency consumption and lipoproteins serum levels in the population of an urban area, Brazil

OBJECTIVE: To identify the association between food group consumption frequency and serum lipoprotein levels among adults. METHODS: The observations were made during a cross-sectional survey of a representative sample of men and women over 20 years old living in Cotia county, S. Paulo, Brazil. Data on food frequency consumption, serum lipids, and other covariates were available for 1,045 adults. Multivariate analyses adjusted by age, gender, body mass index, waist-to-hip ratio, educational level, family income, physical activity, smoking, and alcohol consumption were performed. RESULTS: Consumption of processed meat, chicken, red meat, eggs and dairy foods were each positively and significantly correlated with LDL-C, whereas the intake of vegetables and fruits showed an inverse correlation. Daily consumption of processed meat, chicken, red meat, eggs, and dairy foods were associated with 16.6 mg/dl, 14.5 mg/dl, 11.1 mg/dl, 5.8 mg/dl, and 4.6 mg/dl increase in blood LDL-C, respectively. Increases of daily consumption of fruit and vegetables were associated with 5.2 mg/dl and 5.5 mg/dl decreases in LDL-C, respectively. Alcohol beverage consumption showed a significant positive correlation with HDL-C. CONCLUSIONS: Dietary habits in the study population seem to contribute substantially to the variation in blood LDL and HDL concentrations. Substantially CHD risk reduction could be achieved with dietary changes.

Detection of anti-Giardia lamblia serum antibody among children of day care centers

OBJETIVES: To detect anti-Giardia lamblia serum antibodies in healthy children attending public day care centers and to assess serological tests as tools for estimating the prevalence of G. lamblia in endemic areas. METHODS: Three separate stool specimens and filter paper blood samples were collected from 147 children ranging from 0 to 6 years old. Each stool sample was processed using spontaneous sedimentation and zinc sulfate flotation methods. Blood samples were tested by indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) for Giardia IgG. RESULTS AND CONCLUSIONS: Of 147 individuals tested, 93 (63.3%) showed Giardia cysts in their feces. Using IIF and ELISA, serum antibodies were detected in 93 (63.3%) and 100 (68%) samples , respectively. Sensitivity of IIF and ELISA was 82% and 72%, respectively. However, ELISA revealed to be less specific (39%) than IIF (70%). IIF also showed a higher concordance with microscopic examination than ELISA.

Body mass index for predicting hyperglycemia and serum lipid changes in Brazilian adolescents

OBJECTIVE: To determine the best cut-offs of body mass index for identifying alterations of blood lipids and glucose in adolescents. METHODS: A probabilistic sample including 577 adolescent students aged 12-19 years in 2003 (210 males and 367 females) from state public schools in the city of Niterói, Southeastern Brazil, was studied. The Receiver Operating Characteristic curve was used to identify the best age-adjusted BMI cut-off for predicting high levels of serum total cholesterol (>150mg/dL), LDL-C (>100mg/dL), serum triglycerides (>100mg/dL), plasma glucose (>100mg/dL) and low levels of HDL-C (< 45mg/dL). Four references were used to calculate sensitivity and specificity of BMI cut-offs: one Brazilian, one international and two American. RESULTS: The most prevalent metabolic alterations (>50%) were: high total cholesterol and low HDL-C. BMI predicted high levels of triglycerides in males, high LDL-C in females, and high total cholesterol and the occurrence of three or more metabolic alterations in both males and females (areas under the curve range: 0.59 to 0.67), with low sensitivity (57%-66%) and low specificity (58%-66%). The best BMI cut-offs for this sample (20.3 kg/m² to 21.0 kg/m²) were lower than those proposed in the references studied. CONCLUSIONS: Although BMI values lower than the International cut-offs were better predictor of some metabolic abnormalities in Brazilian adolescents, overall BMI is not a good predictor of these abnormalities in this population.

Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE). As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80) than the latter (1:80 to 1:160). Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

An ELISA method using serum derived HDAg for the sorological detection of HDV antigens and antibodies

One of the main difficulties related to the detection of the Hepatitis Delta Virus (HDV) antigen and antibody has been the source of the needed HD antigen since HDV containing human and animal livers are very difficult to obtain and since yield is low. This fact prompted us to try to use the serum of patients in the acute phase of HDV infection as a source of HDAg and turn to enzyme immunoassays (EIA) instead of RIA for the sake of easiness and economy in the amount of HDAg needed. The antigen for EIA was obtained from patients during the acute phase of HDV infection and the antibody from patients who have been carriers for many years. For the detection of the antigen, a sandwich type method was employed, whereas for the antibody a competition assay was developed. In order to assess the relative specificity and sensibility of the test, the antibody assay was compared to a commercial RIA (C. RIA, Abbott) and to a non-commercial RIA (NC RIA). Forty-two sera were tested by the two methods and only in two cases discrepant results were obtained. Its is concluded that: 1) sera from patients in the acute and chronic phases of HDV infection can be used as source of both antigen and antibody, for immunoassays; 2) EIA and RIA have comparable relative specificity and sensibility and 3) EIA is easier to perform, cheaper, non-hazardous, has a longer shelf-life and saves scarce HDAg.

Comparison of serum hepatitis B virus replication markers in patients with chronic hepatitis B: studies on HBeAg/Anti-HBe system, viral dna polymerase and HBV-DNA

The detection of HBV-DNA in serum by molecular hybridization is the most sensitive and specific marker of replication and infectivity of hepatitis B virus and currently is proposed as a routine diagnostic technique in the follow-up of HBV - related diseases. Comparing different techniques already described, we found that direct spotting of serum samples on nitrocellulose membranes under vacuum filtration, followed by denaturing and neutralizing washes is more practical, simple, sensible and reproducible. DNA polymerase assay using phosphonoformic acid as specific viral inhibitor has shown 86.8% of concordance with HBV-DNA detection, and so, it is an useful alternative in the follow-up of hepatitis B chronic patients. We found 19.2% HBeAg positive samples with no other markers of viral replication and no anti-HBe positive sample had detectable HBV-DNA. Discordance between the 2 systems have been extensively described, and we confirm this for the first time in our country. Molecular biological techniques are essential to determine the replication status of chronic hepatitis B patients.

Simplification of Immune Adherence Hemagglutination test for detection of rabies antibodies in human serum

In the present work the immune adherence hemagglutination test (IAHA) was standardized in a simplified procedure. This test showed good reproducibility, better than the classical mice serum neutralization test (SN). The tests showed high correlation degree: high titers in one test corresponded to high titers in the other one, and the same occured with low titers. The IAHA test is extremely simple, fast to perform, and of low cost when compared to tests such as SN or indirect immunofluorescense (IIF). It also proved to be useful in less sophisticated laboratories or even as a screening test for the titration of rabies antibodies.

Total serum IgE and parasite: specific IgG and IgA antibodies in human strongyloidiasis

Total serum IgE, and Strongyloides - specific IgG and IgA antibodies were studied in 27 patients with parasitologically proven strongyloidiasis. Clinical manifestations in this case series were investigated by a restrospective study of the patient's records. Total serum IgE levels were elevated (greater than 250 IU/ml) in 59% of the patients (mean concentration = 1364 IU/ml). Parasite - specific IgG and IgA antibodies were detected by ELISA in the serum of 23 (85.2%) and 21 (77.8%) patients, respectively. Elevated serum IgE and clinical manifestations were not useful indexes of the presence of strongyloidiasis. On the other hand, our results support the view that serologic tests, particularly ELISA for detecting Strongyloides - specific IgG antibodies, can be usefully exploited for diagnostic purposes in strongyloidiasis.

Malaria serology: performance of six Plasmodium falciparum antigen extracts and of three ways of determining serum titers in IgG and IgM-ELISA

The study evaluated six Plasmodium falciparum antigen extracts to be used in the IgG and IgM enzyme-linked immunosorbent assays (ELISA), for malaria diagnosis and epidemiological studies. Results obtained with eighteen positive and nine negative control sera indicated that there were statistically significant differences among these antigen extracts (Multifactor ANOVA, p< 0.0001). Urea, sodium deoxycholate and Zwittergent antigen extracts performed better than did the three others, their features being very similar for the detection of IgG antibodies. Urea, alkaline and sodium deoxycholate antigen extracts proved to be better than the others for the detection of IgM antibodies. A straight line relationship was found between the optical densities (or their respective log 10) and the log 10 of antibody dilutions, with a very constant slope. Thus serum titers could be determined by direct titration and by two different equations, needing only one serum dilution. For IgM antibody detections, log 10 expression gave results that better correlated with direct titration (95% Bonferroni). For IgG antibody detections, the titer differences were not significant. The reproducibility of antibody titers and antigen batches was also evaluated, giving satisfactory results.

CALIBRATION AND USE OF AN IN-HOUSE ANTI-MEASLES IgG STANDARD SERUM

For the purpose of research a large quantity of anti-measles IgG working reference serum was needed. A pool of sera from five teenagers was prepared and named Alexandre Herculano (AH). In order to calibrate the AH serum, 18 EIA assays were performed testing in parallel AH and the 2nd International Standard 1990, Anti-Measles Antibody, 66/202 (IS) in a range of dilutions (from 1/50 to 1/25600). A method which compared parallel lines resulting from the graphic representation of the results of laboratory tests was used to estimate the power of AH relative to IS. A computer programme written by one of the authors was used to analyze the data and make potency estimates. Another method of analysis was used, comparing logistic curves relating serum concentrations with optical density by EIA. For that purpose an existing computer programme (WRANL) was used. The potency of AH relative to IS, by either method, was estimated to be 2.4. As IS has 5000 milli international units (mIU) of anti-measles IgG per millilitre (ml), we concluded that AH has 12000 mIU/ml.

AN ELISA SUITABLE FOR THE DETECTION OF RABIES VIRUS ANTIBODIES IN SERUM SAMPLES FROM HUMAN VACCINATED WITH EITHER CELL-CULTURE VACCINE OR SUCKLING-MOUSE-BRAIN VACCINE

An indirect ELISA for determination of post-vaccination rabies antibody was applied. Purified rabies virus was used as antigen to coat plates, and staphylococcal protein A linked with horseradish peroxidase was used for detecting IgG antibody in human sera. Sera from humans, vaccinated with cell-culture vaccine or suckling-mouse-brain vaccine, were examined. ELISA results were compared to those obtained from the virus neutralization test. The mean and standard deviation of OD were determined for 126 negative sera (pre-vaccination) and for 73 sera from vaccinated persons showing antibody titers lower than 0.5 IU/ml. Results were defined as ELISA -positive, -negative or -doubtful. Establishment of a doubtful region reduced the number of sera otherwise classified as positive (false-positive sera). In this way, the sensitivity, specificity and agreement values were respectively 87.5%, 92.4% and 88.5%. No significant differences were observed in these values when the group vaccinated with cell-culture vaccine and the group vaccinated with suckling-mouse-brain vaccine were compared. It was shown that much of the disagreement between the values obtained by neutralization test and ELISA occurred in sera obtained at the beginning of the immunization process, and was probably due to the presence of IgM in the serum samples, detected only by the former test. This ELISA method can be used as a screening test in rabies laboratories regardless of the kind of vaccine used for immunization.

Total IgE detection in paired cerebrospinal fluid and serum samples from patients with neurocysticercosis

Neurocysticercosis (NC), the presence of Taenia solium metacestodes in tissues, is the most frequent and severe parasitic infection of the central nervous system. We investigated the presence of total IgE by an automated chemiluminescence assay in 53 paired cerebrospinal fluid (CSF) and serum samples from patients with NC (P) and in 40 CSF samples from individuals with other neurological disorders as the control group (C). Total IgE concentration ranged from 1.2 to 6.6 IU/ml (mean = 1.4 IU/ml, standard deviation-sd = 1.1 IU/ml) in 28.3% of CSF samples from the P group, a value significantly higher than for the C group (£1.0 IU/ml). The serum samples from the P group showed concentrations ranging from 1.0 to 2330.0 IU/ml (mean = 224.1 IU/ml, sd = 452.1 IU/ml), which were higher than the normal value cited by the manufacturer (<100.0 IU/ml) in 32.1% of the samples. A significant difference was observed in CSF samples from the P and C groups (p = 0.005) and in serum samples from the P group compared to the normal value (p = 0.005), with sera showing more frequent abnormal results.