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.

Entamoeba histolytica: fecal antigen capture immunoassay for the diagnosis of enteric amebiasis by a monoclonal antibody

Amebiasis continues to be of epidemiological importance in underdeveloped countries. Clinical diagnosis and epidemiological setting in a region are based on the fecal microscopic identification of cysts or trophozoites. This procedure requires well trained personnel, is laborious, of low sensitivity and frequently yields false-positives results. The present study was designed to develop an immuno-enzymatic fecal 96 kDa antigen capture test (COPROELISA-Eh) more sensitive and specific than microscopic diagnosis of amebiasis. Triplicates of 177 stool samples processed by the formol-ether concentration method, were defined as positive or negative by three experienced microscopic observers. Another aliquot was submitted to the antigen capture test by a monoclonal antibody against a specific membrane antigen of pathogenic strains of Entamoeba histolytica. Optical densities were interpreted as positive when they exceeded the mean value of negative samples plus two standard deviations. COPROELISA-Eh showed a 94.4% sensitivity, 98.3% specificity, 96.2% positive predictive value and 97.6% negative predictive value for the detection of E. histolytica in feces. COPROELISA-Eh is more sensitive and specific than microscopic examination, does not require specially trained personnel and allows the simultaneous processing of a large number of samples.

Immune responses induced by a Leishmania (Leishmania) amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects

In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L.(L.) amazonensis and L. (Viannia) braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10) against L. (L.) amazonensis after vaccination using Bacille Calmette-Guerin (BCG) as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-g, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasis

Histoplasmin reaction. Comparison of a polysaccharide antigen to the filtrate antigen

This work was planned by taking into account all the knowledge accumulated from the immunological study of paracoccidioidomycosis. It aimed at comparing a polysaccharide antigen from Histoplasma capsulatum to a classic histoplasmin with the help of intradermal tests of delayed type of hypersensitivity. Tests were applied to 115 individuals in Santo Amaro, a town in the state of São Paulo. Positive results using classic histoplasmin were obtained in 46.0% cases whereas positive results using the polysaccharide antigen at its hightest concentration were obtained in 51.30% cases. The major conclusion in this investigation is that it is possible to use the polysaccharide antigen as histoplasmin instead of the filtrate antigen

Cryo-Microtome sections of coproculture larvae of Strongyloides stercoralis and Strongyloides ratti as antigen sources for the immunodiagnosis of human strongyloidiasis

Cryo-microtome sections of larvae of Strongyloides stercoralis and S. ratti respectively obtained from human and rat feces cultures, were used as antigens. Fluoresceinate conjugates against human IgG were employed at the ideal titer of 10 for S. stercoralis and 100 for S. ratti. The sensitivity of the indirect immunofluorescence reaction (IIF) was 94.4% and 92.5% and the specificity 94.2% and 97.1% for the two specific larval antigens, respectively. Sera from 123 persons (54 from carriers of S. stercoralis infections and 69 from controls) were submitted to the reaction. The titers of different sera varied from 20 to 2560. There was a significant linear correlation (r = 0.85 p £ 0.001) between the antibodies from the two species of larval antigens. We conclude that both antigens may be used in the IIF reaction for the diagnosis of human strongyloidiasis. Due to the feasibility of safe and low-cost mass production of S. ratti larvae in the laboratory with a considerable economy of conjugate, their utilization in the serum diagnosis of human strongyloidiasis is recommended

Presence of Circulating Levels of Interferon-g, Interleukin-10 and Tumor Necrosis Factor-a in Patients with Visceral Leishmaniasis

Experimental murine L. major infection is characterized by the expansion of distinct CD4+ T cell subsets. The Th1 response is related to production of IFN-g and resolution of infection, whereas Th-2 response with production of IL-4 and IL-10 and dissemination of infection. The objective of this study was to measure the circulating levels of IFN-g, IL-10 and TNF-a in patients with visceral leishmaniasis (VL) before, during and at the end of therapy and to examine the association between cytokine levels and activity of VL. Fifteen patients with VL were evaluated. The cytokine determinations were done by using the enzyme-linked immunoassay (ELISA) before, during and at the end of therapy. At baseline, we detected circulating levels of IFN-g in 13 of 15 patients (median = 60 pg/ml); IL-10 in 14 of 15 patients (median = 141.4 pg/ml); and TNF-a in 13 of 14 patients (median = 38.9 pg/ml). As patients improved, following antimonial therapy, circulating levels of IL-10 showed an exponential decay (y = 82.34 e–0,10367x, r = –0.659; p < 0.001). IFN-g was no longer detected after 7/14 days of therapy. On the other hand, circulating levels of TNF-a had a less pronounced decay with time on therapy, remaining detectable in most patients during the first seven days of therapy (y = 36.99-0.933x, r = –0.31; p = 0.05). Part of the expression of a successful response to therapy may, therefore, include reduction in secretion of inflammatory as well as suppressive cytokines. Since IL-10 and IFN-g are both detected prior to therapy, the recognized cellular immune depression seen in these patients may be due to biological predominance of IL-10 (type 2 cytokine), rather than lack of IFN-g (type 1 cytokine) production.

RESEARCH OF ANTIGEN AND ANTIBODIES FROM RETROVIRUSES, CMV AND HBV AMONG PRISONERS OF THE PENITENTIARY COMPLEX OF THE REGION OF CAMPINAS, SP, BRAZIL

Some viruses of the families Retroviridae, such as Human T Lymphotropic Virus (HTLV); Herpesviridae as the Cytomegalovirus (CMV) and Hepadnaviridae such as the Hepatitis B Virus (HBV) are liable to be co-transmitted with the Human Immunodeficiency Virus (HIV). Since prisoners are exposed to several and important risk factors involved in the transmission of HIV and the above mentioned viruses, male inmates from the penitentiary complex of Campinas, SP, Brazil, including HIV + and HIV - ones, were examined for the presence of HTLV-I and/or II antibodies; IgG and IgM anti-CMV antibodies, and the research of the superficial hepatitis B antigen (HBsAg). The presence of anti-HTLV-I and/or II was determined by the Western Blot (WB) technique, whereas IgG and IgM anti-CMV and the search of HBsAg were carried out by the Microparticle Enzyme Immunoassay (MEIA-Abbott Lab).With regard to anti-HTLV-I and/or II, 58.3% (14/24-Number of positive reactions/number of sera examined) were reactive among the anti-HIV positive sera. Conversely, only 12.5% (3/24) among the HIV- negative sera showed positive reactions to HTLV-I and/or II antibodies. When looking for IgG anti-CMV percentages of 97.7% (43/44) and 95% (38/40) were obtained for anti-HIV positive and negative sera, respectively. As to IgM anti-CMV antibodies 11.36% (5/44) and 2.5% (1/40) of reactive sera were found for anti-HIV positive and negative, respectively. The HBsAg was found in 12.8% (5/39) of the sera which were anti-HIV positive.

OBTENTION OF RABIES ANTIGEN THROUGH BHK21 CELLS ADHERED TO MICROCARRIERS

Four rabies antigen batches were produced from virus suspensions resulting from BHK21 cells adhered to microcarriers (Cytodex 1), inoculated and cultured in a bioreactor. In parallel the methodology of production of rabies virus through cultures of BHK21 cells in monolayers in bottles was used. The results obtained showed that infecting titles were 106.69 DL50/mL and 107.28 DL50/mL for suspensions cultured in bottles and in the bioreactor, respectively. The viral suspension volumes collected were on average 11,900 per batch from the bioreactor and 800mL per bottle. Ten horses were immunized with the antigen produced in the bioreactor. The means of antirabies antibody titers found were 240 and 212 IU/mL after the initial and the first booster doses, respectively. Rabies antigen with satisfactory infecting titers can be obtained on a large scale by culturing in a bioreactor inoculated BHK21 cells adhered to microcarriers.

STANDARDIZATION OF PROCEDURES OF Plasmodium falciparum ANTIGEN PREPARATION FOR SEROLOGIC TESTS

The objective of the present study is to standardize the technical variables for preparation and storage of Plasmodium falciparum and of antigen components extracted with the amphoteric detergent Zwittergent. P. falciparum obtained from in vitro culture was stored at different temperatures and for different periods of time. For each variable, antigen components of the parasite were extracted in the presence or absence of protease inhibitors and submitted or not to later dialysis. Products were stored for 15, 30 and 60 days at different temperatures and immunological activity of each extract was determined by SDS-PAGE and ELISA using positive or negative standard sera for the presence of IgG directed to blood stage antigens of P. falciparum. Antigen extracts obtained from parasites stored at -20oC up to 10 days or at -70oC for 2 months presented the best results, showing well-defined bands on SDS-PAGE and Western blots and presenting absorbance values in ELISA that permitted safe differentiation between positive and negative sera.

Characterization of rotavirus P genotypes circulating among paediatric inpatients in Northern Brazil

Between November 1992 and August 1993, twenty-eight rotavirus-positive stool samples obtained from paediatric inpatients in Belém, Brazil, aged less than four years, were tested by RT-PCR to determine the P genotype specificities. With the exception of 7 non-diarrhoeic children, all patients were either diarrhoeic at admission or developed diarrhoea while in hospital. Rotavirus strains with the gene 4 alleles corresponding to P1B[4] and P1A[8] types (both of which bearing G2 specificity) predominated, accounting for 78.6% of the strains. While only one P2A[6] type strain - with (mixed) G1 and 4 type specificities - was detected, the gene 4 allele could not be identified in 4 (14.3%) of the strains. Most (81%) of the specimens were obtained from children during their first 18 months of life. Rotavirus strains bearing single P1B[4] type-specificity were identified in both diarrhoeic (either nosocomial, 28.6% or community-acquired diarrhoea, 28.6%) and non-diarrhoeic (42.8%) children. P1A[8] gene 4 allele, on the other hand, was detected only among diarrhoeic children, at rates of 57.1% and 42.9% for nosocomial- and- community acquired diarrhoea, respectively. Mixed P1A[8],1B[4] type infection was identified in only one case of community-acquired diarrhoea.

Atypical disseminated cutaneous histoplasmosis in an immunocompetent child, caused by an "aberrant" variant of Histoplasma capsulatum var. capsulatum

A case of atypical disseminated cutaneous histoplasmosis in a five-year old, otherwise healthy child, native and resident in São Paulo metropolitan area is reported. Cutaneous lesions were clinically atypical. Histologic examination disclosed a granulomatous reaction but no fungal structures could be demonstrated by specific staining nor by immunohistochemical reaction. The fungus was isolated from biopsy material on two different occasions, confirming diagnosis of an unusual fungal infection. The fungus, originally thought to be a Sepedonium sp. due to the large sized, hyaline or brownish colored tuberculated macroconidia and to lack of dimorphism (yeast form at 37 °C) produce H and M antigens, visualized by the immunodiffusion with rabbit anti-Histoplasma capsulatum hyperimmune serum. Patient’s serum sample was non reactive with H. capsulatum antigen by immunodiffusion, counterimmunoelectrophoresis and complement fixation tests, and immunoenzymatic assay failed to detect the specific circulating antigen. This serum was tested negative by double immunodiffusion when antigen obtained from one of the isolated samples was used. Both cultures were sent to Dr. Leo Kaufman, Ph.D. (Mycoses Immunodiagnostic Laboratory, CDC-Atlanta/USA), who identified them as H. capsulatum by the exoantigen and gen-probe tests. Both clinic and mycologic characteristics of the present case were atypical, suggesting the fungus isolated is an “aberrant variant” of H. capsulatum var. capsulatum, as described by SUTTON et al. in 199719. Treatment with itraconazole 100 mg/day led to cure within 90 days

Detection of Toxoplasma gondii soluble antigen, SAG-1(p30), antibody and immune complex in the cerebrospinal fluid of HIV positive or negative individuals

Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45% of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19% of the CSF samples and 62% were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.