Comparison on the performance of Leishmania major-like and Leishmania braziliensis braziliensis as antigen for new world leishmaniasis IgG-immunofluorescence test

The performance of an antigen of L. major-like promastigotes for the serological diagnosis of mucocutaneous leishmaniasis in the IgG-immunofluorescent test was compared to that of an antigen of L. braziliensis braziliensis. Each antigen was used to test two hundred and twenty-four sera of etiologies such as mucocutaneous leishmaniasis, deep mycoses, toxoplasmosis, malaria, Chagas' disease, visceral leishmaniasis, anti-nuclear factor, schistosomaiasis, rheumatoid factor and normal controls. Agreement between responses to each antigen was high: 77.2% of leishmaniases sera agreed on a positive or a negative result to both antigens and 91.1 % of control sera. Cross reactivity was restricted to Chagas' disease sera, visceral leishmaniasis, anti-nuclear factor and paracoccidiodomycosis. The quantitative response of leishmaniasis and Chagas' disease sera to both antigens was evaluated by a linear regression; although the y-intercept and the slope were different for each antigen, neither was better than the other in the disclosure of anti-Leishmania antibodies. In the case of Chagas' disease sera the L. major-like antigen was better than L. b. braziliensis' to disclose cross-reacting antibodies.

Slaughterhouses fungal burden assessment: a contribution for the pursuit of a better assessment strategy

In slaughterhouses, the biological risk is present not only from the direct or indirect contact with animal matter, but also from the exposure to bioaerosols. Fungal contamination was already reported from the floors and walls of slaughterhouses. This study intends to assess fungal contamination by cultural and molecular methods in poultry, swine/bovine and large animal slaughterhouses. Air samples were collected through an impaction method, while surface samples were collected by the swabbing method and subjected to further macro- and micro-scopic observations. In addition, we collected air samples using the impinger method in order to perform real-time quantitative PCR (qPCR) amplification of genes from specific fungal species, namely A. flavus, A. fumigatus and A. ochraceus complexes. Poultry and swine/bovine slaughterhouses presented each two sampling sites that surpass the guideline of 150 CFU/m3. Scopulariopsis candida was the most frequently isolated (59.5%) in poultry slaughterhouse air; Cladosporium sp. (45.7%) in the swine/bovine slaughterhouse; and Penicillium sp. (80.8%) in the large animal slaughterhouse. Molecular tools successfully amplified DNA from the A. fumigatus complex in six sampling sites where the presence of this fungal species was not identified by conventional methods. This study besides suggesting the indicators that are representative of harmful fungal contamination, also indicates a strategy as a protocol to ensure a proper characterization of fungal occupational exposure.

Antibodies against non-structural c100/3 and structural core antigen of hepatitis C virus (HCV) in hemodialysis patients

Two groups of patients undergoing hemodialysis (HD) maintenance were evaluated for their antibody response to non-structural c100/3 protein and structural core protein of hepatitis C virus (HCV). Forty-six patients (Group 1) never presented liver abnormalities during HD treatment, while 52 patients (Group 2) had either current or prior liver enzyme elevations. Prevalence rates of 32.6% and 41.3% were found for anti-c100/3 and anti-HCV core antibodies, respectively, in patients with silent infections (Group 1). The rate of anti-c100/3 in patients of Group 2 was 71.15% and reached 86.5% for anti-HCV core antibodies. The recognition of anti-c100/3 and anti-core antibodies was significantly higher in Group 2 than in Group 1. A line immunoassay composed of structural and non-structural peptides was used as a confirmation assay. HBV infection, measured by the presence of anti-HBc antibodies, was observed in 39.8% of the patients. Six were HBsAg chronic carriers and 13 had naturally acquired anti-HBs antibodies. The duration of HD treatment was correlated with anti-HCV positivity. A high prevalence of 96.7% (Group 2) was found in patients who underwent more than 5 years of treatment. Our results suggest that anti-HCV core ELISA is more accurate for detecting HCV infection than anti-c100/3. Although the risk associated with the duration of HD treatment and blood transfusion was high, additional factors such as a significant non-transfusional spread of HCV seems to play a role as well. The identification of infective patients by more sensitive methods for HCV genome detection should help to control the transmission of HCV in the unit under study.

Malaria serology: performance of six Plasmodium falciparum antigen extracts and of three ways of determining serum titers in IgG and IgM-ELISA

The study evaluated six Plasmodium falciparum antigen extracts to be used in the IgG and IgM enzyme-linked immunosorbent assays (ELISA), for malaria diagnosis and epidemiological studies. Results obtained with eighteen positive and nine negative control sera indicated that there were statistically significant differences among these antigen extracts (Multifactor ANOVA, p< 0.0001). Urea, sodium deoxycholate and Zwittergent antigen extracts performed better than did the three others, their features being very similar for the detection of IgG antibodies. Urea, alkaline and sodium deoxycholate antigen extracts proved to be better than the others for the detection of IgM antibodies. A straight line relationship was found between the optical densities (or their respective log 10) and the log 10 of antibody dilutions, with a very constant slope. Thus serum titers could be determined by direct titration and by two different equations, needing only one serum dilution. For IgM antibody detections, log 10 expression gave results that better correlated with direct titration (95% Bonferroni). For IgG antibody detections, the titer differences were not significant. The reproducibility of antibody titers and antigen batches was also evaluated, giving satisfactory results.

Alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) applied to dot-ELISA

The alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) was assessed in dot-ELISA for the diagnosis of Chagas' disease. Serum samples (355) from chagasic and non-chagasic patients were studied, and IgG antibodies to ASEA were found in all patients with chronic Chagas' disease. In non-chagasic patients 95.6% were negative, except for those with leishmaniasis (visceral and mucocutaneous), and some patients from control group reacted in low titers. The data indicate that dot-ELISA using ASEA is suitable for seroepidemiologic surveys to be employed in endemic areas for Chagas' disease.

Schistosoma mansoni: identification of a 46KDa antigen of the schistosomular surface by monoclonal antibody

An IgG2a subclass monoclonal antibody, C6G9, was obtained by immunization of BALB/c mice with Schistosoma mansoni egg antigens. With this monoclonal antibody, it was possible to identify a schistosomular antigen with a molecular weight of 46 kilodaltons (KDa), and its expression being evaluated by means of indirect immunofluorescence. The antigen persisted in the integument of the developing schistosomulum, for at least 96 hours post-transformation. The monoclonal antibody also reacted with the cercaria surface, but not with that of adult worm. The C6G9 was also able to mediate significant levels of cytotoxicity in the presence of complement for newly transformed schistosomula.

Pattern of mycobacterial antigen detection in leprosy

Immunohistochemistry reaction (Peroxidase anti-peroxidase - PAP) was carried out on fifty-two skin biopsies from leprosy patients with the purpose to identify the antigenic pattern in mycobacteria and to study the sensitivity of this method. Five different patterns were found: bacillar, granular, vesicular, cytoplasmatic and deposits, classified according to the antigenic material characteristics. Deposits (thinely particulate material) appeared more frequently, confirming the immunohistochemistry sensitivity to detect small amounts of antigens even when this material is not detected by histochemical stainings.

Leishmania braziliensis: isolation of carbohydrate-containing antigen and possibility of its use in the immunodiagnosis of American Cutaneous Leishmaniasis

Leishmania braziliensis is a causative agent of American Cutaneous Leishmaniasis (ACL). The 034-JCG strain, isolated from a patient from the northern region of Paraná State, Brazil, was cultivated in Blood Agar Base medium, lyophilized and submitted to phenol-water extraction. The extract was treated with RNase I. The carbohydrate containing-antigen (Ag-CHO) was immunogenic to rabbits and showed at least a fraction with some negative charge at pH 8.2. This antigen showed cross-reactivity with the phenol-water extract of the growth medium used for the culture of promastigotes and with the surface antigens of promastigotes. Its composition is: 24.3% of total sugars, from which 11.2% of galactose, 7.5% of mannose and 5.6% of ribose. Protein content was 5.4% and phosphate 18.5%. The antigenic activity was maintained after: repeated freezing-thawing; lyophilization; heating at 100ºC for 30 minutes; treatment with RNase, trichloroacetic acid and sodium metaperiodate. The precipitin line obtained is Periodic Acid Schiff positive. The application of the Ag-CHO in counterimmunoelectrophoresis reaction for the immunodiagnosis of ACL showed 60% sensitivity, and no cross-reaction with the five sera of Chagas' disease patients tested. The use of this antigen in a more sensitive technique, with more samples of sera, may improve these results.

Bacterial antigen detection in cerebrospinal fluid by the latex agglutination test

Eighty purulent cerebrospinal fluid (CSF) samples from patients with clinical evidence of meningitis were studied using the Directigen latex agglutination (LA) kit to determine the presence of bacterial antigen in CSF. The results showed a better diagnostic performance of the LA test than bacterioscopy by Gram stain, culture and counterimmunoelectrophoresis (CIE), as far as Neisseria meningitidis groups B and C, and Haemophilus influenzae type b are concerned, and a better performance than bacterioscopy and culture considering Streptococcus pneumoniae. Comparison of the results with those of culture showed that the LA test had the highest sensitivity for the Neisseria meningitidis group C. Comparing the results with those of CIE, the highest levels of sensitivity were detected for N. meningitidis groups B and C. Regarding specificity, fair values were obtained for all organisms tested. The degree of K agreement when the LA test was compared with CIE exhibited better K indices of agreement for N. meningitidis groups B and C.

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.

Detection of Cryptococcus neoformans capsular polysaccharide antigen in asymptomatic HIV-infected patients

Serum samples from 242 HIV-positive persons were studied for the detection of capsular polysaccha-ride antigen of Cryptococcus neoformans; 193 of these patients presented less than 300 CD4+ cells/µl of blood and 49 patients had more than 300 CD4+ cells/µl. None of them had symptoms or signs characteristic of cryptococcosis. The capsular antigen of C. neofarmans was detected by latex agglutination technique with pronase pre-treatment (IMMY, Crypto-Latex Antigen Detection System, Immunomycologics Inc., OK, USA); in 61% of the samples, ELISA technique was also used (Premier, Cryptococcal Antigen, Meridian Diagnostic Inc., Cincinatti, Oh, USA). The comparative study of both methods showed that the results obtained were similar in 96.9% of the cases. The capsular antigen was detected in 13 out of 193 (6.7%) patients with less than 300 CD4+ cells/µl. Cryptococcosis was confirmed mycologically in 3 of these 13 cases (23%) by the isolation of C. neoformans in CSF or blood cultures. Three patients, who had presented negative results of both tests for capsular antigen, suffered disseminated cryptococcosis 4 to 8 months later. The predictive diagnostic value of capsular antigen detection of C. neoformans seems tobe low and we believe that it should not be done routinely in asymptomatic HIV-positive persons.

Fungal infections in neutropenic patients: a 8-year prospective study

In this paper we report a eight-year prospective study designed to further characterize incidence, epidemiology, specific syndromes, treatment and prognosis associated with fungal infections in neutropenic patients. During the study period 30 fungal infections were diagnosed in 30 patients among 313 episodes of fever and neutropenia (10%). There were 15 cases of candidiasis, 5 pulmonary aspergillosis, 3 sinusitis by Aspergillus fumigatus, 5 infections by Fusarium sp., one infection by Trichosporon sp., and one infection due to Rhodotorula rubra. Blood cultures were positive in 18 cases (60%). The predisposing factors for fungal infection in multivariate analysis were the presence of central venous catheter (p<0.001), longer duration of profound (<100/mm³) neutropenia (p<0.001), the use of corticosteroids (p<0.001), gram-positive bacteremia (p=0.002) and younger age (p=0.03). In multivariate analysis only recovery of the neutropenia (p<0.001) was associated with good prognosis whereas the diagnosis of infection by Fusarium sp. (p=0.006) was strongly associated with a poor outcome. The death rate was 43%. There was no statistically significant difference in the death rate between patients who did receive (52%) or did not receive (50%) antifungal treatment. Identifying patients at risk, specific syndromes and prognostic factors may help to reduce the high mortality associated with disseminated fungal infections in neutropenic patients.