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There is no consensus on the diagnostic algorithms for many scenarios of Trypanosoma cruzi infection, which hinders the establishment of governmental guidelines in endemic and non-endemic countries. In the acute phase, parasitological methods are currently employed, and standardised surrogate molecular tests are being introduced to provide higher sensitivity and less operator-dependence. In the chronic phase, IgG-based serological assays are currently used, but if a single assay does not reach the required accuracy, PAHO/WHO recommends at least two immunological tests with different technical principles. Specific algorithms are applied to diagnose congenital infection, screen blood and organ donors or conduct epidemiological surveys. Detecting Chagas disease reactivation in immunosuppressed individuals is an area of increasing interest. Due to its neglect, enhancing access to diagnosis of patients at risk of suffering T. cruzi infection should be a priority at national and regional levels. Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Alonso Padilla, Julio. Universidad de Barcelona; España Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Picado, Albert. Foundation For Innovative New Diagnostics; Suiza

Background Chagas disease (CD) has significant global health impact, but safe, effective treatments remain elusive. The nitroimidazole fexinidazole is a potential treatment. Methods This double-blind, randomized, placebo-controlled, dose-finding, proof-of-concept study was conducted in Bolivia. Adults with serologically confirmed chronic indeterminate CD and positive PCR were randomly assigned to 1 of 6 fexinidazole regimens (1200 or 1800 mg/day for 2, 4, or 8 weeks) or placebo. Target recruitment was 20 patients/arm. The primary endpoint was sustained parasitological clearance by serial negative qPCR from end of treatment (EOT) until 6 months follow-up in the intention-to-treat (ITT) population. Follow-up was extended to 12 months. Results Enrollment was interrupted after 4/47 patients presented with transient asymptomatic grade 3 and 4 neutropenia. Treatment of ongoing patients was stopped in all patients administered >2 weeks. A total of 40 patients received treatment with fexinidazole from 3 days to 8 weeks. Delayed-onset neutropenia (n = 8) and increased liver enzymes (n = 8) were found in fexinidazole patients vs none in the placebo arm. In the ITT analysis, sustained parasitological clearance from EOT to 12 months follow-up varied between 66.7% (1200 mg–2 week) and 100.0% (1800 mg–2 week). Rapid, sustained clearance of parasitemia was observed in all treated patients with available data, but not in any patients in the placebo group, at 12 months (P = .0056). Further exploratory exposure-response analysis suggested low dosages of fexinidazole may be safe and effective. Conclusions Further evaluation is needed to establish fexinidazole’s minimum effective dosage and risk–benefit relationship. Results suggest potential for effective treatment regimens Clinical Trials Registration NCT02498782.

Background The role that the genetic diversity of natural Trypanosoma cruzi populations plays in response to trypanocidal treatment of chronic Chagas disease (CD) patients remains to be understood. We analysed the genetic polymorphisms of parasite bloodstream populations infecting chronic CD patients enrolled in the E1224 clinical trial. Methods A total of 506 baseline and post-treatment follow-up samples from 188 patients were analysed. T. cruzi satellite DNA (satDNA) was amplified and sequenced using cruzi1/cruzi2 primers, and samples with TcI/III, TcII, TcIV or hybrid satDNA sequences were identified. Minicircle signatures were obtained after kinetoplast DNA amplification using 121/122 primers and restriction enzyme digestion. Genetic distances between baseline and post-treatment minicircle signatures were estimated using the Jaccard coefficient. Results At baseline, 74.3% TcII, 17.9% hybrid and 7.8% TcI/III satDNA sequences were found, whereas at the end of follow-up the distribution was 55.2% TcII, 35.2% hybrid and 9.5% TcI/III. The placebo arm was the treatment group with the highest variation of satDNA sequences between baseline and post-treatment follow-up. Genetic distances between baseline and post-treatment minicircle signatures were similar among all treatment arms. No association between minicircle signature variability and satDNA type distribution was found. Conclusions Genetic variability of T. cruzi bloodstream populations during post-treatment follow-up did not differ from that observed during chronic infection in the absence of treatment, suggesting that there were no selection events of E1224-resistant parasite populations. This is the first report documenting the genetic polymorphism of natural T. cruzi populations in chronic patients in the context of clinical trials with trypanocidal drugs.

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, is considered a neglected tropical disease by the World Health Organization. Congenital transmission of CD is an increasingly relevant public health problem. It progressively becomes the main transmission route over others and can occur in both endemic and non-endemic countries. Though most congenitally infected newborns are asymptomatic at birth, they display higher frequencies of prematurity, low birth weight, and lower Apgar scores compared to uninfected ones, and some suffer from severe symptoms. If not diagnosed and treated, infected newborns are at risk of developing disabling and life-threatening chronic pathologies later in life. The success or failure of congenital transmission depends on interactions between the parasite, the placenta, the mother, and the fetus. We review and discuss here the current knowledge about these parameters, including parasite virulence factors such as exovesicles, placental tropism, potential placental defense mechanisms, the placental transcriptome of infected women, gene polymorphism, and the maternal and fetal/neonatal immune responses, that might modulate the risk of T. cruzi congenital transmission.

Currently, evaluation of drug efficacy for Chagas disease remains a controversial issue with no consensus. In this work, we evaluated the parasitological efficacy of Nifurtimox treatment in 21 women with chronic Chagas disease from an area of endemicity in Chile who were treated according to current protocols. Under pre- and posttherapy conditions, blood (B) samples and xenodiagnosis (XD) samples from these patients were subjected to analysis by real-time PCR targeting the nuclear satellite DNA of Trypanosoma cruzi (Sat DNA PCR-B, Sat DNA PCR-XD) and by PCR targeting the minicircle of kinetoplast DNA of T. cruzi (kDNA PCR-B, kDNA PCR-XD) and by T. cruzi genotyping using hybridization minicircle tests in blood and fecal samples of Triatoma infestans feed by XD. In pretherapy, kDNA PCR-B and kDNA PCR-XD detected T. cruzi in 12 (57%) and 18 (86%) cases, respectively, whereas Sat DNA quantitative PCR-B (qPCR-B) and Sat DNA qPCR-XD were positive in 18 cases (86%) each. Regarding T. cruzi genotype analysis, it was possible to observe in pretherapy the combination of TcI, TcII, and TcV lineages, including mixtures of T. cruzi strains in most of the cases. At 13 months posttherapy, T. cruzi DNA was detectable in 6 cases (29.6%) and 4 cases (19.1%) by means of Sat DNA PCR-XD and kDNA PCR-XD, respectively, indicating treatment failure with recovery of live parasites refractory to chemotherapy. In 3 cases, it was possible to identify persistence of the baseline genotypes. The remaining 15 baseline PCR-positive cases gave negative results by all molecular and parasitological methods at 13 months posttreatment, suggesting parasite response. Within this follow-up period, kDNA PCR-XD and Sat DNA qPCR-XD proved to be more sensitive tools for the parasitological evaluation of the efficacy of Nifurtimox treatment than the corresponding PCR methods performed directly from blood samples.

A new member of the ras gene family was characterized from Neurospora crassa cDNA libraries. The clone designated NC-ras codes for a polypeptide containing 213 amino acids ( M r 24000). This polypeptide is 84% homologous to the H-ras-1 domain comprising the first 80 amino acids and 60% homologous to the next 84 residues. The NC-ras polypeptide contains all the well-known sequences involved in the interaction with GTP/GDP, the recognition of the Y13-259 neutralizing antibody, the ‘effector site’ for interaction with GAP proteins, and the CAAX acylation motif in the COOH-terminal.

A doença de Chagas, causada pelo protozoário Trypanosoma cruzi, atinge cerca de 18 milhões de pessoas nas três Américas. O parasito apresenta estrutura populacional complexa, com extensa variabilidade intraespecífica. Todavia, a análise conjunta de um grande número demarcadores suporta a divisão do táxon em pelo menos duas linhagens: T. cruzi I, ligada ao ciclo silvestre e T. cruzi II, ligada ao ciclo doméstico de transmissão.Em seres humanos, a doença de Chagas apresenta-se com manifestações clínicas muito variáveis na sua fase crônica, com parasitemia escassa e um curso clínico imprevisível, podendo variar de uma ausência total dos sintomas até s severas manifestações cardiovasculares e/ou gastrintestinais.Apesar de estar cada vez mais claro que a doença de Chagas provavelmente é resultante da interação complexa entre parasitos e hospedeiros, os motivos pelos quais diferentes indivíduos apresentam formas clínicas distintas permanecem desconhecidos. Para que estas questões sejam elucidadas, existe a necessidade de um melhor entendimento da contribuição de fatores genéticos de parasitos e hospedeiros e da maneira de como eles se interagem para resultar na patologia da doença.Neste aspecto, no presente trabalho, procuramos desenvolver metodologias de análise moleculares complementares que possibilitassem a adequada caracterização molecular de parasitos ehospedeiros bem como de suas interações.Inicialmente, com o objetivo de contribuir no entendimento da estrutura populacional do parasito, analisamos cinco locos de microssatélites, a região D7 da subunidade 24S do rDNA e polimorfismos de seqüências dos genes mitocondriais citocromo oxidase subunidade II, citocromoB e NADH desidrogenase em 75 cepas do parasito. Baseando-se nos dados de microssatélites as cepas puderam ser divididas em quatro grupos correspondendo à linhagem T. cruzi I (MDScluster A), T. cruzi II (MDS-cluster C), T. cruzi (MDS-cluster B) e cepas híbridas (MDS-cluster BH).Os dois primeiros grupos correspondem aos tipos mitocondriais A e C respectivamente, enquanto que os outros dois pertencem ao tipo mitocondrial B. Os dados obtidos através da tipagem por microssatélites e rDNA 24S foram analisados conjuntamente por reconstituição haplotípica através do programa PHASE, resultando em 141 haplótipos, claramente distribuídos em três haplogrupos (X,Y e Z).Todas as cepas pertencentes à linhagem T. cruzi I (MDS-cluster A) apresentam os dois haplótipos originados do haplogrupo Z (Z/Z), as cepas da linhagem T. cruzi II (MDS-cluster C) são Y/Y, e aquelas pertencentes ao MDS-cluster B (T. cruzi) apresentam haplótipos X/X. As cepas agrupadas no MDS-cluster BH apresentam haplótipos X/Y, confirmando a sua natureza híbrida. Baseando-se nestes resultados, propusemos a existência de três linhagens ancestrais do Trypanosoma cruzi, as quais foram denominadas T. cruzi I, T. cruzi II 5 e T. cruzi III. No mínimo dois eventos de hibridação envolvendo T. cruzi II e T. cruzi III resultaram em progênies viávéis. Nos dois eventos a linhagem doadora de mitocôndria (conforme indicado pelo tipo mitocondrial das cepas híbridas) foi a linhagem T. cruzi III e a linhagem receptora foi a linhagem T. cruzi II.Segundo, através de uma nova abordagem resultante da combinação da amplificação por heminested PCR e a análise em aparelho de PCR em tempo real, obtivemos sensibilidade e poder de resolução suficientes para a tipagem do rDNA 24S de T. cruzi diretamente emamostras de tecidos cronicamente infectados. Em vinte e sete amostras coração, esôfago e cólon, provenientes de 25 pacientes originados dos estados de Minas Gerais e Goiás, demonstramos a presença de apenas do rDNA tipo 1, bem correlacionado com linhagem T. cruzi II. Estes dados confirmam evidências anteriores de que esta linhagem seria a responsável pela doença ao menos nestas regiões endêmicas. Adicionalmente, caracterizamos geneticamente os parasitos presentesna corrente sanguínea e em uma biopsia cerebral de um paciente Chagásico e portador da síndrome de imunodeficiência adquirida. Análises dos genes de rDNA 24S e de mini-éxon, juntamente com perfis de microssatélites e de LSSP / PCR, indicam que havia uma maior diversidade clonal do parasito no cérebro em comparação com o sangue e que as populações eram geneticamente distintas, pois no sangue estava presente a linhagem T. cruzi II enquanto queno cérebro estava uma população híbrida.Finalmente, com o objetivo de melhor estudar o fenômeno da distribuição diferenciada de T. cruzi nos diversos tecidos hospedeiros, utilizamos o modelo murino da doença, para o qual jáhavia evidências de que o tropismo tecidual poderia estar correlacionado com o haplótipo de MHC (Também denominada H-2) dos camundongos. Para tanto, em abordagens in vivo e ex vivo,infectamos animais congênicos para a região H-2 [C57BLK/sJ (H-2d), C57BL/6 (H-2b), BALB/c (H-2d) e BALB/B10 (H-2b)] com uma mistura artificial de duas cepas de T. cruzi [JG (T. cruzi II) e Col1.7G2 (T. cruzi I)]. Em todos os experimentos, a prevalência de uma cepa em relação à outra no tecido cardíaco foi associada ao haplótipo de H-2, onde a colonização pela cepa Col1.7G2 foi favorecida nos camundongos de haplótipo H-2b, independentemente do background genéticodos animais. O conjunto dos resultados apresentados nesta tese de doutorado estabiliza a noção de que, tanto a variação genética do T. cruzi como dos seus hospedeiros, influenciam no curso da doença de Chagas. Chagas disease, which is caused by Trypanosoma cruzi, afflicts more than 18 million people in the American continent. Both a complex populational structure and a extensive intraespecific variability are observed in this parasite, but a great number of genetic andbiochemical markers support the taxon subdivision into at least two major lineages: T. cruzi I - associated to the wild transmission cycles; and the T. cruzi II - associated to the domestic transmission cycles. The human disease shows variable presentation in the chronic phase, withlow parasitism levels and undeterminable clinical routes, ranges from completely asymptomatic to massive manifestations. The disease results from complex interactions between parasites andhosts, but the reasons for the different patterns of clinical manifestations remain elusive. To clarify these questions it is necessary to understand better both the parasites and hosts genetic factors and how they interact during the pathogenesis. In order to investigate the structure and evolution of Trypanosoma cruzi populations we profiled 75 strains of the parasite with five nuclear microsatellite loci, 24S RNA genes, andsequence polymorphisms in the mitochondrial cytochrome oxidase subunit II, cytochrome B and NADH dehydrogenase subunit 1 genes. A multidimensional scaling plot (MDS) based in microsatellite data divided the parasites into four clusters corresponding to T. cruzi I (MDS-clusterA), T. cruzi II (MDS-cluster C), a third group of T. cruzi strains (MDS-cluster B), and hybrid strains (MDS-cluster BH).The first two clusters matched respectively mitochondrial clades A and C, while the other two belonged to mitochondrial clade B. The 24S rDNA and microsatellite profiling data were analyzed by the haplotype reconstruction program PHASE. We identified 141 haplotypes thatwere clearly distributed into three haplogroups (X, Y, and Z). All strains belonging to T. cruzi I (MDS-cluster A) were Z/Z, the T. cruzi II strains (MDS-cluster C) were Y/Y, and those belonging to MDS-cluster B (unclassified T. cruzi) had X/X haplogroup genotypes. The strains grouped in the MDS-cluster BH were X/Y, confirming their hybrid character. Based on these results we propose three ancestral lineages that we call, respectively, T. cruzi I, T. cruzi II and T. cruzi III. At least twohybridization events involving T. cruzi II and T. cruzi III produced evolutionarily viable progeny. In both events, the mitochondrial donor (as identified by the mitochondrial clade of the hybrid strains) was T. cruzi III and the mitochondrial recipient was T. cruzi II. By using a new molecular tool resulting of the combination of heminested PCR amplification and real time PCR we obtained the sensitiveness and resolution of typing the T. cruzi 24S directly from chronically infected tissues. In twenty-seven hearts, esophagus and colon samples of twenty-five patients from Minas Gerais and Goiás states we detected only the rDNA allele type 1, which correlates well to the T. cruzi II 7 lineage. These data confirm previous evidences of T. cruzi II being responsible for the disease, at least in that geographic region. Furthermore, we genotyped the parasites in the blood stream and in brain biopsy from a Chagasic patient with AIDS. Analyses of rDNA 24S and mini-exon genes and of microsatellite and LSSP/PCR profiles indicate a bigger clonal diversity at brain than at the blood stream and that the two populations were genetically diverse, since in the blood we found the T. cruzi II lineage while a hybrid population was found in the brain. With the aim of investigating further the T. cruzi differential distribution into host tissues, we used the murine model of thedisease, where there is evidence of a correlation between tissue tropism and MHC haplotypes (denominated H-2 in mice). In order to study this we used both in vivo and ex vivo procedures. First we infected mice congenic for the H-2 region [C57BLK/sJ (H-2d), C57BL/6 (H-2b), BALB/c (H-2d)and BALB/B10 (H-2b)] with an artificial mixture of two parasites [JG (T. cruzi II) and Col1.7G2 (T. cruzi I)]. For all experiments, the prevalence of a strain in detriment to the other into cardiac tissue is correlated to the H-2 haplotype, were the Col1.7G2 strain colonization is improved by the H-2bhaplotype, regardless of the genetic background. The results presented in this work reinforce the notion that the genetic variation in T. cruziand its host influence the course of the Chagas disease.