Development and Validation of a Novel Diagnostic Test for Human Brucellosis Using a Glyco-engineered Antigen Coupled to Magnetic Beads.

Brucellosis is a highly contagious zoonosis and still a major human health problem in endemic areas of the world. Although several diagnostic tools are available, most of them are difficult to implement especially in developing countries where complex health facilities are limited. Taking advantage of the identical structure and composition of the Brucella spp. and Yersinia enterocolitica O:9 O-polysaccharide, we explored the application of a recombinant Y. enterocolitica O:9-polysaccharide-protein conjugate (OAg-AcrA) as a novel antigen for diagnosis of human brucellosis. We have developed and validated an indirect immunoassay using OAg-AcrA coupled to magnetic beads. OAg-AcrA was produced and purified with high yields in Y. enterocolitica O:9 cells co-expressing the oligosaccharyltransferase PglB and the protein acceptor AcrA of Campylobacter jejuni without the need for culturing Brucella. Expression of PglB and AcrA in Y. enterocolitica resulted in the transfer of the host O-polysaccharide from its lipid carrier to AcrA. To validate the assay and determine the cutoff values a receiver-operating characteristic analysis was performed using a panel of characterized serum samples obtained from healthy individuals and patients of different clinical groups. Our results indicate that, using this assay, it is possible to detect infection caused by the three main human brucellosis agents (B. abortus, B. melitensis and B. suis) and select different cutoff points to adjust sensitivity and specificity levels as needed. A cutoff value of 13.20% gave a sensitivity of 100% and a specificity of 98.57%, and a cutoff value of 16.15% resulted in a test sensitivity and specificity of 93.48% and 100%, respectively. The high diagnostic accuracy, low cost, reduced assay time and simplicity of this new glycoconjugate-magnetic beads assay makes it an attractive diagnostic tool for using not only in clinics and brucellosis reference laboratories but also in locations with limited laboratory infrastructure and/or minimally trained community health workers.Fil: Ciocchini, Andres Eduardo. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina;Fil: Rey Serantes, Diego A.. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina;Fil: Melli, Luciano Jorge. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina;Fil: Iwashkiw, Jeremy A.. University of Alberta . Department of Biological Sciences . Alberta Glycomics Centre; Estados Unidos de América;Fil: Deodato, Bettina. Hospital Múñiz. Unidad de Enfermedades Infecciosas; Argentina;Fil: Wallach, Jorge. Hospital Múñiz. Unidad de Enfermedades Infecciosas; Argentina;Fil: Feldman, Mario F. University of Alberta . Department of Biological Sciences . Alberta Glycomics Centre; Estados Unidos de América;Fil: Ugalde, Juan E. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina;Fil: Comerci, Diego J. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina

Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins

Background: TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3–5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen. Methods/Principle findings: Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis. Conclusion/Significance: This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications.Fil: Lobo, Mabel Maite. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Balouz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Melli, Luciano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Carlevaro, Giannina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Cortina, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Camara, María de los Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Canepa, Gaspar Exequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Carmona, Santiago Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Altcheh, Jaime Marcelo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Campetella, Oscar Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ciocchini, Andres Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Agüero, Fernan Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Mucci, Juan Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Development and evaluation of a novel VHH-based immunocapture assay for high-sensitivity detection of Shiga toxin type 2 (Stx2) in stool samples

Shiga toxin (Stx)-producing Escherichia coli (STEC) is the main cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening clinical complication characterized by hemolytic anemia, thrombocytopenia, and acute renal failure that mainly affects children. A relevant feature of STEC strains is the production of Stx, and all of them express Stx1 and/or Stx2 regardless of the strain serotype. Therefore, Stx detection assays are considered the most suitable methods for the early detection of STEC infections. Single-domain antibodies from camelids (VHHs) exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis. In this work, we have exploited VHH technology for the development of an immunocapture assay for Stx2 detection. Thirteen anti- Stx2 VHHs previously obtained from a variable-domain repertoire library were selected and evaluated in 130 capture-detection pair combinations for Stx detection. Based on this analysis, two VHHs were selected and a double VHH-based biotinstreptavidin capture enzyme-linked immunosorbent assay (ELISA) with spectrophotometric detection was developed and optimized for Stx2 detection. This assay showed an excellent analytical and clinical sensitivity in both STEC culture supernatants and stool samples even higher than the sensitivity of a commercial ELISA. Furthermore, based on the analysis of stool samples, the VHH-based ELISA showed high correlation with stx2 detection by PCR and a commercial rapid membrane-based immunoassay. The intrinsic properties of VHHs (high target affinity and specificity, stability, and ease of expression at high yields in recombinant bacteria) and their optimal performance for Stx detection make them attractive tools for the diagnosis of HUS related to STEC (STEC-HUS).Fil: Melli, Luciano Jorge. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Zylberman, Vanesa. Inmunova; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hiriart, Yanina. Inmunova; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lauche, Constanza E.. Inmunova; ArgentinaFil: Baschkier, Ariela. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Pardo, Romina. Inmunova; ArgentinaFil: Miliwebsky, Elizabeth. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Chinen, Isabel. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Rivas, Marta. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Goldbaum, Fernando A.. Inmunova; ArgentinaFil: Ugalde, Juan Esteban. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Comerci, Diego José. Inmunova; Argentina. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ciocchini, Andres Eduardo. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

Improving bioreactor production of a recombinant glycoprotein in Escherichia coli: Effect of specific growth rate on protein glycosylation and specific productivity

In the last decades bacterial glycoengineering emerged as a new field as the result of the ability to transfer the Campylobacter jejuni N- glycosylation machinery into Escherichia coli for the production of recombinant glycoproteins that can be used as antigens for diagnosis, vaccines, and therapeutics. However, the identification of critical parameters implicated in the production process and its optimization to jump to a productive scale is still required. In this study, we developed a dual expression glycosylation vector for the production of the recombinant glycoprotein AcrA-O157, a novel antigen that allows the serodiagnosis of the infection with enterohemorrhagic E. coli O157 in humans. Volumetric productivity was studied in different culture media and found that 2xYP had 6.9-fold higher productivity than the extensively used LB. Subsequently, bioreactor batch and exponential-fed-batch cultures were designed to determine the influence of the specific growth rate (μ) on AcrA-O157 glycosylation efficiency, production kinetics, and specific productivity. At μ max , AcrA glycosylation with O157-polysaccharide and the specific synthesis rate were maximal, constituting the optimal physiological condition for AcrA-O157 production. Our findings should be considered for the design, optimization, and scaling up of AcrA-O157 production and other recombinant glycoproteins attractive for industrial applications.Fil: Caillava, Ana Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ortiz, Gastón Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Melli, Luciano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ugalde, Juan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ciocchini, Andres Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Comerci, Diego José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

A recombinant O-polysaccharide-protein conjugate approach to develop highly specific monoclonal antibodies to Shiga toxin-producing Escherichia coli O157 and O145 serogroups

Shiga toxin-producing Escherichia coli (STEC) is the major etiologic agent of hemolytic-uremic syndrome (HUS). The high rate of HUS emphasizes the urgency for the implementation of primary prevention strategies to reduce its public health impact. Argentina shows the highest rate of HUS worldwide, being E. coli O157 the predominant STEC-associated HUS serogroup (>70%), followed by E. coli O145 (>9%). To specifically detect these serogroups we aimed at developing highly specific monoclonal antibodies (mAbs) against the O-polysaccharide (O-PS) section of the lipopolysaccharide (LPS) of the dominant STEC-associated HUS serogroups in Argentina. The development of hybridomas secreting mAbs against O157 or O145 was carried out through a combined immunization strategy, involving adjuvated-bacterial immunizations followed by immunizations with recombinant O-PS-protein conjugates. We selected hybridoma clones that specifically recognized the engineered O-PS-protein conjugates of O157 or O145 serogroups. Indirect ELISA of heat-killed bacteria showed specific binding to O157 or O145 serogroups, respectively, while no cross-reactivity with other epidemiological important STEC strains, Brucella abortus, Salmonella group N or Yersinia enterocolitica O9 was observed. Western blot analysis showed specific recognition of the sought O-PS section of the LPS by all mAbs. Finally, the ability of the developed mAbs to bind the surface of whole bacteria cells was confirmed by flow cytometry, confocal microscopy and agglutination assays, indicating that these mAbs present an exceptional degree of specificity and relative affinity in the detection and identification of E. coli O157 and O145 serogroups. These mAbs may be of significant value for clinical diagnosis and food quality control applications. Thus, engineered O-PS specific moieties contained in the recombinant glycoconjugates used for combined immunization and hybridoma selection are an invaluable resource for the development of highly specific mAbs.Fil: Castillo, Daniela Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Rey Serantes, Diego A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Melli, Luciano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ciocchini, Andres Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ugalde, Juan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Comerci, Diego José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Cassola, Alejandro Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Development of improved enzyme-based and lateral flow immunoassays for rapid and accurate serodiagnosis of canine brucellosis

Brucellosis is a widespread zoonotic disease caused by Brucella spp. Brucella canis is the etiological agent of canine brucellosis, a disease that can lead to sterility in bitches and dogs causing important economic losses in breeding kennels. Early and accurate diagnosis of canine brucellosis is central to control the disease and lower the risk of transmission to humans. Here, we develop and validate enzyme and lateral flow immunoassays for improved serodiagnosis of canine brucellosis using as antigen the B. canis rough lipopolysaccharide (rLPS). The method used to obtain the rLPS allowed us to produce more homogeneous batches of the antigen that facilitated the standardization of the assays. To validate the assays, 284 serum samples obtained from naturally infected dogs and healthy animals were analyzed. For the B. canis-iELISA and B. canis-LFIA the diagnostic sensitivity was of 98.6%, and the specificity 99.5% and 100%, respectively. We propose the implementation of the B. canis-LFIA as a screening test in combination with the highly accurate laboratory g-iELISA. The B. canis-LFIA is a rapid, accurate and easy to use test, characteristics that make it ideal for the serological surveillance of canine brucellosis in the field or veterinary laboratories. Finally, a blind study including 1040 serum samples obtained from urban dogs showed a prevalence higher than 5% highlighting the need of new diagnostic tools for a more effective control of the disease in dogs and therefore to reduce the risk of transmission of this zoonotic pathogen to humans.Fil: Cortina, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Novak, Analia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Melli, Luciano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Elena, Sebastián. Ministerio de Agricultura, Ganadería, Pesca y Alimento. Servicio Nacional de Sanidad y Calidad Agroalimentaria; ArgentinaFil: Corbera, Natalia Leonor. Provincia de Buenos Aires. Instituto Veterinario "Dr. Juan E. Romero"; ArgentinaFil: Romero, Juan E.. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias; ArgentinaFil: Nicola, Ana M.. Ministerio de Agricultura, Ganadería, Pesca y Alimento. Servicio Nacional de Sanidad y Calidad Agroalimentaria; ArgentinaFil: Ugalde, Juan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Comerci, Diego José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Comisión Nacional de Energía Atómica; ArgentinaFil: Ciocchini, Andres Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

The trypomastigote small surface antigen from Trypanosoma cruzi improves treatment evaluation and diagnosis in pediatric chagas disease

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. Assessment of parasitological cure upon treatment with available drugs relies on achieving consistent negative results in conventional parasitological and serological tests, which may take years to assess. Here, we evaluated the use of a recombinant T. cruzi antigen termed trypomastigote small surface antigen (TSSA) as an early serological marker of drug efficacy in T. cruzi-infected children. A cohort of 78 pediatric patients born to T. cruzi-infected mothers was included in this study. Only 39 of the children were infected with T. cruzi, and they were immediately treated with trypanocidal drugs. Serological responses against TSSA were evaluated in infected and noninfected populations during the follow-up period using an in-house enzymelinked immunosorbent assay (ELISA) and compared to conventional serological methods. Anti-TSSA antibody titers decreased significantly faster than anti-whole parasite antibodies detected by conventional serology both in T. cruzi-infected patients undergoing effective treatment and in those not infected. The differential kinetics allowed a significant reduction in the required follow-up periods to evaluate therapeutic responses or to rule out maternal-fetal transmission. Finally, we present the case of a congenitally infected patient with an atypical course in whom TSSA provided an early marker for T. cruzi infection. In conclusion, we showed that TSSA was efficacious both for rapid assessment of treatment efficiency and for early negative diagnosis in infants at risk of congenital T. cruzi infection. Based upon these findings we propose the inclusion of TSSA for refining the posttherapeutic cure criterion and other diagnostic needs in pediatric Chagas disease.Fil: Balouz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Melli, Luciano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Volcovich, Romina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Moscatelli, Guillermo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Moroni, Samanta. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: González, Nicolás. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Ballering, Griselda. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Bisio, Margarita María Catalina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ciocchini, Andres Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Altcheh, Jaime Marcelo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

A bacterial engineered glycoprotein as a novel antigen for diagnosis of bovine brucellosis.

Brucellosis is a highly contagious zoonosis that affects livestock and human beings. Laboratory diagnosis of bovine brucellosis mainly relies on serological diagnosis using serum and/or milk samples. Although there are several serological tests with different diagnostic performance and capacity to differentiate vaccinated from infected animals, there is still no standardized reference antigen for the disease. Here we validate the first recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of bovine brucellosis. This antigen can be produced in homogeneous batches without the need of culturing pathogenic brucellae; all characteristics that make it appropriate for standardization. An indirect immunoassay based on the detection of anti O-polysaccharide IgG antibodies in bovine samples was developed coupling OAg-AcrA to magnetic beads or ELISA plates. As a proof of concept and to validate the antigen, we analyzed serum, whole blood and milk samples obtained from non-infected, experimentally infected and vaccinated animals included in a vaccination/infection trial performed in our laboratory as well as more than 1000 serum and milk samples obtained from naturally infected and S19-vaccinated animals from Argentina. Our results demonstrate that OAg-AcrA-based assays are highly accurate for diagnosis of bovine brucellosis, even in vaccinated herds, using different types of samples and in different platforms. We propose this novel recombinant glycoprotein as an antigen suitable for the development of new standard immunological tests for screening and confirmatory diagnosis of bovine brucellosis in regions or countries with brucellosis-control programs.Fil: Ciocchini, Andres Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); ArgentinaFil: Rey Serantes, Diego A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); ArgentinaFil: Melli, Luciano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); ArgentinaFil: Guidolin, Leticia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); ArgentinaFil: Iwashkiw, Jeremy. University of Alberta; CanadáFil: Elena, Sebastian. Ministerio de Agricultura, Ganadería, Pesca y Alimento. Servicio Nacional de Sanidad y Calidad Agroalimentaria; ArgentinaFil: Franco, Cristina. Ministerio de Agricultura, Ganadería, Pesca y Alimento. Servicio Nacional de Sanidad y Calidad Agroalimentaria; ArgentinaFil: Nicola, Ana M. Ministerio de Agricultura, Ganadería, Pesca y Alimento. Servicio Nacional de Sanidad y Calidad Agroalimentaria; ArgentinaFil: Feldman, Mario F. University of Alberta; CanadáFil: Comerci, Diego José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentina. Comisión Nacional de Energía Atómica; ArgentinaFil: Ugalde, Juan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentin

Receiver-operating characteristic (ROC) analysis of glycoconjugate-magnetic beads assay results.

<p>A) ROC plot. The analysis was carried out considering as positive controls sera of patients with culture-confirmed brucellosis and culture-negative/serologically-positive patients with clinical diagnosis of brucellosis (138 sera), and as negative controls serum samples from blood-donors, healthy individuals occupational-exposed to the pathogen, patients with febrile syndrome and patients with other diseases (350 sera). AUC, area under the ROC curve. Values in parentheses indicate the 95% confidence interval. B) Plot of the diagnostic sensitivity (Se) and specificity (Sp) of the assay as a function of the cutoff value. The dot vertical line indicates the cutoff value that concurrently optimizes Se and Sp (cutoff = 14.18%, Se = 99.28% and Sp = 99.43%). The dash vertical lines indicate the cutoff values for which maximal Se or Sp is achieved (cutoff = 13.20%, Se = 100% and Sp = 98.57%; cutoff = 16.15%, Se = 93.48% and Sp = 100%).</p