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.

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.

COMPARISON OF A COMMERCIAL ENZYME IMMUNOASSAY WITH PLAQUE REDUCTION NEUTRALIZATION FOR MATERNAL AND INFANT MEASLES ANTIBODY MEASUREMENT

The most practicable assay for measurement of measles IgG (mIgG) in large numbers of sera is an enzyme immunoassay (EIA). To assess how EIA results would agree with those by the gold standard method of plaque reduction neutralization (PRN) we compared the results from the two methods in 43 pairs of maternal and umbilical cord sera, and sera from the corresponding infants when aged 11 - 14 months. In maternal-cord sera, the differences between mean antibody levels by EIA or PRN were not statistically significant, though in individual sera, differences could be large. However, agreement was less good for infants sera, in which levels of mIgG were very low. The conclusions of a study of transplacental transport of mIgG would not be affected by the use of either technique. When studying waning immunity in infants, PRN should be the method of choice, while results from studies using EIA should be interpreted with caution.

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.

hsp65 PCR-restriction enzyme analysis (PRA) for identification of mycobacteria in the clinical laboratory

More than 70 species of mycobacteria have been defined, and some can cause disease in humans, especially in immunocompromised patients. Species identification in most clinical laboratories is based on phenotypic characteristics and biochemical tests and final results are obtained only after two to four weeks. Quick identification methods, by reducing time for diagnosis, could expedite institution of specific treatment, increasing chances of success. PCR restriction-enzyme analysis (PRA) of the hsp65 gene was used as a rapid method for identification of 103 clinical isolates. Band patterns were interpreted by comparison with published tables and patterns available at an Internet site (http://www.hospvd.ch:8005). Concordant results of PRA and biochemical identification were obtained in 76 out of 83 isolates (91.5%). Results from 20 isolates could not be compared due to inconclusive PRA or biochemical identification. The results of this work showed that PRA could improve identification of mycobacteria in a routine setting because it is accurate, fast, and cheaper than conventional phenotypic identification.

Importance of immunoglobulin E (IgE) in the protective mechanism against gastrointestinal nematode infection: looking at the intestinal mucosae

This review discusses experimental evidences that indicate the IgE participation on the effector mechanisms that leads to gastrointestinal nematode elimination. Data discussed here showed that, for most experimental models, the immune response involved in nematode elimination is regulated by Th-2 type cytokines (especially IL-4). However, the mechanism(s) that result in worm elimination is not clear and might be distinct in different nematode species. Parasite specific IgE production, especially the IgE produced by the intestinal mucosae or associated lymphoid organs could participate in the intestinal elimination of Trichinella spiralis from infected rats. Intestinal IgE may also be important to the protective mechanism developed against other gastrointestinal nematodes that penetrate the murine duodenum mucosa tissue, such as Strongyloides venezuelensis and Heligmosomoides polygyrus. At least in Trichinella spiralis infected rats, the results indicated that intestinal IgE might work independently from mast cell degranulation for worm elimination.

Role of iron in the nitric oxide-mediated fungicidal mechanism of IFN-gamma-activated murine macrophages against Paracoccidioides brasiliensis conidia

Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50U/mL of IFN-gamma or treated with 35 µM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS0(4). The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56%, respectively) in comparison with non-activated or untreated Mthetas (80%). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.

Portal cd4+ and cd8+ t lymphocyte correlate to intensity of interface hepatitis in chronic hepatitis C

BACKGROUND: The pathogenesis of chronic hepatitis C is still a matter of debate. CD4+ and CD8+ T lymphocytes (TL) are typically observed within the portal and periportal spaces of affected livers, but their functional role in hepatitis C progression has not been fully elucidated. METHODS: CD4+ and CD8+ TL were quantified by immunohistochemistry in portal and periportal spaces of 39 liver biopsies from patients with chronic hepatitis C. They were associated to demographic data, histological parameters, laboratory findings of patients and hepatitis C genotypes. RESULTS: There was high numbers of CD4+ and CD8+ TL from which the density of CD4+ T was higher than CD8+ TL in portal and periportal spaces. CD4+ and CD8+ TL were directly correlated to intensity of interface hepatitis. CD8+ TL correlated to serum enzyme levels. CONCLUSION: The high numbers of CD4+ and CD8+ TL in portal and periportal spaces and their correlation to interface hepatitis suggest that hepatitis C evolution depends on the action of intrahepatic T lymphocytes, lending support to the notion of an immune-mediated mechanism in the pathogenesis of chronic hepatitis C.

THE COPPER INTERFERENCE WITH THE MELANOGENESIS OF Cryptococcus neoformans

Melanin is a pigment produced by laccase, a phenoloxydase enzyme, and is related to the virulence of Cryptococcus neoformans as it is also considered an adaption mechanism to environmental conditions and protection against UV radiation, phagocytic system attack and antifungal drugs. Laccase synthesis is stimulated by several factors, including copper metabolism. The current study shows C. neoformans strains with higher melanization intensity when grown in L-dopa medium supplemented with different concentrations of copper sulfate. This increase shows that melanization rates may be enhanced in the presence of copper ions and may also enhance the virulence of C. neoformans in infected patients that present increasing copper concentrations in serum, such as those with HIV. The virulence of these strains may also be increased in the environment, where this metal is available as CuSO4 in algicidal and fungicidal compounds.

Two competitive enzyme immunoassays for the detection of IgG class antibodies to hepatitis a antigen

Two competitive enzyme immunoassays (EIA) techniques were developed: in the first (COMP-1), test sera were added together with HAV antigen on anti-HAV IgG-coated wells followed by an anti-HA VHRP conjugate; in the second (COMP-2), test sera and anti-HA VHRP conjugate competed for HAV epitopes previously adsorbed to anti-HA V IgG-coated wells. Both procedures used tetramethylbenzidine (TMB) as a substrate. Both competitive tests were shown to be reproducible and suitable for routine diagnosis and research purposes.

Diagnostic tests for amoebic liver abscess: comparison of enzyme - linked immunosorbent assay (Elisa) and counterimmunoelectrophoresis (CIE)

The liver abscess is the most frequent extraintestinal complication of intestinal amoebiasis: its diagnosis is suggested by the clinical picture but it must be confirmed by paraclinic tests. Themost stringent diagnosis requires identification of E. histolytica. But this is possible only in a few cases. Serological tests greatly improve the diagnosis of this severe complication of amoebiasis. We compared the Enzyme Linfed Immunosorbent Assay and the Counterimmunoeletrophoresis techniques. Both techniques were used to detect amoebic antibodies in 50 control patients, 30 patients with liver abscess and 30 patients with intestinal amoebiasis. All the sera from control patients gave negative results iin both techniques. When analysing the sera from patients with intestinal amoebiasis, 10% of them were positive by ELISA but non by CIE. The sera of patients with liver abscess, we found that 90% were positive by the ELISA method and 66.6% by the CIE technique. In patients with amoebic liver abscess, the results showed that the ELISA was more sensitive than the CIE, as it presented a higher sensitivity (100%) than that of the CIE technique (66%).

Evaluation of Leishmania antigens preparation and storage for use in enzyme immunoassays

The influence of time and temperature on the storage of an alkaline antigen of L.major-like and L.(V.) braziliensis promastigotes added or not of a proteases inhibitor (PMSF) was evaluted by means of an IgG-ELISA. Antibodies in assays using L. major-like antigen stored at -20oC for 6 monsths had a statistically lower geometric mean titer (GMT) and different 95% confidence interval limits (CL) than antigens stored otherwise, as assessed by the "t" statistic. The PMSF L. major-like antigen after storage for 6 months at a temperature of 4oC had the same GMT and 95% CL displayed at time zero as well as when storage for 4 and 6 months at -20oC. Significant diferences were not found when L.(V.) braziliensis antigens were stored at times and temperatures mentioned; the PMSF antigen stored for 2 months at -70oC resulted in a lower serum GMT and 95% CL than any other, as assessed by the "t" statistic. Antigen performance did not show any statistical difference associated to the addition of PMSF within the same species; the largest difference between antigens was that between PMSF-L. (V.) braziliensis and L. major-like without PMSF.

Dot enzyme-linked immunosorbent assay (dot-ELISA) for schistosomiasis diagnosis using dacron as solid-phase

Dacron and nitrocellulose were evaluated as matrices for the dot enzyme linked immunosorbent assay (dot-ELISA) for schistosomiasis and compared to indirect immunofluorescence (IMF). Titration of sera from 18 schistosomiasis patients against soluble worm antigen preparation (SWAP) was carried out and sera from healthy individuals from non-endemic areas were used as controls. The IMF was less sensitive than the dot-ELISAs, although the difference was not statistically significant (p > 0.05). The dot-ELISA based on nitrocellulose was as sensitive as that using dacron. Stability did not differ between nitrocellulose and dacron. Specificity was lower when dacron was used than when nitrocellulose was used, although the difference was not statistically significant (p > 0.05). In conclusion, this work showed that nitrocellulose and dacron performed similarly in dot-ELISA, suggesting that they may be used alternatively in population surveillance in endemic areas.

Evaluation of an enzyme immunoassay for clinical diagnosis of neurocysticercosis in symptomatic patients

INTRODUCTION: Neurocysticercosis is an infection of the human central nervous system caused by the metacestode larvae of Taenia solium. Neurocysticercosis is the most common parasitic disease in developing countries. Epilepsy is the most common clinical manifestation. Difficulties in confirming the diagnosis motivated the evaluation of the enzyme-linked immunosorbent assay on cerebral spinal fluid (CSF). METHODS: Twenty-two patients with NCC and 44 control patients were studied. CSF was analyzed using a commercial ELISA kit developed for NCC. Sensitivity and specificity were measured and a multivariate logistic regression was performed. RESULTS: Sensitivity and specificity of ELISA were 31.8% and 100%, respectively, with accuracy of 77.3%. Only the size of the lesions proved to be important for performance of the test. CONCLUSIONS: The results showed that ELISA contributes to the diagnosis of neurocysticercosis if the result is negative or if the patient has a lesion of 2 cm or more.