Preliminary multiplex microarray IgG immunoassay for the diagnosis of toxoplasmosis and rubella

BACKGROUND During pregnancy, toxoplasmosis and rubella can cause serious damage to the mother and the foetus through vertical transmission. Early diagnosis enables implementation of health measures aimed at preventing vertical transmission and minimising damage caused by these diseases. OBJECTIVE Here, we report the development of a multiplex assay for simultaneous detection of IgG antibodies produced during toxoplasmosis and rubella infection. METHODS This assay is based on xMap technology. Initially, by singleplex assays, we evaluated the following antigens: one Toxoplasma gondii lysate; two antigenic extracts of T. gondii (TOX8131 and TOX8122); fragments of T. gondii antigens [SAG-1 (amino acids 45-198), GRA-7 (24-100), GRA-1 (57-149), ROP-4, and MIC-3 (234-306)]; two chimeric antigens composed of fragments of SAG-1, GRA-7, and P35 (CTOX and CTOXH); and fragments of Rubella virus antigens [E-1 (157-176, 213-239, 374-390), E-2 (31-105), and C (1-123)]. FINDINGS A multiplex assay to simultaneously diagnose toxoplasmosis and rubella was designed with the best-performing antigens in singleplex and multiplex assays, which included CTOXH, T. gondii lysate, TOX8131, E-1, and E-2. The multiplex assay showed 100% sensitivity and specificity for anti-T. gondii IgG detection and 95.6% sensitivity and 100% specificity for anti-R. virus IgG detection. MAIN CONCLUSIONS We found that, despite the difficulties related to developing multiplex systems, different types of antigens (extracts and recombinant proteins) can be used to develop high-performance diagnostic tests. The assay developed is suitable to screen for prior T. gondii and R. virus infections, because it is a rapid, high-throughput, low-cost alternative to the current standard diagnostic tools, which require multiple individual tests.

Viability study of a multiplex diagnostic platform for Chagas disease

A new multiplex assay platform was evaluated to detect Trypanosoma cruzi infection using the recombinant antigensCRA, FRA, CRAFRA fusion and parasite lysate. The antigens presented different sensitivity and specificity in a singleplex test when compared to a serial dilution of two pools comprising 10 positive serum samples and one pool of 10 negative samples. The recombinant protein CRA presented lower sensitivity (55%) in contrast to the 100% specificity and sensitivity of FRA, CRAFRA and T. cruzi lysate. These antigens also showed good results in a duplex test and the duplex test with CRAFRA/T. cruzi lysate showed better performance with 100% specificity and sensitivity, as well as a lower cut-off value in comparison to the other duplex test, FRA/T. cruzi lysate. Hence, when the antigens were used in duplex format, both tests showed decreased cut-off values and no interference between different bead sets, resulting in increasing sensitivity and specificity. The results of these multiplex tests show that they could be an alternative to singleplex detection for Chagas disease, and also indicate the necessity of using multiplex diagnostic tools to increase the sensitivity and specificity for diagnostic tests. Emerging data from the T. cruzi genome and from its ORFeome project will also allow the identification of new antigens for this disease detection application.

Caracterização genômica da bactéria endossimbiótica do tripanosomatídeo Crithidia deanei e de suas DNA topoisomerases / Genomic characterization of the endosymbiotic bacteria of the trypanosome Crithidia deanei and its DNA topoisomerases

O principal objeto de estudo do presente trabalho são as DNA topoisomerases do tipo II do endossimbionte,devido ao seu papel essencial tanto nos processos replicação e transcrição,como também na recombinação e segregação dos cromossomos bacterianos.Existem duas topoisomerases do tipo II em bactérias:a DNA girase e DNA topoisomerase IV. A DNA girase introduz superenovelamento negativo no DNA circular,enquanto a topo IV tem como sua principal função a decatenação do DNA durante a segregação dos cromossomos.Para esses estudos foi gerada uma biblioteca genômica do endossimbionte em pUC18 e seqüenciados 7.500 clones inicialmente,abrangendo uma cobertura de 1,2 MB. As seqüências obtidas foram analisadas através do algoritmo BLAST.A análise mostrou que o genoma do endossimbionte apresenta uma grande homologia com os genomas de bactérias do gênero Bordetella,mas diferem no conteúdo A+T,que no genoma do endossimbionte é de aproximadamente 70por cento,enquanto que no gênero Bordetella é de apenas 32por cento.Esta estratégia permitiu encontrar as seqüências dos genes que codificam as subunidades A(GyrA)e B(GyrB)da DNA girase. Identificamos também o gene que codifica a topoisomerase III(topo III),mas não fomos capazes de identificar os genes que codificam as enzimas topo IV e I(topoisomerase I),que formam juntamente com a DNA girase e topo III o repertório de topoisomerases encontrado em outras bactérias.Baseados nas seqüências dos genes gyrA e gyrB foram desenhados iniciadores para amplificação dos alvos através de PCR.Os produtos das amplificações foram clonados para expressão em E.coli.As proteínas recombinantes foram purificadas para a produção de antissoros policlonais.Ensaio de western blot...preditas a partir da seqüência dos genes gyrA e gyrB.Análise por imunofluorescência mostrou que os antissoros contra as subunidades A e B da DNA girase reconhecem antígenos específicos do endossimbionte,inoculados por todo o citoplasma da bactéria.Os genes gyrA e gyrB também...