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 £ 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

An Evaluation of clinical, serologic, anatomopathologic and immunohistochemical findings for fifteen patients with mucosal leishmaniasis before and after treatment

Treatment of mucosal leishmaniasis (ML) can be controlled by clinical examination and by serologic titers by the indirect immunofluorescence serologic reaction (IISR). We studied the correlation between the presence of antigen in tissue determined by immunohistochemistry, the IISR titers and the anatomopathologic findings in fifteen patients with ML before and after healing of the lesions as determined by otorhinolaryngologic evaluation, and evaluated these parameters to determine which of them could be useful during follow-up. Tissue antigens became negative in four patients (group A) after treatment, with a statistically significant reduction or negativity of IISR titers (p<0.05). This did not occur in patients in whom the antigen persisted after treatment (group B), suggesting that serologic follow-up should be performed together with the search for tissue antigen, a combination which, to our knowledge, has not been used in previous studies. The negativity of tissue antigens and the behavior of IIRS titers in group A patients probably indicate a lower possibility of recurrence. Upon anatomopathologic examination the inflammatory process was found to persist after treatment even in group A, suggesting that the permanence of inflammatory activity even in clinically healed lesions is possibly correlated with the presence of the antigen or of some unknown factor.

SIMULTANEOUS INFECTION WITH DENGUE 1 AND 2 IN A BRAZILIAN PATIENT

Dengue outbreaks have occurred in several Brazilian States since 1986 involving serotypes 1 (DEN-1) and 2 (DEN-2). In view of the few cases of double infection documented in the literature, we report here a case of simultaneous infection with DEN-1 and DEN-2 in a patient residing in the municipality of Miranda, State of Mato Grosso do Sul, Western region of Brazil. DEN-1 was introduced in this State in 1989 and DEN-2 in 1996, both of them circulating in some municipalities. This double infection was identified by virus isolation and by indirect immunofluorescence using monoclonal antibodies and confirmed by the polymerase chain reaction (PCR). This is the first documented case of simultaneous infection with serotypes DEN-1 and DEN-2 in Brazil.

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.

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 a-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.®) - 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.

Toxoplasma gondii infection in Brazilian domestic outpatient cats

The occurrence of Toxoplasma antibodies in domestic outpatient cats in the city of São Paulo was evaluated using the indirect immunofluorescence assay. A total of 248 blood samples obtained from male and female cats seen at the Veterinary Teaching Hospital at the University of São Paulo between February 1996 and January 1997 were tested. Of these, 17.7% were positive, with a 64 titer being detected in most animals. The frequence of Toxoplasma antibodies was significantly higher in older cats, those fed raw meat and those with free access to the outdoor environment. There was no significant difference in reactivity between males and females. We conclude that diet and free access to the outdoor environment were equally important as predisposing factors to the risk of Toxoplasma infection.

Parasitological and immunological diagnoses of strongyloidiasis in immunocompromised and non-immunocompromised children at Uberlândia, state of Minas Gerais, Brazil

Parasitological and immunological diagnoses were part of a study conducted among 151 children, 83 immunocompromised (IC) and 68 non-immunocompromised (non-IC) aged from zero to 12, seen at the University Hospital, Universidade Federal de Uberlândia, State of Minas Gerais, Brazil, from February, 1996, to June, 1998. Three fecal samples from each child were analyzed for the parasitological diagnosis by Baermann-Moraes and Lutz methods. The immunological diagnosis to detect IgG and IgM antibodies was carried out by the indirect immunofluorescence antibody test (IFAT) with cryo-microtome sections of Strongyloides stercoralis and Strongyloides ratti larvae as antigens and by the ELISA test with an alkaline extract of S. ratti as the antigens. Of the 151 children 5 (3.31%) were infected with larvae of S. stercoralis (2 cases IC, 2.41%, and 3 cases non-IC, 4.41%). The IFAT-IgG detected 7 (8.43%) serum samples positive among IC, and 2 (2.94%) cases among non-IC. The ELISA-IgG test detected 10 (12.05%) serum samples positive among IC, and 1 (1.47%) case among non-IC. The IFAT-IgM detected 6 (7.22%) positive cases among IC, and 3 (4.41%) cases among non-IC. ELISA-IgM test detected 10 (12.05%) positive cases among IC, and 3 (4.41%) cases among non-IC. It was concluded that the immunological tests can help in the diagnosis of strongyloidiasis in immunocompromised children.

First isolation of dengue 3 in Brazil from an imported case

The authors report the isolation of dengue 3 virus for the first time in Brazil. The patient, resident in Limeira-SP, traveled to Nicaragua on May 16th, 1998, where he stayed for two months. Starting on August 14th he had fever, headache, myalgia, arthralgia, retro-orbital pain and diarrhea. He returned to Brazil on August 16th and was hospitalized in the next day. The patient had full recovery and was discharged on August 20th. The virus was isolated in C6/36 cell culture inoculated with serum collected on the 6th day after the onset of the symptoms. The serotype 3 was identified by indirect immunofluorescence assays performed with type-specific monoclonal antibodies. This serotype was further confirmed by polymerase chain reaction analysis. The introduction of a new dengue serotype in a susceptible population is a real threat for the occurrence of severe forms of the disease. The isolation and identification of dengue virus are important in order to monitoring the serotypes circulating in Brazil and to take the measures necessary to prevent and control an epidemic.

Borrelia burgdorferi antibodies in dogs from Cotia county, São Paulo State, Brazil

Dogs sera samples collected from Cotia County, São Paulo were tested using indirect immunoenzymatic test (ELISA) in order to study Lyme disease serology in dogs. ELISA method was standardized and G39/40 North American strain of Borrelia burgdorferi was used as antigen. Positive results were confirmed employing the Western blotting technique. Because of the possibility of cross-reactions, sera were also tested for different serological strains of Leptospira interrogans and L. biflexa using microscopic sera agglutination test. Twenty-three of 237 (9.7%) serum samples were positive in the ELISA; 20 of them (86.9%) were confirmed by the Western blotting, what suggests that Cotia may be a risk area for Lyme disease. Although 4 samples (1.7%) were positive for Lyme disease and leptospirosis, no correlation was found between the results (X² = 0.725; p = 0.394) what suggests absence of serological cross reactivity.

Experimental infection and horizontal transmission of Bartonella henselae in domestic cats

In order to study B. henselae transmission among cats, five young cats were kept in confinement for two years, one of them being inoculated by SC route with B. henselae (10(5) UFC). Only occasional contact among cats occurred but the presence of fleas was observed in all animals throughout the period. Blood culture for isolation of bacteria, PCR-HSP and FTSZ (gender specific), and BH-PCR (species-specific), as well as indirect immunofluorescence method for anti-B. henselae antibodies were performed to confirm the infection of the inoculated cat as well as the other naive cats. Considering the inoculated animal, B. henselae was first isolated by blood culture two months after inoculation, bacteremia last for four months, the specific antibodies being detected by IFI during the entire period. All contacting animals presented with bacteremia 6 months after experimental inoculation but IFI did not detect seroconversion in these animals. All the isolates from these cats were characterized as Bartonella (HSP and FTSZ-PCR), henselae (BH-PCR). However, DNA of B. henselae could not be amplified directly from peripheral blood by the PCR protocols used. Isolation of bacteria by blood culture was the most efficient method to diagnose infection compared to PCR or IFI. The role of fleas in the epidemiology of B. henselae infection in cats is discussed.

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.

Long term evaluation of etiological treatment of Chagas disease with benznidazole

The aim of this article is to present an investigation of cure rate, after long follow up, of specific chemotherapy with benznidazole in patients with both acute and chronic Chagas disease, applying quantitative conventional serological tests as the base of the criterion of cure. Twenty one patients with the acute form and 113 with one or other of the various chronic clinical forms of the disease were evaluated, after a follow up period of 13 to 21 years, for the acute, and 6 to 18 years, for the chronic patients. The duration of the acute as well as the chronic disease, a condition which influences the results of the treatment, was determined. The therapeutic schedule was presented, with emphasis on the correlation between adverse reactions and the total dose of 18 grams, approximately, as well as taking into consideration precautions to assure the safety of the treatment. Quantitative serological reactions consisting of complement fixation, indirect immunofluorescence, indirect hemagglutination, and, occasionally, ELISA, were used. Cure was found in 76 per cent of the acute patients but only in 8 per cent of those with chronic forms of the disease. In the light of such contrasting results, fundamentals of the etiological therapy of Chagas disease were discussed, like the criterion of cure, the pathogenesis and the role of immunosuppression showing tissue parasitism in long standing chronic disease, in support of the concept that post-therapeutic consistently positive serological reactions mean the presence of the parasite in the patient's tissues. In relation to the life-cycle of T. cruzi in vertebrate host, there are still some obscure and controversial points, though there is no proof of the existence of resistant or latent forms. However, the finding over the last 15 years, that immunosuppression brings about the reappearance of acute disease in long stand chronic patients justifies a revision of the matter. Facts were quoted in favor of the treatment of chronic patients.

Visceral leishmaniasis caused by Leishmania (Viannia) braziliensis in a patient infected with human immunodeficiency virus

The current article reports the case of a 19-month-old-girl, from the state of Minas Gerais, Brazil, with visceral leishmaniasis, by Leishmania (Viannia) braziliensis, and Human Immunodeficiency Virus (HIV) co-infection. The child's mother and father, aged 22 and 27 years old, respectively, were both HIV positive. The child was admitted to the General Pediatric Center, in Belo Horizonte, presenting high fever, fatigue, weight loss and enlargement of liver and spleen. Indirect immunofluorescent test revealed a titer of 1:320 for Leishmania. Such result was confirmed by the presence of amastigotes in bone marrow aspirate samples and culture of promastigote forms. Parasites were identified as being Leishmania (Viannia) braziliensis through PCR, using a L. braziliensis complex primer and a generic primer, followed by hibridization. Specific leishmaniasis therapy (GlucantimeÒ antimonial) was intravenously administered.

Chagas disease in the Brazilian Amazon: IV. a new cross-sectional study

A new conglomerate family sample of 194 dwellings with 996 resident persons were studied in the town of Barcelos, State of Amazonas, in order to re-evaluate the risk of Chagas disease. During the survey the persons were interviewed and in this occasion we showed to them a collection of Panstrongylus, Rhodnius and Triatoma, asking if they recognized and eventually have been bitten by this kind of bugs. At this time we collected 500 ul of blood in microtainer® tubes from 886 interviewed persons who gave permission after informed consent. A screening test for T. cruzi antibodies based on agglutination of colored polymer particles, sensitized with three different synthetic peptides of T. cruzi (ID-PaGIA Chagas Test)®, showed 13.2% of sera positivity, but only 6.8% were confirmed by indirect immunofluorescence, and ELISA with purified T. cruzi antigens. Two hundred and six interviewed persons (20.7%) recognized the triatomines, as "piaçavas' lice" and 62 (30%) confirmed that have been bitten by the bugs, 25.8% of them had a positive serology for T. cruzi infection. Electrocardiographic alterations were shown in 9.3% of the seropositives and in 11.9% of the seronegative cases. This was considered not statistically significant.