Cláudio Lúcio Rossi

Evaluation of serological markers for the immunodiagnosis of acute acquired toxoplasmosis

The detection of specific IgM antibodies has been the most frequently used serological marker for diagnosing recent toxoplasmosis. However, the persistence of specific IgM antibodies in some patients and the use of tests with a low specificity have complicated the interpretation of serological results when toxoplasmosis is suspected. The purpose of the present study was to determine the value of newer serological techniques in the diagnosis of acute acquired toxoplasmosis. Sixty-four sera, 31 from patients with Toxoplasma gondii infection and 33 from patients with latent infection, were tested. Anti-T. gondii IgA was measured by two antibody capture ELISA tests (Platelia Toxo IgA and ETI-TOXOK A) and an automated direct ELISA (IMx Toxo IgA); all three assays detected antibody levels compatible with a recent infection in sera from all 31 patients with acute toxoplasmosis. However, significant levels of IgA were also detected with high frequency by all three assays in sera from patients with latent infection. IgE antibodies detected by IgE immunosorbent agglutination assay (ISAGA) were present in 26 (84%) of 31 patients with acute toxoplasmosis and in sera from two subjects with latent infection taken >1 year after the beginning of the clinical symptoms of infection. Thirty (97%) of 31 patients with a recent T. gondii infection and 15 (45%) of 33 subjects with latent infection had an AC/HS pattern compatible with acute toxoplasmosis. The avidity of T. gondii IgG was evaluated by two methods. One method was based on the titration of each serum sample and calculation of the titres, in the absence and presence of urea, in relation to a defined cut-off value. In the other method, a single serum dilution was used and the absorbances of the reactions in the presence and absence of urea were compared. The titration method was more sensitive for diagnosing recent primary infection; all 31 sera from patients with acute toxoplasmosis had avidity indices compatible with acute toxoplasmosis by the titration method, whereas with the single dilution method, sera from four patients had equivocal results. In the 33 individuals with latent infection, similar results were obtained with the two avidity methods; only one serum sample had a non-compatible avidity value with the titration method. The results obtained in the present study show that the current serological markers used for diagnosing acute acquired toxoplasmosis have significant limitations. The data suggest that determination of the avidity of T. gondii-specific IgG by the titration method in patients with detectable IgM antibodies defines most accurately the stage of infection by T. gondii.

Chemical Modifications on Polystyrene Latex: Preparation and Characterization for Use in Immunological Applications

Polystyrene latex microspheres are efficient supports for immunological reactions. In the presence of suitable functional groups these microspheres may react by covalent binding with antigens or antibodies and have been extensively used in immunoassay technique. By using the emulsion polymerization and diazotization methods, a kind of functionalized latex was prepared. The preparation of this latex consisted of six syntheses of polystyrene followed by nitration, amination and diazotization. A narrow distribution of particle size was obtained in all the cases. The synthesis that presented 98% of monomer conversion and the largest particle size diameter 0.2 ± 0.05 μm was chosen for the subsequent chemical treatments. The chemical characterizations of every step were performed by Fourier transform infrared and Raman spectroscopies. The particle size was determined by a submicrometer particle analyzer and scanning electron microscopy. The polydiazostyrene latex obtained was sensitized using bovine serum albumin (BSA) and when it was submitted to agglutination assay in the presence of a rabbit serum containing specific antibodies to BSA demonstrated efficient results.

A Simple, Rapid Enzyme-linked Immunosorbent Assay for Evaluating Immunoglobulin G Antibody Avidity in Toxoplasmosis

This article describes an enzyme-linked immunosorbent assay (ELISA) for evaluating the avidity of Toxoplasma-specific immunoglobin (Ig) G. The test was standardized with the Falcon assay screening test (F.A.S.T) ELISA system using 6 M urea in phosphate-buffered saline for dissociating low-avidity antibodies after the antigen-antibody interaction. The avidity indices for 25 serum samples from patients with a recent Toxoplasma infection ranged from 10 to 40% (mean index = 25.3%), whereas for sera from 25 subjects with preexisting Toxoplasma immunity, the indices ranged from 58 to 94% (mean index = 73.1%). In sequential serum samples obtained over a period of up to 15 months from three patients exhibiting a persistent IgM antibody response to T. gondii, the avidity indices gradually increased during the course of infection. Avidity indices compatible with a recent infection were found in serum samples taken during the first 3 months after the onset of toxoplasmic lymphadenopathy, whereas indices compatible with the presence of a long-term infection were found in serum samples taken 7 or more months after the onset of toxoplasmic lymphadenopathy. The simplicity and short assay time (less than 1 h) make the test a potentially useful tool in assessing the avidity of Toxoplasma-specific IgG.

Serological diagnosis of toxoplasmosis: usefulness of IgA detection and IgG avidity determination in a patient with a persistent IgM antibody response to Toxoplasma gondii

We report the detection of specific IgA antibodies and the determination of IgG avidity in sequential serum samples from a patient exhibiting significant levels of Toxoplasma-specific IgM antibodies for seven years after the onset of the clinical symptoms of toxoplasmosis. IgM antibodies were detected by an indirect immunofluorescence test and by three commercial enzyme-linked immunosorbent assays (ELISA). Anti-T. gondii IgA was quantified by the alpha-capture ELISA technique using a commercial kit. As defined by the manufacturer of the IgA ELISA test used, most patients with acute toxoplasmosis have antibody levels > 40 arbitrary units per ml (AU/mL). At this cut-off level, the patient still had a positive ELISA result (45 AU/mL) in a serum sample taken one year after the beginning of clinical manifestations. The IgG avidity-ELISA test was performed with the Falcon assay screening test (F.A.S.T.(R)) - ELISA system. Avidity indices compatible with a recent Toxoplasma infection were found only in serum samples taken during the first 5 months after the onset of the clinical symptoms of toxoplasmosis. These results show that the interpretation of positive IgM results as indicative of recently acquired toxoplasmosis requires additional laboratory confirmation either by other tests or by the demonstration of a significant rise in the antibody titers in sequential serum samples.

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 retrospective study of the patients 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.

Evaluation of Taenia solium and Taenia crassiceps cysticercal antigens for the serodiagnosis of neurocysticercosis

We evaluated the usefulness of seven cysticercal antigen extracts, four from Taenia solium cysticerci (whole parasite‐TsoW, membrane‐TsoMe, vesicular fluid‐TsoVF and scolex‐TsoSc) and three from T. crassiceps cysticerci (whole parasite‐TcraW, membrane‐TcraMe and vesicular fluid‐TcraVF), for serodiagnosis of neurocysticercosis with an enzyme‐linked immunosorbent assay (ELISA). Cysticercus‐specific IgG were screened in serum samples from 23 patients with neurocysticercosis, 32 patients with other infections and 48 healthy persons. The best results were obtained with the TsoVF‐ELISA (91.3% sensitivity; 96.2% specificity) and TcraVF‐ELISA (91.3% sensitivity; 95% specificity). The ELISA done with whole parasite and membrane extracts from cysts of T. solium and T. crassiceps and the scolex extract from T. solium cysts showed a low performance in terms of sensitivity, ranging from 47.8% to 73.9%. None of the antigen preparations from T. solium and T. crassiceps cysticerci used in this study showed outstanding performance for the serodiagnosis of neurocysticercosis. However, considering the results obtained with the seven antigen preparations, vesicular fluid from T. solium and T. crassiceps cysticerci may be useful for detecting specific antibodies in sera from patients with neurocysticercosis.

Polyarteritis Nodosa and Cytomegalovirus: Diagnosis by Polymerase Chain Reaction

Abstract: We investigated the occurence of an active cytomegalovirus (CMV) infection in patients with polyarteritis nodosa (PAN). Eleven patients with PAN were screened for the presence of CMV-DNA in their blood using the polymerase chain reaction (PCR). Serum anti-CMV IgG and anti-CMV IgM antibodies were determined by enzyme-linked immunosorbent assays (ELISA). The ELISA for IgM was negative in all cases whereas that for IgG was positive in eight cases. Only one patient was positive for CMV-DNA by PCR. He presented with myalgia, polyarthralgia, fever and weight loss, suggesting PAN activity. CMV infection was uncommon in our series of patients with PAN, despite disease activity and immunosuppressor therapy. The finding of a transient CMV infection in one case at the beginning of PAN activity suggests that CMV may be involved in the pathogenesis of PAN.

Avaliação de preparações antigênicas de Strongyloides stercoralis para o imunodiagnóstico da estrongiloidíase

Aqueous-soluble (AS) antigens from larvae of Strongyloides stercoralis, extracted with phosphate- buffered saline, are traditionally used for serodiagnosis of strongyloidiasis. To identify sources of antigensfor use in serodiagnosis, residual particulates from parasite larvae after aqueous extraction were solubilized with Tris-buffered 8M urea, yielding a urea-soluble (US) antigen fraction. Both AS and US antigens from S. stercoralis were evaluated by an enzyme-linked immunosorbent assay. No significalive differences were observed between AS and US antigens from the parasite regarding specific antigenic activity and cross-reactivity. lmmunoassays are highly dependent on the antigen for sensitivity and specificity. Crude extracts from S. stercoralis should be further studied, mainly in relation to antigenic fractions which could provide even more sensitive and specific results. Studies of fractionation of S. stercoralis must take into account the antigen yield of both the crude extract and fractions, since larvae of parasite are normally difficult to obtain. Considering this aspect, the results from this study are very useful, since the extraction with urea subslantialty increased the amounts of antigenic materiais normally obtained with the classical aqueous extraction.

A quantitative enzyme-linked immunosorbent assay (ELISA) for the immunodiagnosis of neurocysticercosis using a purified fraction from Taenia solium cysticerci

A quantitative enzyme-linked immunosorbent assay (ELISA) for the immunodiagnosis of neurocysticercosis is described. The ELISA was standardized using a purified Taenia solium cysticerci fraction (PCF) obtained by ion exchange chromatography. The ELISA using PCF (PCF-ELISA) and a qualitative ELISA using a whole extract from T. solium cysticerci (WEC-ELISA) were used to screen for cysticercus-specific IgG antibodies in cerebrospinal fluid (CSF) samples from 57 patients with neurocysticercosis and 50 patients with other neurologic disorders. The sensitivity of both assays was 95%, whereas the specificities of PCF-ELISA and WEC-ELISA were 100% and 92%, respectively. The excellent sensitivity and specificity of the PCF-ELISA make this assay a potentially useful tool in screening for antibodies against T. solium cysticerci.

Simultaneous monitoring of CMV and human herpesvirus 6 infections and diseases in liver transplant patients: one-year follow-up

OBJECTIVE: The aim of this study was to simultaneously monitoring cytomegalovirus and human herpesvirus 6 active infections using nested-polymerase chain reaction and, together with clinical findings, follow the clinical status of patients undergoing liver transplant. INTRODUCTION: The human β-herpesviruses, including cytomegalovirus and human herpesvirus 6, are ubiquitous among human populations. Active infections of human herpesvirus 6 and cytomegalovirus are common after liver transplantation, possibly induced and facilitated by allograft rejection and immunosuppressive therapy. Both viruses affect the success of the transplant procedure. METHODS: Thirty patients submitted to liver transplant at the Liver Transplant Unit, at the Gastro Center, State University of Campinas, SP, Brazil, were studied prospectively from six months to one year, nested-polymerase chain reaction for cytomegalovirus and human herpesvirus 6 DNA detections. Two or more consecutive positive nested-polymerase chain reaction were considered indicative of active infection. RESULTS: Active infection by cytomegalovirus was detected in 13/30 (43.3%) patients, median time to first cytomegalovirus detection was 29 days after transplantation (range: 0-99 days). Active infection by human herpesvirus 6 was detected in 12/30 (40%) patients, median time to first human herpesvirus 6 detection was 23.5 days after transplantation (range: 0-273 days). The time-related appearance of each virus was not statistically different (p = 0.49). Rejection of the transplanted liver was observed in 16.7% (5/30) of the patients. The present analysis showed that human herpesvirus 6 and/or cytomegalovirus active infections were frequent in liver transplant recipients at our center. CONCLUSIONS: Few patients remain free of betaherpesviruses after liver transplantation. Most patients presenting active infection with more than one virus were infected sequentially and not concurrently. Nested-polymerase chain reaction can be considered of limited value for clinically monitoring cytomegalovirus and human herpesvirus 6.