Atypical rotavirus among diarrhoeic children living in Belém, Brazil

Atypical rotaviruses were detected in faeces from two diarrhoeic children living in Belém, Pará, Brazil. Rotavirus particles were detected by electron microscopy and the RNA electrophoresis showed patterns which were compatible with group C rotaviruses. Tests for the presence of group A antigen by enzyme-linked-immunosorbent assay (ELISA) were negative. The two children had three successive rotavirus infection and in both cases the atypical strains were excreted at the time of the third infection, causing a mild and short-lasting disease.

Prevalence of human T cell leukemia virus-I (HTLV-I) antibody among populations living in the Amazon region of Brazil (preliminary report)

Forty-tree (31.4%) out of 137 serum samples obtained from two Indian communities living in the Amazon region were found to be positive for HTLV-I antibody, as tested by enzyme-linked immunosorbent assay (Elisa). Eighty-two sera were collected from Mekranoiti Indians, yielding 39% of positivity, whereas 11 (20.0%) or the 55 Tiriyo serum samples had antibody to HTLV-I. In addition, positive results occurred in 10 (23.2%) out of 43 sera obtained from patients living in the Belem area, who were suffering from cancer affecting different organs. Five (16.7%) out of 30 Elisa positive specimens were also shown to be positive by either Western blot analysis (WB) or indirect immunogold electron microscopy (IIG-EM).

Prevalence of Trypanosoma cruzi infection in blood banks of seven departments of Bolivia

Trypanosoma cruzi infection was studied in 1,298 sera samples of blood banks from 7 capital departments of Bolivia, using the immunofluorescence test (IFI) and Enzyme Linked Immunosorbent Assay (Elisa). The percentages of positivity in these 7 departments have an average of 28% and are distributed as follows: Sta. Cruz 51%, Tarija 45%, Cochabamba 28%, Sucre 39%, La Paz 4.9%, Oruro 6% and Potosi 24%. The prevalence is related with the altitude levels of the different departments. However in Potosi (3,945 m) we found a 24% of prevalence, probably due to the proximity of endemic valleys to the city. The authors suggest a strict control in blood donors since there exists a great risk of infection

Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production.

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.

Marine biotoxins in the Catalan littoral: could biosensors be integrated into monitoring programmes?

Aquest article descriu els sensors enzimàtics i immunosensors electroquímics que s’han desenvolupat als nostres grups per a la detecció de la biotoxina marina àcid okadaic (OA), i discuteix la possibilitat d’integrar-los en programes de seguiment. Els sensors enzimàtics per a OA que es presenten es basen en la inhibició de la proteïna fosfatasa (PP2A) per aquesta toxina i la mesura electroquímica de l’activitat enzimàtica mitjançant l’ús de substrats enzimàtics apropiats, electroquímicament actius després de la seva desfosforació per l’enzim. Els immunosensors electroquímics descrits en aquest article es basen en un enzimoimmunoassaig sobre fase sòlida competitiu indirecte (ciELISA), amb fosfatasa alcalina (ALP) o peroxidasa (HRP) com a marcatges, i un sistema de reciclatge enzimàtic amb diaforasa (DI). Els biosensors presentats aquí s’han aplicat a l’anàlisi de dinoflagel·lats, musclos i ostres. Les validacions preliminars amb assaigs colorimètrics i LC-MS/MS han demostrat la possibilitat d’utilitzar les bioeines desenvolupades per al cribratge preliminar de biotoxines marines en mostres de camp o de cultiu, que ofereixen informació complementària a la cromatografia. En conclusió, tot i que encara cal optimitzar alguns paràmetres experimentals, la integració dels biosensors a programes de seguiment és viable i podria proporcionar avantatges respecte a altres tècniques analítiques pel que fa al temps d’anàlisi, la simplicitat, la selectivitat, la sensibilitat, el fet de poder ser d’un sol ús i l’efectivitat de cost. This article describes the electrochemical enzyme sensors and immunosensors that have been developed by our groups for the detection of marine biotoxin okadaic acid (OA), and discusses the possibility of integrating them into monitoring programmes. The enzyme sensors for OA reported herein are based on the inhibition of immobilised protein phosphatase 2A (PP2A) by this toxin and the electrochemical measurement of the enzyme activity through the use of appropriate enzyme substrates, which are electrochemically active after dephosphorylation by the enzyme. The electrochemical immunosensors described in this article are based on a competitive indirect Enzyme- Linked ImmunoSorbent Assay (ciELISA), using alkaline phosphatase (ALP) or horseradish peroxidase (HRP) as labels, and an enzymatic recycling system with diaphorase (DI). The biosensors presented herein have been applied to the analysis of dinoflagellates, mussels and oysters. Preliminary validations with colorimetric assays and LC-MS/MS have demonstrated the possibility of using the developed biotools for the preliminary screening of marine biotoxins in field or cultured samples, offering complementary information to chromatography. In conclusion, although optimisation of some experimental parameters is still required, the integration of biosensors into monitoring programmes is viable and may provide advantages over other analytical techniques in terms of analysis time, simplicity, selectivity, sensitivity, disposability of electrodes and cost effectiveness.

Evaluation of three serological tests for the detection of human plague in northeast Brazil

The passive haemagglutination (PHA) test, enzyme-linked immunosorbent assay (ELISA) and the dot enzyme-immunosorbent assay (DOT-ELISA) were used to detect the levels of IgG antibodies against the Fraction 1 (F1) antigen of Yersinia pestis in sera of plague-infected patients from Northeast Brazil. Twenty three selected PHA-positive sera of subjects with bacteriological confirmation of plague were also positive in the DOT-ELISA but only 19 were detected by the conventional ELISA technique. Another group of 186 serum samples from subjects diagnosed as plague-infected by clinical and epidemiological parameters, but PHA-negative, were screened with DOT-ELISA and 11 gave positive results. The specificity of the assays on the serological detection of plague was confirmed in inhibition tests using purified F1 antigen. These results suggest that DOT-ELISA can be an useful, simple and more sensitive alternative for the serodiagnosis of plague in Northeast Brazil.

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

A high-throughput test to detect C.E.R.A. doping in blood.

C.E.R.A., a continuous erythropoietin receptor activator, is a new third-generation erythropoiesis-stimulating agent (ESA) that has recently been linked with abuse in endurance sports. In order to combat this new form of doping, we examined an enzyme-linked immunosorbent assay (ELISA) designed to detect the presence of C.E.R.A. in serum samples. The performance of the assay was evaluated using a pilot excretion study that involved six subjects receiving C.E.R.A. Validation data demonstrated an excellent reproducibility and ensured the applicability of the assay for anti-doping purposes. To maximize the chances of detecting the drug in serum samples, we propose the use of this specific ELISA test as a high-throughput screening method, combined with a classic isoelectric focusing test as a confirmatory assay. This strategy should make C.E.R.A. abuse relatively easy to detect, thereby preventing the future use of this drug as a doping agent.

Visceral leishmaniasis in Teresina, state of Piauí, Brazil: preliminary observations on the detection and transmissibility of canine and sandfly infections

A Leishmania donovani-complex specific DNA probe was usedto confirm the widespread dissemination of amastigotes in apparently normal skinof dogs with canine visceral leishmaniasis. When Lutzomyia longipalpis were fed on abnormal skin of five naturally infected dogs 57 of 163 (35 per cent) fliesbecame infected: four of 65 flies (6 per cent) became infected when fed on apparently normal skin. The bite of a single sandfly that had fed seven days previouslyon a naturally infected dog transmitted the infection to a young dog from a non-endemic area. Within 22 days a lesion had developed at the site of the infectivebite (inner ear): 98 days after infection organisms had not disseminated throughout the skin, bone marrow, spleen or liver and the animal was still serologically negative by indirect immunofluorescence and dot-enzyme-linked immunosorbent assay. When fed Lu. longipalpis were captured from a kennel with a sick dog known to be infected, 33 out of 49 (67 per cent) of flies contained promastigotes. In contrast only two infections were detected among more than 200 sandflies captured in houses. These observations confirm the ease of transmissibility of L.chagasi from dog to sandfly to dog in Teresina. It is likely that canine VL is the major source of human VL by the transmission route dog-sandfly-human. the Lmet2 DNA probe was a useful epidemiological tool for detecting L. chagasi in sandflies.

Naturally acquired antibodies against the major merozoite surface coat protein (MSP-1) of Plasmodium falciparum acquired by residents in an endemic area of Colombia

A preliminary baseline epidemiological malaria survey was conducted in the village of Punta Soldado, Colombia. Parasite prevalence and density as well as serological data were obtained from 151 asymptomatic children and adults. Fifty individuals were infected with Plasmodium falciparum. The mean parasite density was 184 parasites/mm3. Greater than 90 of the sample population were P. falciparum antibody positive as detected by the indirect immunofluorescent antibody test (IFAT). The enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against the major merozoite surface protein (MSP-1) of P. falciparum. In this population, anti-MSP-1 antibody concentration is acquired in an age dependent manner with equal immunogenicity to both the N- and C-terminal regions of the molecule. Infection at the time of sampling was associated with a higher anti-MSP-1 antibody concentration than that found in non-infected individuals. Further studies are planned to assess the role of immune and non-immune factors in limiting the number of cases of severe malaria seen in this population.

Validity of serology for American Trypanosomiasis with eluates from filter paper

This study evaluates whether blood collected on filter paper kept at 4 degrees C and tested at different intervals of time (1, 7, 15, 30 and 60 days after collection) would present similar results when compared to the serum samples and whether the type of filter paper influences the results. Eluates from filter paper samples were tested for Trypanosoma cruzi antibodies using indirect immunofluorescence antibody test (IFAT), indirect haemagglutination (IHA) and enzyme-linked immunosorbent assay (ELISA) as reference, the antibody titer in sera. Analysis of data showed that results obtained with IFAT, IHA (cut off point = 1:40) and ELISA in sera had similar sensitivity and good concordance among reactions. The use of a multiple linear regression model indicated that titer fall in eluates occurs up to the 7th day after the collection, and it is more marked for samples with lower antibodies titers. However, no significant differences were observed by IFAT, IHA (cut off point = 1:20) and ELISA in the proportion of positive reactions between sera and eluates. The results also showed that Melitta, Klabin or Whatman (reference) filter papers could be indicated for surveys, since they have shown similar capacity of maintenance of anti-T. cruzi immunoglobulins.

Rotaviruses as a cause of nosocomial, infantile diarrhoea in Northern Brazil: pilot study

Faecal samples were obtained from 190 children, aged 0 to 5 years, admitted to a public hospital in Belém, Pará, Brazil. These patients were placed in a pediatric ward with 40 beds distributed in six rooms. Case were classified into three groups: (a) nosocomial: children who developed gastroenteritis 72 hr or later after admission; (b) community-acquired: patients admitted either with diarrhoea or who had diarrhoea within 72 hr following admission; (c) non-diarrhoeic: those children who had no diarrhoea three days before and three days after collection of formed faecal sample. Specimens were routinely processed for the presence of rotaviruses, bacteria and parasites. Rotaviruses were detected through enzyme-linked immunosorbent assay (ELISA) and subsequently serotyped/electrophoretyped. Rotaviruses were the most prevalent enteropathogens among nosocomial cases, accounting for 39 % (9/23) of diarrhoeal episodes; on the other hand, rotaviruses ocurred in 8.3 % (11/133) and 9 % (3/34) of community-acquired and non-diarrhoeic categories, respectively. Mixed infections involving rotavirus and Giardia intestinalis and rotavirus plus G. intestinalis and Entamoeba histolytica were detected in frequencies of 8.6 and 4.3 %, respectively, in the nosocomial group. The absence of bacterial pathogens in this category, and the unusual low prevalence of these agents in the other two groups may reflect the early and routine administration of antibiotics following admission to this hospital. Rotavirus serotype 2 prevailed over the other types, accounting for 77.8 % of isolates from nosocomial diarrhoeal episodes. In addition, at least five different genomic profiles could be observed, of which one displayed an unusual five-segment first RNA cluster. Dehydration was recorded in all cases of hospital-acquired, rotavirus-associated diarrhoea, whereas in only 57 % of nosocomial cases of other aetiology. It was also noted that nosocomial, rotavirus-associated diarrhoeal episodes occur earlier (7 days), following admission, if compared with those hospital-acquired cases of other aetiology (14 days).

Use of the 2,3-diacyl-trehalose and the purified protein derivative in the serodiagnosis of tuberculosis in AIDS

The effect of the human immunodeficiency virus (HIV) infection on IgG production against purified protein derivative (PPD) and 2,3-diacil-trehalose (SL-IV) was investigated by an enzyme-linked immunosorbent assay (ELISA) test. Comparison between the antigens showed that immunocompetent patients produce preferentially antibodies to SL-IV than to PPD (73.3% versus 63.3%). Combination of these results showed an increase of the sensitivity to 80%, which decreased over the spectrum of immunodepression caused by HIV. In the tuberculous HIV seropositive group the sensitivities of SL-IV and PPD were 36.4% versus 40% and 0% versus 22.2% in the tuberculosis/acquired immunodeficiency syndrome (TB/AIDS) group. Combination of these results gave respectively 54.5% and 20%, showing that serological tests have limited value for diagnosis of tuberculosis in HIV infected patients. High antibody levels were observed in HIV seropositive asymptomatic group, but only two individuals were positive for both antigens. In the follow up, one of them developed tuberculous lymphadenitis, indicating that further work is needed to access the value of serological tests in predicting tuberculosis in HIV infected individuals.

The use of polyvinyl alcohol glutaraldehyde as solid-phase in ELISA for plague

Discs of polyvinyl alcohol cross-linked with glutaraldehyde were synthesized under acid catalysis (H2SO4). Then, the antigen F1 purified from Yersinia pestis was covalently linked to this modified polymer. Afterwards, an enzyme-linked immunosorbent assay (ELISA) was established for the diagnosis of plague in rabbit and human. The best conditions for the method were achieved by using 1.3 ¼g of F1 prepared in 0.067 M phosphate buffer, pH 7.2, containing 1 M NaCl (PBS); anti-IgG peroxidase conjugate diluted 6,000 times and as a blocking agent 3% w/v skim milk in PBS. The titration of positive rabbit serum according to this procedure detected antibody concentrations up to 1:12,800 times. The present method, the conventional ELISA and passive haemagglutination assay are compared.

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.