Evaluation of the enzyme-linked-immuno-electro-diffusion-assay (ELIEDA) for the diagnosis of Schistosoma mansoni infection with low worm burden

An immunoprecipitation technique, ELIEDA (enzyme-linked-immuno-electro-diffusion assay), was evaluated for the diagnosis of Schistosoma mansoni infection with low worm burden. One hundred of serum samples from patients excreting less than 600 eggs per gram of feces (epg), with unrelated diseases and clinically healthy subjects were studied. In patients with egg counts higher than 200 epg, the sensitivities of IgM and IgG ELIEDA were 1.000 and 0.923, respectively, not differing from other Serologic techniques, such as indirect hemaglutination (IHAT), immunofluorescence (IFT) tests and immuno-electrodiffusion assay (IEDA). However in patients with low egg counts (< 100 epg), the IgG ELIEDA provided better results (0.821) than IgM ELIEDA (0.679), showing sensitivity that did not differ from that of IgG IFT (0.929), but lower than that of IgM IFT (0.964). However, its sensivity was higher than that found with IHAT (0.607) and IEDA (0.536). The specificity of IgG ELIEDA was comparable to that of other techniques. The data indicate that IgG ELIEDA might be useful for the diagnosis of slight S. mansoni infections, and the cellulose acetate membrane strips can be stored for further retrospective studies.

Cyclophosphamide effect on paracoccidioidomycosis in the rat

Paracoccidioidomycosis is an endemic fungal disease widely distributed throughout Latin America. The potent immunosuppressor cyclophosphamide (CY) has been used to modulate host immune response to Paracoccidioides brasiliensis in an experimental model. Inbred male Buffalo/Sim rats weighing 250-300 g were inoculated with 5 x 10(6) P. brasiliensis cells of the yeast phase form by intracardiac route. One group of animals was treated with 20 mg/kg body weight at days +4, +5, +6, +7, +11 and +12 post-infection (pi.), while a control group was infected alone. No mortality was recorded in either group. Treated rats presented: a) a decrease in granuloma size, which contained less fungal cells; b) a lack of specific antibodies up to 35 days pi., and c) a significant increase in the footpad swelling test (DTH) against paracoccidioidin. Splenic cell transfer from CY-treated P. brasiliensis-infected donors to recipients infected alone led to a significant increase in DTH response in the latter versus untreated infected controls. Likewise, in treated infected recipients transferred with untreated infected donor spleen cells, footpad swelling proved greater than in controls. Thus, it would seem that each successive suppressor T lymphocyte subset belonging to the respective cascade may be sensitive to repeated CY doses administered up to 12 days pi.. Alternatively, such CY schedule may induce the appearance of a T cell population capable of amplifying DTH response.

Enzyme linked immunosorbent assay for rubella antibodies: a simple method of antigen production. A preliminary report

A simple method of rubella antigen production by treatment with sodium desoxycholate for use in enzyme immunoassay (IMT-ELISA) is presented. When this assay was compared with a commercial test (Enzygnost-Rubella, Behring), in the study of 108 sera and 118 filter paper blood samples, 96.9% (219/226) overall agreement and correlation coefficient of 0.90 between absorbances were observed. Seven samples showed discordant results, negative by the commercial kit and positive by our test. Four of those 7 samples were available, being 3 positive by HI.

EVALUATION OF A RAPID SCREENING ASSAY FOR BACTERIAL IDENTIFICATION (DOT-ELISA) IN FECAL SAMPLES FROM CHILDREN

With the objective of standardizing a Dot Enzyme-Linked Immunosorbent Assay (Dot-ELISA) to detect antigens of fecal bacterial enteropathogens, 250 children, aged under 36 months and of both sexes, were studied; of which 162 had acute gastroenteritis. The efficacy of a rapid screening assay for bacterial enteropathogens (enteropathogenic Escherichia coli "EPEC", enteroinvasive Escherichia coli "EIEC", Salmonella spp. and Shigella spp.) was evaluated. The fecal samples were also submitted to a traditional method of stool culture for comparison. The concordance index between the two techniques, calculated using the Kappa (k) index for the above mentioned bacterial strains was 0.8859, 0.9055, 0.7932 and 0.7829 respectively. These values express an almost perfect degree of concordance for the first two and substantial concordance for the latter two, thus enabling this technique to be applied in the early diagnosis of diarrhea in infants. With a view to increasing the sensitivity and specificity of this immunological test, a study was made of the antigenic preparations obtained from two types of treatment: 1) deproteinization by heating; 2) precipitation and concentration of the lipopolysaccharide antigen (LPS) using an ethanol-acetone solution, which was then heated in the presence of sodium EDTA

Immunoglobulin Fc receptors in clinical strains of Staphylococcus aureus do not confer resistance to Phagocytosis in an in vitro assay

Staphylococcus aureus binds Immunoglobulin G (IgG) on its external surface due to the presence of specific receptors for the Fc domain of this immunoglobulin. This mechanism represents a kind of camouflage against phagocytic cells. In order to confirm that possibility an in vitro evaluation of the phagocytic activity of leukocytes polymorpho-nuclear (PMN) against strains of Staphylococcus aureus was done, comparing 18 strains isolated from clinical samples and 16 from healthy individuals. The presence of Fc receptors was evaluated by haemagglutination (HA) with erythrocytes group A after incubation of the strains with IgG anti blood group A. Phagocytosis of S. aureus was carried out by mixing live bacteria with a suspension of human PMN and incubating at 37 °C for 1 h; survivors were counted as colony forming units by plating. The strains from clinical specimens showed higher HA than those from healthy individuals (p = 0.01); but the former were killed more efficiently than the latter (80-90% and 40%, respectively). It is may be possible that S. aureus showed different behavior in vivo, where could express other virulence factors to prevent the action of phagocytes.

Detection of a Giardia lamblia coproantigen by using a commercially available immunoenzymatic assay, in Belo Horizonte, Brazil

It is known that fecal examination to detect Giardia lamblia cysts or trophozoites produces a high percentage of false-negative results. A commercially available immunoenzymatic assay (ProSpecT Giardia Microplate Assay, Alexon, Inc., BIOBRÁS) to detect G. lamblia specific coproantigen was evaluated for the first time in Brazil. A total of 90 specimens were tested. Each specimen was first tested as unpreserved stool, and then it was preserved in 10% Formalin to be tested 2 months later. The assay was able to identify all the 30 positive patients (sensitivity = 100.0%) by visual or spectrophotometric examination in the unpreserved specimens and was negative in 57 of the 60 patients without G. lamblia (specificity = 95.0%). The assay identified 27 of the 30 positive patients (sensitivity = 90.0%) and was negative in 59 of the 60 negatives (specificity = 98.3%) in the preserved stools according to both readings. A marked difference was observed in the optical densities in both groups, preserved and unpreserved stools, when the G. lamblia-positive specimens were compared to the negative or positive for other intestinal parasites than G. lamblia. The assay seems a good alternative for giardiasis diagnosis, especially when the fecal examination was repeatedly negative and the patient presents giardiasislike symptoms.

Efficiency of indirect immunofluorescence assay as a confirmatory test for the diagnosis of human retrovirus infection (HIV-1 and HTLV-I/II) in different at risk populations

We compared the indirect immunofluorescence assay (IFA) with Western blot (Wb) as a confirmatory method to detect antibodies anti retrovirus (HIV-1 and HTLV-I/II). Positive and negative HIV-1 and HTLV-I/II serum samples from different risk populations were studied. Sensitivity, specificity, positive, negative predictive and kappa index values were assayed, to assess the IFA efficiency versus Wb. The following cell lines were used as a source of viral antigens: H9 ( HTLV-III b); MT-2 and MT-4 (persistently infected with HTLV-I) and MO-T (persistently infected with HTLV-II). Sensitivity and specificity rates for HIV-1 were 96.80% and 98.60% respectively, while predictive positive and negative values were 99.50% and 92.00% respectively. No differences were found in HIV IFA performance between the various populations studied. As for IFA HTLV system, the sensitivity and specificity values were 97.91% and 100% respectively with positive and negative predictive values of 100% and 97.92%. Moreover, the sensitivity of the IFA for HTLV-I/II proved to be higher when the samples were tested simultaneously against both antigens (HTLV-I-MT-2 and HTLV-II-MO-T). The overall IFA efficiency for HIV-1 and HTLV-I/II-MT-2 antibody detection probed to be very satisfactory with an excellent correlation with Wb (Kappa indexes 0.93 and 0.98 respectively). These results confirmed that the IFA is a sensitive and specific alternative method for the confirmatory diagnosis of HIV-1 and HTLV-I/II infection in populations at different levels of risk to acquire the infection and suggest that IFA could be included in the serologic diagnostic algorithm.

Diagnosis of hepatitis C virus in Brazilian blood donors using a reverse transcriptase nested polymerase chain reaction: comparison with enzyme immunoassay and recombinant protein immunoblot assay

Screening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT) serum levels generally prevents the transmission of hepatitis C virus (HCV) by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA) screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT)-nested polymerase chain reaction (PCR) to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2) results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value). ALT results were expressed as the ALT quotient (qALT), calculated dividing the ALT value of the samples by the maximum normal value (53UI/l) for the method. Donors (n=178) were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA > or = 3, ALT<1), group B (EIA > or = 3, ALT>1), group C (1<=EIA<3, ALT<1), group D (1<=EIA<3, ALT>1) and group E (EIA<=0.7). HCV sequences were detected by RT-nested PCR, using primers for the most conserved region of viral genome. RIBA-2 was applied to the same samples. In group A (n=6), all samples were positive by RT-nested PCR and RIBA-2. Among 124 samples in group B, 120 (96.8%) were RIBA-2 positive and 4 (3.2%) were RIBA-2 indeterminate but were seropositive for antigen c22.3. In group B, 109 (87.9%) of the RIBA-2 positive samples were also RT-nested PCR positive, as well as were all RIBA-2 indeterminate samples. In group C, all samples (n=9) were RT-nested PCR negative: 4 (44.4%) were also RIBA-2 negative, 4 (44.4%) were RIBA-2 positive and 1 (11.1%) was RIBA-2 indeterminate. HCV-RNA was detected by RT-nested PCR in 3 (37.5%) out of 8 samples in group D. Only one of them was also RIBA-2 positive, all the others were RIBA-2 indeterminate. All of the group E samples (controls) were RT- nested PCR and RIBA-2 negative. Our study suggests a strong relation between anti-HCV EIA-2 ratio > or = 3 and detectable HCV-RNA by RT-nested PCR. We have also noted that blood donors with RIBA-2 indeterminate presented a high degree of detectable HCV-RNA using RT-nested PCR (75%), especially when the c22.3 band was detected.

Descending pain modulation in a Kaolin-induced hydrocephalus rat model

Pain transmission at the spinal cord is modulated by descending actions that arise from supraspinal areas which collectively form the endogenous pain control system. Two key areas involved of the endogenous pain control system have a circunventricular location, namely the periaqueductal grey (PAG) and the locus coeruleus (LC). The PAG plays a crucial role in descending pain modulation as it conveys the input from higher brain centers to the spinal cord. As to the LC, it is involved in descending pain inhibition by direct noradrenergic projections to the spinal cord. In the context of neurological defects, several diseases may affect the structure and function of the brain. Hydrocephalus is a congenital or acquired disease characterized by an enlargement of the ventricles which leads to a distortion of the adjacent tissues, including the PAG and LC. Usually, patients suffering from hydrocephalus present dysfunctions in learning and memory and also motor deficits. It remains to be evaluated if lesions of the periventricular brain areas involved in pain control during hydrocephalus may affect descending pain control and, herein, affect pain responses. The studies included in the present thesis used an experimental model of hydrocephalus (the rat injected in the cisterna magna with kaolin) to study descending modulation of pain, focusing on the two circumventricular regions referred above (the PAG and the LC). In order to evaluate the effects of kaolin injection into the cisterna magna, we measured the degree of ventricular dilatation in sections encompassing the PAG by standard cytoarquitectonic stanings (thionin staining). For the LC, immunodetection of the noradrenaline-synthetizing enzyme tyrosine hydroxylase (TH) was performed, due to the noradrenergic nature of the LC neurons. In general, rats with kaolin-induced hydrocephalus presented a higher dilatation of the 4th ventricle, along with a tendency to a higher area of the PAG. Due to the validated role of detection the c-fos protooncogene as a marker of neuronal activation, we also studied neuronal activation in the several subnuclei which compose the PAG, namely the dorsomedial, dorsolateral, lateral and ventrolateral (VLPAG) parts. A decrease in the numbers of neurons immunoreactive for Fos protein (the product of activation of the c-fos protooncogene) was detected in rats injected with kaolin, whereas the remaining PAG subnuclei did not present changes in Fos-immunoreactive nuclei. Increases in the levels of TH in the LC, namely at the rostral parts of the nucleus, were detected in hydrocephalic animals. The following pain-related parameters were measured, namely 1) pain behavioural responses in a validated pain inflammatory test (the formalin test) and 2) the nociceptive activation of spinal cord neurons. A decrease in behavioral responses was detected in rats with kaolin-induced hydrocephalus was detected, namely in the second phase of the test (inflammatory phase). This is the phase of the formalin test in which the motor behaviour is less important, which is important since a semi-quantitative analysis of the motor performance of rats injected with kaolin indicates that these animals may present some motor impairments. Collectively, the results of the behavioral studies indicate that rats with kaolin-induced hydrocephalus exhibit hypoalgesia. A decrease in Fos expression was detected at the superficial dorsal layers of the spinal cord in rats with kaolin-induced hydrocephalus, further indicating that hydrocephalus decreases nociceptive responses. It remains to be ascertained if this is due to alterations in the PAG and LC in the rats with kaolin-induced hydrocephalus, which may affect descending pain modulation. It remains to be evaluated what are the mechanisms underlying the increased pain inhibition at the spinal dorsal horn in the hydrocephalus rats. Regarding the VLPAG, the decrease in neuronal activity may impair descending modulation. Since the LC has higher levels of TH in rats with kaolininduced hydrocephalus, which also appears to increase the noradrenergic innervation in the spinal dorsal horn, it is possible that an increase in the release of noradrenaline at the spinal cord accounts for pain inhibition. Our studies also determine the need to study in detail patients with hydrocephalus namely in what concerns their thresholds to pain and to perform imaging studies focused on the structure and function of pain control areas in the brain.

Immunofluorescence assay reactivity patterns of serum samples presenting indeterminate Western blot results for antibodies to HIV-1 and HTLV-I/II in Cordoba, Argentina

Serum samples (n: 110) from blood donors and high risk individuals from Cordoba, Argentina with indeterminate HIV-1 and HTLV-I/II Wb profiles were studied for specific antibodies to HTLV-I/II and HIV-1 by indirect immunofluorescence assay (IFA) and for the presence or absence of HIV-1 and HTLV-I/II specific bands by Wb. This study was carried out in order to characterize their putative reactions with HIV-1 and HTLV-I/II proteins and to resolve the retrovirus infection status of these individuals. Results indicated that blood donors sera displaying indeterminate HIV-1 or HTLV-I/II Wb patterns were not immunoreactive to HTLV-I/II and HIV-1 on IFA. However, a high rate of indeterminate HIV-1 and HTLV-I/II Wb samples from high risk individuals had positive HTLV-I/II and HIV-1 IFA results respectively. Our study supports the growing evidence that HTLV-HIV indeterminate seroreactivity in low risk population is due to a cross reaction against nonviral antigens, and in high risk populations the indeterminate samples show serological cross-recognition between HIV-1 proteins and HTLV-I/II proteins on Wb. These results point out the necessity to investigate the HTLV-I/II reactivity in indeterminate HIV-1 samples and viceversa in order to confirm the diagnosis. Finally, this study shows the potential usefulness of IFA in elucidating the status of HIV-1 and HTLV-I/II infection of individuals with indeterminate Wb profiles, thus enabling resolution of retrovirus infection status.

An indirect immunofluorescence assay to detect antibodies against St. Louis Encephalitis virus

An in house indirect immmunofluorescence assay ( IFA ) in relation to neutralization (NT) reference test, was assessed as a fast and cheap method to carry out serological surveys for St. Louis Encephalitis virus (SLE). Sera obtained from 213 blood donors were analyzed by both tests. The prevalence of seropositivity obtained with IFA was lower than (30.98%) that observed on NT (41.78%). The relative specificity rate of IFA was 96.77% whereas its relative sensitivity rate was 69.66%. Kappa index showed a good correlation between both tests. The results indicate that neutralization assay is still the serological test with the highest sensitivity and specificity relative rates for detecting antibodies against SLE virus. Nevertheless, the IFA could be useful as an alternative test in order to learn the circulation of the Flavivirus genus in a certain area.

Rat hepatocyte invasion by Listeria monocytogenes and analysis of TNF-alpha role in apoptosis

Listeria monocytogenes, etiological agent of severe human foodborne infection, uses sophisticated mechanisms of entry into host cytoplasm and manipulation of the cellular cytoskeleton, resulting in cell death. The host cells and bacteria interaction may result in cytokine production as Tumor Necrosis Factor (TNF) alpha. Hepatocytes have potential to produce pro-inflammatory cytokines as TNF-alpha when invaded by bacteria. In the present work we showed the behavior of hepatocytes invaded by L. monocytogenes by microscopic analysis, determination of TNF-alpha production by bioassay and analysis of the apoptosis through TUNEL technique. The presence of bacterium, in ratios that ranged from 5 to 50,000 bacteria per cell, induced the rupture of cellular monolayers. We observed the presence of internalized bacteria in the first hour of incubation by electronic microscopy. The levels of TNF-alpha increased from first hour of incubation to sixth hour, ranging from 0 to 3749 pg/mL. After seven and eight hours of incubation non-significant TNF-alpha levels decrease occurred, indicating possible saturation of cellular receptors. Thus, the quantity of TNF-alpha produced by hepatocytes was dependent of the incubation time, as well as of the proportion between bacteria and cells. The apoptosis rate increased in direct form with the incubation time (1 h to 8 + 24 h), ranging from 0 to 43%, as well as with the bacteria : cells ratio. These results show the ability of hepatocyte invasion by non-hemolytic L. monocytogenes, and the main consequences of this phenomenon were the release of TNF-alpha by hepatocytes and the induction of apoptosis. We speculate that hepatocytes use apoptosis induced by TNF-alpha for release bacteria to extracellular medium. This phenomenon may facilitate the bacteria destruction by the immune system.