Investigation on the association between thyroid tumorigeneses and herpesviruses.

Herpesviruses have been associated with various human malignancies and with thyroid autoimmunity. Aiming to investigate the presence of these viruses in thyroid nodules, we analyzed serum and thyroid tissue from 183 patients (83 benign and 100 malignant thyroid nodules). We also obtained 104 normal thyroid tissues extracted from the contralateral lobe of these patients. We used ELISA to screen the serology of all patients and a real-time quantitative PCR to analyze thyroid tissue viral load in antibody-positive patients. In addition, the presence of herpesviruses was tested by histological analysis in 20 EBV-positive tissues using the expression of LMP-1 by immunohistochemistry (IHC) and EBER by in situ hybridization (ISH). There was no evidence of HSV-2 or CMV DNA, but we found EBV DNA sequences in 29 (16%) thyroid tissue samples. We also found 7 positive EBV cases out of 104 normal tissues. Viral load was higher in tumors than in their respective normal tissues (p = 0.0002). ISH analysis revealed EBER expression in 11 out of 20 (52%) EBV-positive tissues, mostly in malignant cases (8/11, 73%). The presence of high EBV copy numbers in thyroid tumors and the expression of EBER only in malignant cases suggest an association between EBV and thyroid malignancies. However, we did not find any association between the presence of EBV and/or its viral load and any clinical or pathological tumor feature. Further studies aiming to clarify the mechanisms of EBV infection in thyroid cells are necessary to support a possible role in the development of thyroid cancer.

Effect of vitamin E supplementation on antibody levels against malondialdehyde modified LDL in hyperlipidemic hamsters.

OBJECTIVE: The aim of this work was to investigate the effect of vitamin E (VE) supplementation on the formation of autoantibodies against oxidized low density lipoproteins (LDL) in a hyperlipidemic animal model. METHODS: Thirty-four male hamsters (Mesocricetus auratus), (4 weeks old) were divided into three groups: Group A (n=9) was fed with standard rodent chow; group B (n=13) was fed with a standard rodent chow plus 2% cholesterol and 10% butter and group C (n=12) was fed with the same diet plus 0.2% (w/w) VE. Blood samples were collected by intracardiac puncture and antibody levels were determined in each animal at 4 weeks of age and after 20 weeks of experimental diet. A modified ELISA technique was used to analyze the modulation of autoantibody titers against an epitope of oxidized LDL in serum samples. Antigens prepared for the ELISA tests were characterized using spectrofluorimetry. Serum VE levels were determined in the lipidic fractions by HPLC. RESULTS: The groups fed with cholesterol-fat enriched diet presented a three-fold increase in total serum cholesterol and two-fold increase in serum triglycerides compared to the control group. VE supplementation played no role in serum cholesterol and serum triglyceride concentrations but led to a decreased autoantibody (anti-LDL-malondialdehyde) formation (P<0.05). CONCLUSIONS: Our results show that VE supplementation leads to a lower production of autoantibodies against oxidized LDL, suggesting a protective effect of VE against in vivo oxidation of LDL particles, in a dose-dependent manner.

Use of three immunological techniques for the detection of Toxoplasma spIgA antibodies in acute toxoplasmosis.

AIMS: To assess the performance and efficacy of three immunological techniques for the detection of Toxoplasma specific IgA antibodies in acute toxoplasmosis. METHODS: The following techniques were used to examine 128 serum samples (51 cases of acute toxoplasmosis, 50 cases of heterologous infections, and 27 healthy controls): direct enzyme linked immunosorbent assay (ELISA), antibody capture ELISA, and antibody capture agglutination. RESULTS: Direct ELISA had a sensitivity of 98% and a specificity of 97%, antibody capture ELISA of 100% and 99%, respectively, and antibody capture agglutination had sensitivity and specificity of 100%. CONCLUSIONS: All three immunological techniques performed well with similar efficacy. Detection of Toxoplasma specific IgA antibodies is a useful diagnostic marker for acute toxoplasmosis.

Capture enzyme-linked immunosorbent assay to detect specific immunoglobulin E in sera of patients with paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is the most frequent systemic mycosis in South America. The disease is characterized by a polyclonal activation of B cells, resulting in hyperimmunoglobulinemia. The production of immunoglobulin (Ig) E in deep mycosis has been related to the severity of the disease. However, the detection of specific IgE in sera of patients is difficult because of the competition with the IgG. We compared a capture and an indirect enzyme-linked immunosorbent assay (ELISA) technique to detect Paracoccidioides brasiliensis IgE. We found that the capture ELISA presented higher performance and lower background values than the indirect assay, resulting in a significant quantitative discrimination between sera from patients with the 2 major clinical forms of PCM. Patients with the juvenile form presented significantly higher levels of P. brasiliensis IgE, as compared with patients with the adult form. The capture ELISA was used in the follow-up of patients receiving treatment, showing that the levels of specific IgE decreased as the patient's clinical conditions improved.

A simple, rapid enzyme-linked immunosorbent assay for evaluating immunoglobin 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.

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.

Polyarteritis nodosa and cytomegalovirus: diagnosis by polymerase chain reaction.

We investigated the occurrence 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.

Erythro-lectin immuno test (Erythro-LIT) in the immunodiagnosis of neurocysticercosis.

1. The effectiveness of the Erythro-Lectin Immuno Test (Erythro-LIT) to detect anticysticercus antibodies was tested using cerebrospinal fluid (CFS) from patients with neurocysticercosis. 2. Both Erythro-LIT and complement fixation (CF) were used to detect antibodies in 36 CSF samples from cysticercotic patients. Erythro-LIT detected anticysticercus antibodies in 35 CSF samples (97%) and CF in 26 (72%). The antibody titers ranged from 1:4 to 1:4096 for Erythro-LIT (mean geometric titer = 282.65) and 1:1 to 1:64 in CF (mean geometric titer = 7.92). 3. When greater than or equal to 1:16 Erythro-LIT titer was used as a significant diagnostic cut-off value, the sensitivity and specificity of Erythro-LIT were 92% and 100%, respectively, for CFS. 4. The high sensitivity and specificity demonstrated here, together with the simplicity and low cost of the test, make the Erythro-LIT a potentially useful method for screening for specific antibodies in neurocysticercosis.

Donated organs as a source of cytomegalovirus (CMV) in renal transplant patients.

Two patients receiving the same cadaver kidney graft were investigated prospectively for cytomegalovirus (CMV) infection using the polymerase chain reaction (PCR) and serologic tests (ELISA and IFI). The data indicate that a strain of CMV was probably transmitted from the same donor to both kidney recipients including one who was seropositive for CMV.

Detection of cytomegalovirus infections by PCR in renal transplant patients.

Cytomegalovirus (CMV) is the single most important infectious agent affecting recipients of organ transplants. To evaluate the incidence and the clinical importance of CMV infection in renal transplants in Brazil, 37 patients submitted to renal allograft transplants were tested periodically for the presence of cytomegalovirus DNA in urine using the polymerase chain reaction (PCR), and for the presence of IgM and IgG antibodies against CMV by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF). The PCR-amplified products were detected by gel electrophoresis and confirmed by dot-blot hybridization with oligonucleotide probes. Thirty-two of the 37 patients (86.4%) were positive by at least one of the three methods. In six patients, PCR was the only test which detected the probable CMV infection. Ten patients had a positive result by PCR before transplantation. In general, the diagnosis was achieved earlier by PCR than by serologic tests. Active infection occurred more frequently during the first four months after transplantation. Sixteen of the 32 patients (50%) with active CMV infection presented clinical symptoms consistent with CMV infection. Five patients without evidence of active CMV infection by the three tests had only minor clinical manifestations during follow-up. Our results indicate that PCR is a highly sensitive procedure for the early detection of CMV infection and that CMV infection in renal transplant patients is a frequent problem in Brazil.

IgM and IgA antibody responses in 12 cases of human acquired toxoplasmosis.

The persistence, in some subjects, of specific IgM antibodies to Toxoplasma gondii for several months after the acute phase of infection has complicated the interpretation of serological test results for toxoplasmosis. Several reports have emphasized the value of the detection of Toxoplasma-specific IgA antibodies for the diagnosis of acute toxoplasmosis. In this article, we report the follow-up profiles of Toxoplasma-specific IgM and IgA antibodies in serum samples obtained from 12 patients at various intervals after the onset of the clinical manifestations of infection. IgM antibodies were detected by the indirect immunofluorescence (IIF) test, antibody capture enzyme-linked immunosorbent assay (cELISA) and enzyme-mediated chemilluminescent technique (CmL). IgA antibodies were quantified by the direct ELISA (dELISA) and cELISA procedures. As defined by the manufacturer of the cELISA test for IgA used, most patients with acute toxoplasmosis have antibody levels > 40 arbritary units per ml (AU/ml). At values > 40 AU/ml, the cELISA for IgA detected significant antibody levels for a shorter time than the other techniques used for IgM and IgA detection. However, IgA levels < or = 40 AU/ml do not exclude the possibility of acute toxoplasmosis since such levels can be reached very soon after infection with T. gondii. The results obtained in the present study show that the serological diagnosis of acute toxoplasmosis may not be such an easy task. Our data suggest that use of the IgA-cELISA concomitantly with IgM antibody screening could permit, in some circumstances, a more efficient diagnosis of acute acquired toxoplasmosis.

Cerebrospinal fluid profiles in acquired immunodeficiency syndrome with and without neurocryptococcosis.

Cryptococcosis is one of the most common fungal infections of the central nervous system (CNS) in AIDS patients and meningoencephalitis or meningitis is a frequently observed manifestation. However, systematic studies of cerebrospinal fluid (CSF) composition from AIDS patients with CNS cryptococcosis have been few. CSF samples from 114 HIV seropositive patients whose clinical complaint suggested CNS involvement, were analyzed; 32 samples from patients diagnosed as having neurocryptococcosis (Group 1) and 82 samples from patients with no identified neurological disfunction (Group 2). Based on cytological and biochemical results, two distinct profiles were observed: Normal (Group 1 = 31%, Group 2 = 39%); Abnormal (Group 1 = 69%, Group 2 = 61%). Lymphocytes were the most frequent cells in both groups. Our CSF cytological and biochemical findings showed that in AIDS patients liquoric abnormalities are quite frequent, non-specific and difficult to interpret. In these circumstances a systematic search to identify the etiologic agent using microbiological and/or immunological assays must be routinely performed.

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 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.

[The evaluation of antigenic preparations of Strongyloides stercoralis for the immunodiagnosis of strongyloidiasis]. / 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 antigens for 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 significative differences were observed between AS and US antigens from the parasite regarding specific antigenic activity and cross-reactivity. Immunoassays 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 substantially increased the amounts of antigenic materials normally obtained with the classical aqueous extraction.

[Evaluation of antigenic fractions of Cysticercus cellulosae for the immunodiagnosis of neurocysticercosis using antibody-lectin conjugates]. / Avaliação de frações antigênicas de cysticercus cellulosae para o imunodiagnóstico da neurocisticercose utilizando conjugados anticorpo-lectina.

In an attempt to find out some fraction with high antigenic activity for the immunodiagnosis of neurocysticercosis a crude extract from Cysticercus cellulosae was fractioned by Sephadex G-200 column chromatography. The protein elution profile revealed two distinct peaks (fractions I and III) and a heterogeneous fraction containing several peaks (fraction II). The crude extract and the fractions were tested by Erythro-Lectin Immuno Test (Erythro-LIT) using cerebrospinal fluid samples from patients with neurocysticercosis. The results of Erythro-LIT antibody titers showed that most of the anticysticercus antibodies recognized antigenic components contained in the fraction II.

Diagnosis of alpha-1-antitrypsin deficiency by DNA analysis of children with liver disease.

BACKGROUND: Alpha-1-antitrypsin deficiency is a genetic disorder which is transmitted in a co-dominant, autosomal form. Alpha-1-antitrypsin deficiency affects mainly the lungs and the liver leading, in the latter case, to neonatal cholestasis, chronic hepatitis or cirrhosis. A precise diagnosis of Alpha-1-antitrypsin deficiency may be obtained by biochemical or molecular analysis. OBJECTIVE: The purpose of this study was to use DNA analysis to examine the presence of an alpha-1-antitrypsin deficiency in 12 children suspected of having this deficiency and who showed laboratory and clinical characteristics of the disease. PATIENTS AND METHODS: Twelve patients, aged 3 months to 19 years, who had serum alpha-1-antitrypsin levels lower than normal and/or had hepatic disease of undefined etiology were studied. The mutant alleles S and Z of the alpha-1-antitrypsin gene were investigated in the 12 children. Alpha-1-antitrypsin gene organization was analyzed by amplification of genome through the polymerase chain reaction and digestion with the restriction enzymes Xmnl (S allele) and Taq-1 (Z allele). RESULTS: Seven of the 12 patients had chronic liver disease of undefined etiology and the other five patients had low serum levels of alpha-1-antitrypsin as well as a diagnosis of neonatal cholestasis and/or chronic liver disease of undefined etiology. Five of the 12 patients were homozygous for the Z allele (ZZ) and two had the S allele with another allele (*S) different from Z. CONCLUSION: These results show that alpha-1-antitrypsin deficiency is relatively frequent in children with chronic hepatic disease of undefined etiology and/or low alpha-1-antitrypsin levels (41.6%). A correct diagnosis is important for effective clinical follow-up and for genetic counseling.

Cytomegalovirus, human herpesvirus-6, and human herpesvirus-7 in adult liver transplant recipients: diagnosis based on antigenemia.

Human herpesvirus (HHV)-6, HHV-7, and cytomegalovirus (CMV) that remain latent after primary infection can be reactivated during immunosuppression following organ transplantation in liver transplant recipients. The aim of this study was to monitor active infections for HHV-6, HHV-7, and CMV among adult liver transplantation recipients using antigenemia detected by an immunoperoxidase staining. Twenty-eight adult liver transplant patients were monitored using antigenemia in blood samples obtained at the time of transplantation, as well as weekly in the first month and once a month for 6 months. Of these patients, 28.5% showed positive CMV antigenemia; 39.2%, HHV-6 antigenemia; and 14.2%, HHV-7 antigenemia. The detection of the three viruses was considered to be independent of one another (P>.05). The results described above showed that few patients remain free of beta herpesviruses after liver transplantation. Most patients were infected sequentially and not concurrently. Antigenemia has been considered useful to detect active HHV-6 and HHV-7 infections. Antigenemia can be more efficiently interpreted when compared with polymerase chain reaction results, although other studies are necessary to establish the reference of HHV-6 and HHV-7 antigenemia.

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.

A rapid, quantitative enzyme-linked immunosorbent assay (ELISA) for the immunodiagnosis of Chagas' disease.

A rapid, quantitative enzyme-linked immunosorbent assay (ELISA) for the immunodiagnosis of Chagas' disease is described. The ELISA was standardized with the Falcon assay screening test (F.A.S.T.) system using a purified Trypanosoma cruzi epimastigote fraction (PEF) obtained by ion exchange chromatography. The ELISA technique and an indirect immunofluorescence (IIF) test were used to search for T.cruzi--specific IgG antibodies in 73 patients with chronic Chagas' disease, 58 patients with heterologous infections and 20 healthy controls. The ELISA exhibited 98.6% sensitivity and 98.7% specificity, whereas the sensitivity and specificity of the IIF test were 94.5% and 96.2%, respectively. The excellent performance with regard to sensitivity and specificity, as well as the short assay time make the F.A.S.T. system--based ELISA with PEF a potentially useful tool in screening for antibodies against T. cruzi.