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Chagas disease (CD) is caused by the parasite Trypanosoma cruzi and affects 6–7 million people worldwide. The diagnosis is still challenging, due to extensive parasite diversity encompassing seven genotypes (TcI-VI and Tcbat) with diverse ecoepidemiological, biological, and pathological traits. Chemotherapeutic intervention is usually effective but associated with severe adverse events. The development of safer, more effective therapies is hampered by the lack of biomarker(s) (BMKs) for the early assessment of therapeutic outcomes. The mammal-dwelling trypomastigote parasite stage expresses glycosylphosphatidylinositol-anchored mucins (tGPI-MUC), whose O-glycans are mostly branched with terminal, nonreducing α-galactopyranosyl (α-Gal) glycotopes. These are absent in humans, and thus highly immunogenic and inducers of specific CD anti-α-Gal antibodies. In search for α-Gal-based BMKs, here we describe the synthesis of neoglycoprotein NGP11b, comprised of a carrier protein decorated with the branched trisaccharide Galα(1,2)[Galα(1,6)]Galβ. By chemiluminescent immunoassay using sera/plasma from chronic CD (CCD) patients from Venezuela and Mexico and healthy controls, NGP11b exhibited sensitivity and specificity similar to that of tGPI-MUC from genotype TcI, predominant in those countries. Preliminary evaluation of CCD patients subjected to chemotherapy showed a significant reduction in anti-α-Gal antibody reactivity to NGP11b. Our data indicated that NGP11b is a potential BMK for diagnosis and treatment assessment in CCD patients.

In the past 5–10 years, Venezuela has faced a severe economic crisis, precipitated by political instability and declining oil revenue. Public health provision has been affected particularly. In this Review, we assess the impact of Venezuela's health-care crisis on vector-borne diseases, and the spillover into neighbouring countries. Between 2000 and 2015, Venezuela witnessed a 359% increase in malaria cases, followed by a 71% increase in 2017 (411 586 cases) compared with 2016 (240 613). Neighbouring countries, such as Brazil, have reported an escalating trend of imported malaria cases from Venezuela, from 1538 in 2014 to 3129 in 2017. In Venezuela, active Chagas disease transmission has been reported, with seroprevalence in children ( less than 10 years), estimated to be as high as 12·5% in one community tested (n=64). Dengue incidence increased by more than four times between 1990 and 2016. The estimated incidence of chikungunya during its epidemic peak is 6975 cases per 100 000 people and that of Zika virus is 2057 cases per 100 000 people. The re-emergence of many vector-borne diseases represents a public health crisis in Venezuela and has the possibility of severely undermining regional disease elimination efforts. National, regional, and global authorities must take action to address these worsening epidemics and prevent their expansion beyond Venezuelan borders. © 2019 Elsevier Ltd

Accurate diagnostics tools and surrogate markers of parasitological response to treatment are priority needs for management of Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a Satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (Tc I) and CL-Brener (Tc VI) stocks similar LOD95 values (0.903 (0.745-1.497) and 0.667 (CI 0.113-3.927) copy numbers/μL, respectively), when synthetic DNA was the standard for quantification, thus allowing direct comparison of loads in samples infected with different DTUs. This standard curve was evaluated in 205 samples from 38 acute oral and 19 chronic CD patients from different geographical areas infected with different genotypes, including samples obtained during treatment follow-up, and high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase III clinical trials, to follow-up patients under treatment or at risk of reactivation and in experimental models using different parasite strains. Fil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Silva Gomess, Natalia Lins. Fundación Oswaldo Cruz; Brasil Fil: Apodaca, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Alarcón de Noya, Belkisyolé. Universidad Central de Venezuela; Venezuela Fil: Díaz Bello, Zoraida. Universidad Central de Venezuela; Venezuela Fil: Quintino Souza, Leticia Rocha. Fundación Oswaldo Cruz; Brasil Fil: Tavares Costa, Alexandre Dias. Fundación Oswaldo Cruz; Brasil Fil: Britto, Constança. Fundación Oswaldo Cruz; Brasil Fil: Moreira, Otacilio Cruz. Fundación Oswaldo Cruz; Brasil 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

We aimed to characterize the genetic constitution of natural T. cruzi populations involved in an Oral Chagas Disease (OCD) outbreak at a rural school of the community of Chichiriviche de la Costa, Venezuela, which affected patients did not respond to the etiological treatment. Peripheral blood samples and/or hemocultures were obtained from twenty-nine OCD patients at time of diagnosis or along nine years of Post-treatment (Tx) follow-up. The IgG serology, T. cruzi discrete typing units (DTU), satellite DNA-qPCR parasitic loads, and minicircle signatures were determined at Pre-Tx and after Tx. The serological titles and parasitic loads changed after treatment, with a significant decrease of IgG titers (Spearman’s r value= -0.961) and median parasite loads from 2.869 [IQR = 2.113 to 3.720] to 0.105 [IQR = -1.147 to 1.761] log10 par eq. /mL at Pre-Tx and Post-Tx, respectively, suggesting infection evolution from acute to chronic phase, without seroconversion or parasitological eradication, which was indicative of treatment failure. All patients were infected with T. cruzi DTU I populations. At Pre-Tx their median Jaccard genetic distances were 0.775 [IQR = 0.708 to 0.882], decreasing in genetic variability towards the end of follow-up (Mann-Whitney U test p= 0.0031). Interestingly, no Post-Tx minicircle signature was identical to its Pre-Tx counterpart population in a same patient, revealing selection of parasite subpopulations between the primary infection and Post-Tx. The parasitic populations isolated from hemocultures showed a lower number of bands in the minicircle signatures with respect to the signatures obtained directly from the patients’ blood samples, demonstrating a process of parasitic selection and reduction of the population variability that initially infected the patients. Decrease of parasitic loads after treatment as well as Pre- and Post-Tx intra-TcI diversity might be a consequence of both, natural evolution of the acute infection to the chronic phase and persistence of refractory populations due to Tx selection. Fil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Díaz Bello, Zoraida. Universidad Central de Venezuela; Venezuela Fil: Alarcón de Noya, Belkisyolé. Universidad Central de Venezuela; Venezuela Fil: Noya González, Oscar O.. Universidad Central de Venezuela; Venezuela 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

Chagas disease is one of the parasitic infections with the greatest socio-economic impact in Latin America. In Venezuela, epidemiological data has shown different sources of infection, such as the vectorial route by oral transmission. Given the importance of the TLR4 gene in the innate immune response triggered by infection with Trypanosoma cruzi, this work analyses the role of TLR4 polymorphisms and its possible effect on cytokine expression. Genomic DNA was extracted from the peripheral blood of patients from the main outbreak of oral Chagas disease in Venezuela (n = 90), as well as from a group of healthy individuals (n = 183). Subsequently, peripheral blood was also extracted from individuals with different TLR4 haplotypes and then stimulated with LPS to determine the cytokine concentration by ELISA. The internalization of TLR4 was evaluated by flow cytometry. In comparison to healthy individuals, the analysis showed a significantly increased frequency of the Asp/Gly genotype in symptomatic patients. Also, observed a correlation of the 299/399 haplotype with a significant decrease in cytokine concentration and disease severity. Finally, the parasites' trypomastigotes cause the internalization or negative regulation of TLR4. The variants of TLR4 associated with low production of cytokines may be a risk factor for chronicity and severity (cardiac involvement) in oral vectorial Chagas disease.

Several diagnostic tools have been developed for clinical and epidemiological assays. RT-PCR and antigen detection tests are more useful for diagnosis of acute disease, while antibody tests allow the estimation of exposure in the population. Currently, there is an urgent need for the development of diagnostic tests for COVID-19 that can be used for large-scale epidemiological sampling. Through a comprehensive strategy, potential 16 mer antigenic peptides suited for antibody-based SARS-CoV-2 diagnosis were identified. A systematic scan of the three structural proteins (S,N and M) and the non-structural proteins (ORFs) present in the SARS-CoV-2 virus was conducted through the combination of immunoinformatic methods, peptide SPOT synthesis and an immunoassay with cellulose-bound peptides (Pepscan). The Pepscan filter paper sheets with synthetic peptides were tested against pools of sera of COVID-19 patients. Antibody recognition showed a strong signal for peptides corresponding to the S, N and M proteins of SARS-CoV-2 virus, but not for the ORFs proteins. The peptides exhibiting higher signal intensity were found in the C-terminal region of the N protein. Several peptides of this region showed strong recognition with all three immunoglobulins in the pools of sera. The differential reactivity observed between the different immunoglobulin isotypes (IgA, IgM and IgG) within different regions of the S and N proteins, can be advantageous for ensuring accurate diagnosis of all infected patients, with different times of exposure to infection. Few peptides of the M protein showed antibody recognition and no recognition was observed for peptides of the ORFs proteins.

Trypanosoma cruzi uses various mechanisms of infection to access humans. Since 1967, food contaminated with metacyclic trypomastigotes has triggered several outbreaks of acute infection of Chagas disease by oral transmission. Follow-up studies to assess the effectiveness of anti-parasitic treatment of oral outbreaks are rather scarce. Here, we report a 10-year laboratory follow-up using parasitological, serological, and molecular tests of 106 individuals infected in 2007 of the largest known outbreak of orally transmitted Chagas disease, which occurred in Caracas city, Venezuela. Before treatment (2007), specific IgA, IgM and IgG, were found in 71% (75/106), 90% (95/106) and 100% (106/106), respectively, in addition to 21% (9/43) parasitemia, Complement Mediated Lysis (CML) in 98% (104/106) and 79% (34/43) parasitic DNA for PCR. Blood culture detected parasitemia up to 18 months post-treatment in 6% (6/106) of the patients. In 2017, the original number of cases in the follow-up decreased by 46% and due to the country's economic situation, not all the trials could be carried out in the entire population. During follow-up, IgA and IgM disappeared promptly, with IgM persisting in 19% (20/104) of the patients three years after treatment. The anti-T. cruzi IgG remained positive 10 years later in 41% (20/49) of the individuals evaluated. CML remained positive seven years later in 79% (65/82) of the cases. PCR positive cases decreased after treatment but progressively recovered, being positive in 69% (32/46) of the individuals evaluated in 2017. The group of children (under 18 years of age) showed the highest PCR positivity with 76% (26/34) of the cases, but their parasitic load tended to diminish, while in adults the parasitic load regained their initial values. The simultaneous evaluation of serological tests and PCR of the patients allowed us to separate patients among responders and non-responders to the anti-parasitic treatment, and this information prompted us to apply a second anti-parasitic treatment in the group of non-responders. In this population not subjected to the like lihood of re-infection, adult patients were more likely to be non-responders when compared to children. These results suggest that rigorous laboratory follow-up with T. cruzi infectious biomarkers is essential to detect cases of parasite persistence.

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects more than 6 million people worldwide. Following a mostly asymptomatic acute phase, the disease progresses to a long-lasting chronic phase throughout which life-threatening disorders to the heart and/or gastrointestinal tract will manifest in about 30% of those chronically infected. During the chronic phase, the parasitemia is low and intermittent, while a high level of anti-T. cruzi antibodies persist for years. These two features hamper post-chemotherapeutic follow-up of patients with the tools available. The lack of biomarkers for timely assessment of therapeutic response discourages a greater use of the two available anti-parasitic drugs, and complicates the evaluation of new drugs in clinical trials. Herein, we investigated in a blinded case-control study the serological reactivity over time of a group of parasite-derived antigens to potentially address follow up of T. cruzi chronically infected subjects after treatment. We tested PFR2, KMP11, HSP70, 3973, F29 and the InfYnity multiplexed antigenic array, by means of serological assays on a multi-national retrospective collection of samples. Some of the antigens exhibited promising results, underscoring the need for further studies to determine their potential role as treatment response biomarkers.

The cryptic parasite Sparganum proliferum proliferates in humans and invades tissues and organs. Only scattered cases have been reported, but S. proliferum infection is always fatal. However, S. proliferum’s phylogeny and life cycle remain enigmatic. To investigate the phylogenetic relationships between S. proliferum and other cestode species, and to examine the mechanisms underlying pathogenicity, we sequenced the entire genomes of S. proliferum and a closely related non–life-threatening tapeworm Spirometra erinaceieuropaei. Additionally, we performed larvae transcriptome analyses of S. proliferum plerocercoid to identify genes involved in asexual reproduction in the host. The genome sequences confirmed that the S. proliferum has experienced a clearly distinct evolutionary history from S. erinaceieuropaei. Moreover, we found that nonordinal extracellular matrix coordination allows asexual reproduction in the host, and loss of sexual maturity in S. proliferum are responsible for its fatal pathogenicity to humans. Our high-quality reference genome sequences should be valuable for future studies of pseudophyllidean tapeworm biology and parasitism., Kikuchi et al. sequence the genome of the fatal tapeworm Sparganum proliferum and a closely related non–life-threatening tapeworm Spirometra erinaceieuropaei, and describe its genomic features suggesting the natural history and molecular mechanisms underlying pathogenicity. Their findings indicate that nonordinal extracellular matrix coordination is important for its asexual reproduction, and suggest that loss of sexual maturity contributes to the fatal pathogenicity of S. proliferum to humans.

In Venezuela, Chagas disease (ChD) is considered a serious health problem, with about 6 million people at risk; and acute outbreaks due to oral transmission of Chagas Disease (OChD) are becoming increasingly important. In 2007 there was a major outbreak of OChD and although patients from this episode were treated with nifurtimox (Lampit®-Bayer), about 70% therapeutic failure was registered. These results led us to examine whether parasite's drug susceptibility was related to this therapeutic failure.The Trypanosoma cruzi parasites were isolated by haemoculture of the peripheral blood drawn from the pre- and post-nifurtimox treated patients infected in the 2007 OChD outbreak at Caracas, Venezuela. The in vitro assays for drug testing were performed by the MTT methodology followed by calculation of inhibitory concentration-50 (ICParasite isolates obtained from the infected patients prior and after nifurtimox treatment when subjected to variable concentrations of the drug showed great heterogeneity in susceptibility with ICThe high heterogeneity in nifurtimox IC

descripción no proporcionada por scopus, ISGlobal work is supported by the Departament d’Universitats i Recerca de la Generalitat de Catalunya, Spain (AGAUR; 017SGR00924) and by the Instituto de Salud Carlos III (ISCIII) RICET Network for Cooperative Research in Tropical Diseases (ISCIII; RD16/0027/0004 - PI1290) and FEDER. MJP research is supported by the Ministry of Health, Government of Catalonia (PERIS 2016-2010 SLT008/18/00132). ICA, JG, and FT are supported by the grant number U01AI129783 from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). ICA is also partly supported by the grant number 5U54MD007592 from the National Institute on Minority Health and Health Disparities (NIMHD), NIH. MCL and MCT were supported by ISCIII RICET grant RD16/0027/0005 - PI1290 and FEDER and by grants SAF2016-81003-R and SAF2016-80998-R from the Spanish “Programa Estatal I+D+i (MINECO)”. AA's work was supported by the Italian Ministry of Health “Fondi Ricerca Corrente - Linea 3, progetto 9” to IRCCS Sacro Cuore Don Calabria Hospital. JR was supported by CONACyT Fossis grant #261006. The Drugs for Neglected Diseases initiative (DNDi) is grateful to its donors, public and private, who have provided funding to DNDi since its inception in 2003. A full list of DNDi's donors can be found at http://www.dndi.org/donate/donors/. FIND is grateful to its donors, public and private, who have helped bring innovative new diagnostics for diseases of poverty. A full list of FIND’s donors can be found at: https://www.finddx.org/partners-donors/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Two old drugs are the only choice against Trypanosoma cruzi and little is known about their secondary effects in the acute stage of oral-transmitted Chagas disease (ChD).A cross-sectional analytical surveillance study was conducted in a sizable cohort of patients seen during the largest acute foodborne ChD microepidemic registered so far. Individuals were treated with benznidazole (BNZ) or nifurtimox (NFX). 'Common Terminology Criteria for Adverse Events' was assessed to categorize side effects according to severity.Out of 176 treatments applied, 79% had one or more adverse effects, which predominated in adults (97.8%) as compared to children (75.5%). Risk of side effects with NFX was significantly higher than BNZ. Four adults and a child treated with NFX had severe side effects (pulmonary infarction, facial paralysis, neutropenia, blurred vision, bone marrow hypoplasia) warranting hospitalization, and drug suspension. Adverse effects frequently reported with NFX were abdominal pain, hyporexia, weight loss, headache, nausea and lymphocytosis, whereas skin rash, neurosensory effects, hyporexia, fatigue, pyrosis, abdominal pain and eosinophilia were observed with BNZ.Frequency and severity of side effects during treatment of acute oral infection by T. cruzi demand direct supervision and close follow-up, even in those asymptomatic, to prevent life-threatening situations.

A Trypanosoma cruzi Loopamp kit was recently developed as a ready-to-use diagnostic method requiring minimal laboratory facilities. We evaluated its diagnostic accuracy for detection of acute Chagas disease (CD) in different epidemiological and clinical scenarios. In this retrospective study, a convenience series of clinical samples (venous blood treated with EDTA or different stabilizer agents, heel-prick blood in filter paper or cerebrospinal fluid samples (CSF)) from 30 infants born to seropositive mothers (13 with congenital CD and 17 noninfected), four recipients of organs from CD donors, six orally–infected cases after consumption of contaminated guava juice and six CD patients coinfected with HIV at risk of CD reactivation (N = 46 patients, 46 blood samples and 1 CSF sample) were tested by T. cruzi Loopamp kit (Tc LAMP) and standardized quantitative real-time PCR (qPCR). T. cruzi Loopamp accuracy was estimated using the case definition in the different groups as a reference. Cohen’s kappa coefficient (κ) was applied to measure the agreement between Tc LAMP (index test) and qPCR (reference test). Sensitivity and specificity of T. cruzi Loopamp kit in blood samples from the pooled clinical groups was 93% (95% CI: 77–99) and 100% (95% CI: 80–100) respectively. The agreement between Tc LAMP and qPCR was almost perfect (κ = 0.92, 95% CI: 0.62–1.00). The T. cruzi Loopamp kit was sensitive and specific for detection of T. cruzi infection. It was carried out from DNA extracted from peripheral blood samples (via frozen EDTA blood, guanidine hydrochloride-EDTA blood, DNAgard blood and dried blood spots), as well as in CSF specimens infected with TcI or TcII/V/VI parasite populations. The T. cruzi Loopamp kit appears potentially useful for rapid detection of T. cruzi infection in congenital, acute and CD reactivation due to HIV infection., Author summary The aim of this study was to evaluate a kit prototype (T. cruzi Loopamp or Tc LAMP) based on loop-mediated isothermal amplification (LAMP) for molecular diagnosis of acute Chagas disease in well-characterized individuals, collected in real life conditions, such as newborns or infants born to Chagas disease mothers aiming to detect congenital Chagas disease, recipients of organs from Chagas disease donors who acquired T. cruzi infection after transplantation, persons with oral Chagas disease acquired after consumption of a contaminated meal, and HIV/T. cruzi coinfected patients at risk of Chagas disease reactivation due to immunosuppression. Different types of clinical samples were tested; peripheral blood samples treated with ethylenediamine tetraacetic acid (EDTA) as anticoagulant or with guanidine hydrochloride or DNAgard stabilizer agents, as well as dried blood spots and cerebrospinal fluid samples. The performance of Tc LAMP was estimated using the case definition in the different groups as a reference and all test kit results were compared to those obtained using standardized duplex real-time PCR (qPCR) in the same clinical samples. Sensitivity and specificity of Tc LAMP in blood samples from the pooled clinical groups were 93% (95% CI: 77–99) and 100% (95% CI: 80–100) respectively. The agreement between Tc LAMP and qPCR was almost perfect (κ index of Cohen = 0.92, 95% CI: 0.62–1.00). Tc LAMP was sensitive and specific for detection of T. cruzi infection in the tested samples, encouraging prospective field studies to validate its use for rapid detection of T. cruzi infection in congenital, acute and CD reactivation due to HIV infection.

Chagas disease (ChD), caused by the protozoan parasite Trypanosoma cruzi, affects millions of people worldwide. Chemotherapy is restricted to two drugs, which are partially effective and may cause severe side effects, leading to cessation of treatment in a significant number of patients. Currently, there are no biomarkers to assess therapeutic efficacy of these drugs in the chronic stage. Moreover, no preventive or therapeutic vaccines are available. In this chapter, we describe the purification of Trypanosoma cruzi trypomastigote-derived glycosylphosphatidylinositol (GPI)-anchored mucins (tGPI-mucins) for their use as antigens for the reliable primary or confirmatory diagnosis and as prognostic biomarkers for early assessment of cure following ChD chemotherapy. We also describe, as an example, the synthesis of a potential tGPI-mucin-derived α-Gal-terminating glycan and its coupling to a carrier protein for use as diagnostic and prognostic biomarker in ChD.

We describe the eleventh major outbreak of foodborne Trypanosoma cruzi transmission in urban Venezuela, including evidence for vertical transmission from the index case to her fetus. After confirming fetal death at 24 weeks of gestation, pregnancy interruption was performed. On direct examination of the amniotic fluid, trypomastigotes were detected. T. cruzi specific-polymerase chain reaction (PCR) also proved positive when examining autopsied fetal organs. Finally, microscopic fetal heart examination revealed amastigote nests. Acute orally transmitted Chagas disease can be life threatening or even fatal for pregnant women and unborn fetuses owing to vertical transmission. There is therefore an urgent need to improve national epidemiologic control measures.

Oral outbreaks of Chagas disease are increasingly reported in Latin America. The transitory presence of Trypanosoma cruzi parasites within contaminated foods, and the rapid consumption of those foods, precludes precise identification of outbreak origin. We report source attribution for 2 peri-urban oral outbreaks of Chagas disease in Venezuela via high resolution microsatellite typing.

Background Chagas disease affects more than 15 million people worldwide. Although vector-borne transmission has decreased, oral transmission has become important. Recently, our group published the clinical and epidemiological characteristics of the largest outbreak of orally transmitted Chagas disease reported till date. Objective: To describe electrocardiographic changes occurring in the study population during the outbreak caused by ingestion of contaminated guava juice. Methods We evaluated 103 positive cases, of which 76 (74%) were aged ≤ 18 years (average age: 9.1 ± 3.1 years) and 27 (26%) were aged > 18 years (average age: 46 ± 11.8 years). All patients underwent clinical evaluations and ECG. If the patients had palpitations or evident alterations of rhythm at baseline, ambulatory ECG monitoring was performed. Results A total of 68 cases (66%; 53 children and 15 adults) had ECG abnormalities. Further, 69.7% (53/76) of those aged ≤ 18 years and 56% (15/27) of those aged >18 years showed some ECG alteration (p = ns). ST-T abnormalities were observed in 37.86% cases (39/103) and arrhythmias were evident in 28.16% cases (29/103). ST alterations occurred in 72% of those aged ≤18 years compared with 19% of th ose aged >18 years (p < 0.0001). Conclusion This study reports the largest number of cases in the same outbreak of acute Chagas disease caused by oral contamination, with recorded ECGs. ECG changes suggestive of acute myocarditis and arrhythmias were the most frequent abnormalities found.

Background Trypanosoma cruzi oral transmission is possible through food contamination by vector's feces. Little is known about the epidemiology and clinical features of microepidemics of orally acquired acute Chagas disease (CD). Methods A case-control, cohort-nested, epidemiological study was conducted during an outbreak of acute CD that affected a school community. Structured interviews were designed to identify symptoms and sources of infection. Electrocardiograms were obtained for all patients. Specific serum antibodies were assessed by immunoenzimatic and indirect hemagglutination tests. In some cases, parasitemia was tested directly or by culture, animal inoculation, and/or a polymerase chain reaction technique. Results Infection was confirmed in 103 of 1000 exposed individuals. Of those infected, 75% were symptomatic, 20.3% required hospitalization, 59% showed ECG abnormalities, parasitemia was documented in 44, and 1 child died. Clinical features differed from those seen in vectorial transmission. The infection rate was significantly higher among younger children. An epidemiological investigation incriminated contaminated fresh guava juice as the sole source of infection. Conclusions This outbreak was unique, because it affected a large, urban, predominantly young, middle-class, otherwise healthy population and resulted in an unprecedented public health emergency. Rapid diagnosis and treatment avoided higher lethality. Food-borne transmission of T. cruzi may occur more often than is currently recognized.

Abdominal ultrasound can be a useful tool for diagnosing periportal fibrosis related to Schistosoma mansoni infection, and also for planning and monitoring the evolution of hepatic morbidity following control measures. We evaluated the standardized ultrasound methodology proposed by the World Health Organization for detecting periportal fibrosis and portal hypertension, among patients from an endemic area in Venezuela, and the impact of praziquantel treatment 3-5 years later. After chemotherapy, complete reversal of periportal lesions was observed in 28.2% of the cases and progression of the disease in 5.1%. Improvement in the hepatic disease started with a reduction in the periportal thickening followed by a decrease in the size of the left hepatic lobe, spleen and mesenteric and spleen veins. Ultrasound confirmed the clinical findings after chemotherapy among the patients with reversal of the disease. However, in patients with more advanced disease, these findings were contradictory. There was no correlation between evolution of the disease seen on ultrasound and age, intensity of infection or serological findings. O ultra-som abdominal pode ser uma ferramenta útil para o diagnóstico da fibrose periportal relacionada à infecção por Schistosoma mansoni, e também para planejar e monitorar a evolução da morbidade hepática após medidas de controle. Nós avaliamos a metodologia padronizada no ultra-som, proposta pela Organização Mundial da Saúde, para a detecção da fibrose periportal e hipertensão porta, em pacientes de área endêmica da Venezuela e o impacto do tratamento com praziquantel 3-5 anos depois. Após quimioterapia, houve reversão completa das lesões periportais em 28,2% dos casos e progressão da patologia em 5,1%. A melhora da patologia hepática começou com a redução do espessamento periportal seguida pela diminuição do tamanho do lobo esquerdo, baço e veias mesentérica e esplênica. O ultra-som confirma os achados clínicos após quimioterapia em pacientes com reversão da patologia; contudo, naqueles com patologia mais avançada, estes achados foram contraditórios. Não houve correlação entre evolução da patologia ultra-sonográfica com idade, intensidade da infecção ou achados sorológicos.

This simple, versatile, reliable, reproducible, multipurpose, and inexpensive technique is based on the adhesion of different antigens to a single nitrocellulose strip using, as template, an acrylic device containing 28 parallel channels. The inclusion of channels containing normal human serum improves the quality control of this assay. Antigen-sensitized nitrocellulose strips are cut perpendicularly to the antigen-rows, exposed to immune sera followed by the appropriate conjugate. Positive signals are recorded using chemiluminescent or precipitable colorimetric substrates. This assay allows the simultaneous qualitative demonstration of antigenicity and immunogenicity of antigens obtained as synthetic peptides, recombinant molecules, or crude preparations, with high sensitivity and specificity. Its major value is based on the rapid and simultaneous comparative evaluation of various antigenic preparations allowing the diagnosis of a variety of infectious, allergic, and autoimmune diseases. It can in general be used to detect any type of antibody or circulating antigen. Some improvements and variants of the original technique are included.

Orally transmitted Chagas disease has become a matter of concern due to outbreaks reported in four Latin American countries. Although several mechanisms for orally transmitted Chagas disease transmission have been proposed, food and beverages contaminated with whole infected triatomines or their faeces, which contain metacyclic trypomastigotes of Trypanosoma cruzi, seems to be the primary vehicle. In 2007, the first recognised outbreak of orally transmitted Chagas disease occurred in Venezuela and largest recorded outbreak at that time. Since then, 10 outbreaks (four in Caracas) with 249 cases (73.5% children) and 4% mortality have occurred. The absence of contact with the vector and of traditional cutaneous and Romana's signs, together with a florid spectrum of clinical manifestations during the acute phase, confuse the diagnosis of orally transmitted Chagas disease with other infectious diseases. The simultaneous detection of IgG and IgM by ELISA and the search for parasites in all individuals at risk have been valuable diagnostic tools for detecting acute cases. Follow-up studies regarding the microepidemics primarily affecting children has resulted in 70% infection persistence six years after anti-parasitic treatment. Panstrongylus geniculatus has been the incriminating vector in most cases. As a food-borne disease, this entity requires epidemiological, clinical, diagnostic and therapeutic approaches that differ from those approaches used for traditional direct or cutaneous vector transmission.

Schistosomiasis low transmission areas as Venezuela, can be defined as those where the vector exists, the prevalence of active cases is under 25%, individuals with mild intensity of infection predominate and are mostly asymptomatic. These areas are the consequence of effective control programs, however, "silent" epidemiological places are difficult to trace, avoiding the opportune diagnosis and treatment of infected persons. Clinic and abdominal ultrasound have not shown to discriminate infected from uninfected persons in areas where besides Schistosoma mansoni, intestinal parasites are the rule. Under these conditions, serology remains as a very valuable diagnostic tool, since it gives a closer approximation to the true prevalence. In this sense, circumoval precipitin test, ELISA-SEA with sodium metaperiodate, and alkaline phosphatase immunoassay joined to coprology allow the identification of the "schistosomiasis cases". In relation to public health, schistosomiasis has been underestimated by the sanitary authorities and the investment on its control is being transferred to other diseases of major social and political relevance neglecting sanitary efforts and allowing growth of snail population. Some strategies of diagnosis and control should be done before schistosomiasis reemergence occurs in low transmission areas.

The previously called “hemoglobinase” Sm32 molecule of the adult worm of Schistosoma mansoni was chemically synthesized in 22 polymeric peptides based on the t-boc strategy. Their immunogenicity was evaluated in rabbits to which a mixture of five to six peptides of 20 amino acids long were given in three doses with Freund's adjuvant. Seventeen peptides were found to be immunogenic, and sera from immunized rabbits corresponding to the molecule from the first 335 amino acids, recognized the 32 kDa native protein from the adult worm antigen by western blot. Of those, the relevant peptides responsible of the recognition of the original molecule corresponded to amino acids 101–120, 121–140 and 244–268, based on inhibition competitive assays. Because Sm32 is one of the excretory and secretory molecules released with the vomitus of the adult worm, it is one of the target antigens for detection in plasma of infected individuals. The production of these polyclonal monospecific antibodies against the synthetic peptides could be of value in the immunodiagnosis of this parasitosis.

American trypanosomiasis is one of the few native parasites of this continent. As a zoonosis, Trypanosoma cruzi infects about 180 species out of 25 families of mammals. Its regular transmission is through triatomines, which can easily transmit parasites either by the skin route (contamination of mammals skin with their feces) or by oral route (ingestion of food contaminated with complete triatomines or their feces) and additionally through haematogenous via (congenital and transfusional) and by tissues (transplants). The oral route, which seems to be the ancestral form of transmission to wild and domestic mammals, has recently become more important after the success achieved in the control of domicile vectors using residual pesticides. From its initial diagnosis in 1967, tens of oral outbreaks have been diagnosed mostly in the Brazilian Amazon and subsequently in other four countries in South America. Environmental imbalance caused by man through the invasion and deforestation of woodlands, results in reduction of biodiversity of mammals as food source for triatomines, affecting the "dilution effect" of T. cruzi in the nature increasing the risk of human infection. On the other hand, triatomines invade houses looking for new blood sources. One of the consequences of domiciliated triatomines is the food contamination spread, especially in home-made juices, which has been the source of infection of most oral outbreaks. Other biotic and abiotic factors help to explain the recent increase of oral transmission outbreaks of Chagas disease, distributed in nine eco-regions of America.

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

Control of schistosomiasis in Venezuela has been a topic of major interest and controversy among the metaxenic parasitosis. A small area of transmission of approximately 15,000 km2 was thought to be eradicated some years ago. However, some epidemiological characteristics of our transmission area have limited the success on the way toward eradication. Since 1945, when the Schistosomiasis Control Program started, the prevalence in the endemic area has decreased from 14% in 1943 to 1.4% in 1996. Until 1982, the surveillance of active cases was based on massive stool examination. Since then, the Schistosomiasis Research Group (SRG) recommended the additional use of serologic tests in the Control Program and the selective or massive chemotherapy depending on serological and parasitological prevalence of each community. At present, the real prevalence is underestimated due to the fact that approximately 80% of the individuals eliminate less than 100 eggs/g of feces. Those persons could be responsible for the maintenance of the foci going on and therefore limiting the impact of the control measures. Efforts of the SRG are being oriented toward improvement of immunodiagnostic tests by using defined antigens (enzymes) and chemically synthesized peptides, derived from relevant molecules of the parasite, either for antibodies or antigens search. On the other hand, introduction of snail competitors has been a biological weapon in the control of the intermediate host in certain areas. However, the recent reinfestation of water courses by Biomphalaria glabrata, the increased prevalence in some areas, together with important administrative changes at the Control Program of the Minister of Health, have arisen new questions and doubts, challenging the eradication strategy proposed during the last decade.

1 Parasitology Lab, Section for Microbiology, Immunology, and Parasitology, Department of Food Safety and Infection Biology, Norwegian University of Life Sciences, Adamstuen-Campus, Oslo, Norway, 2 Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium, 3 Emerging Pathogens Institute and Department of Animal Sciences, University of Florida, Gainesville, Florida, United States of America, 4 Immunology Section, Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela, 5 Biohelmintiasis Section, Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela, 6 Centro para Estudios Sobre Malaria, Instituto de Altos Estudios “Dr. Arnoldo Gabaldon”, Instituto Nacional de Higiene, MPPS, Caracas, Venezuela, 7 Section of Epidemiology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland

Orally transmitted Chagas disease (ChD), which is a well-known entity in the Brazilian Amazon Region, was first documented in Venezuela in December 2007, when 103 people attending an urban public school in Caracas became infected by ingesting juice that was contaminated with Trypanosoma cruzi. The infection occurred 45-50 days prior to the initiation of the sampling performed in the current study. Parasitological methods were used to diagnose the first nine symptomatic patients; T. cruzi was found in all of them. However, because this outbreak was managed as a sudden emergency during Christmas time, we needed to rapidly evaluate 1,000 people at risk, so we decided to use conventional serology to detect specific IgM and IgG antibodies via ELISA as well as indirect haemagglutination, which produced positive test results for 9.1%, 11.9% and 9.9% of the individuals tested, respectively. In other more restricted patient groups, polymerase chain reaction (PCR) provided more sensitive results (80.4%) than blood cultures (16.2%) and animal inoculations (11.6%). Although the classical diagnosis of acute ChD is mainly based on parasitological findings, highly sensitive and specific serological techniques can provide rapid results during large and severe outbreaks, as described herein. The use of these serological techniques allows prompt treatment of all individuals suspected of being infected, resulting in reduced rates of morbidity and mortality.

The development of an immunoassay for detecting circulating antibodies against Trypanosoma cruzi with a good performance appears to be crucial in clinical settings for the management of Chagas disease. Here we propose a new automated ELISA test performed on serum samples using 2 different T. cruzi antigens (a recombinant protein and a T. cruzi whole extract) placed in parallel in separate solid phases. This automated diagnostic tool allows the simultaneous analysis of a large number of sera, by determining the presence of antibodies against both antigens by a single run test, with high sensitivity and specificity. The simultaneous analysis of the reactivity against the 2 antigens in a biparametric modality reduces the percentage of false-negative sera and allows a more accurate diagnosis. Using this multiparametric approach, we propose an effective algorithm for the first step of Chagas diagnosis by performing a single test, with time and cost savings.

Nowadays, the techniques available for chronic Chagas' disease diagnosis are very sensitive; however, they do not allow discrimination of the patient's clinical stages of the disease. The present paper describes that three out of the five different repeats contained in the Trypanosoma cruzi TcCA-2 membrane protein (3972-FGQAAAGDKPPP, 6303-FGQAAAGDKPAP, and 3973-FGQ AAAGDKPSL) are recognized with high sensitivity (>90%) by sera from chronic Chagas' disease patients and that they are not recognized by sera from patients in the acute phase of the disease. A total of 133 serum samples from chagasic patients and 50 serum samples from healthy donors were tested. In addition, sera from 15 patients with different autoimmune diseases, 43 serum samples from patients suffering an infectious disease other than Chagas' disease, and 38 serum samples from patients with nonchagasic cardiac disorders were also included in this study. The residue 3973 peptide shows a specificity of >98%, as it is not recognized by individuals with autoimmune and inflammatory processes or by patients with a nonchagasic cardiomyopathy. Remarkably, the levels of antibody against the 3973 epitope detected by the sera from Chagas' disease patients in the symptomatic chronic phase, involving cardiac or digestive alterations, are higher than those detected by the sera from Chagas' disease patients in the indeterminate phase of the disease. It is suggested that the diagnostic technique described could also be used to indicate the degree of pathology. The amino acids F, Q, and DKP located in the peptide at positions 1, 3, and 8 to 10, respectively, are essential to conform to the immunodominant antigenic epitope., P08-CVI-04037PAI (Junta de Andalucía); BFU2010-1670 from Plan Nacional I+D+i (MICINN); RD06/0021/0014, RD06/0021/1007-ISCIII-RETIC (MICINN); FEDER; FIS 2009SGR385 ISCIII (MICINN)

We evaluated a commercially available immunochromatographic dipstick test to detect Trypanosoma cruzi infection in 366 human serum samples with known serological results from Argentina, Ecuador, Mexico, and Venezuela. One hundred forty-nine of 366 (40.7%) and 171/366 (46.7%) samples tested positive by dipstick and serology, respectively. Dipstick sensitivity was calculated to be 84.8% (range between countries, 77.5 to 95%), and specificity was 97.9% (95.9 to 100%).

Schistosomiasis in America with the exception of Brazil, behaves as a chronic mild disease with few clinical manifestations due to low parasite burden. These features restrict the clinical and parasitological diagnosis. The most commonly used stool examination method, Kato-Katz, becomes insensitive when the majority of individuals excrete less than 100 eggs/g of feces. In view that antigen-detecting techniques have not been able to reveal light infections, the antibody detecting assays remain as a very valuable diagnostic tool for epidemiological surveillance. The Venezuelan Schistosomiasis Research Group (CECOICE) has designed a mass chemotherapy strategy based on sero-diagnosis. Since blood sampling is one of the important limiting factors for large seroepidemiological trials we developed a simple capillary technique that successfully overcame most of the limitations of blood drawing. In this sense, ELISA seems to be the most adequate test for epidemiological studies. Soluble egg Schistosoma mansoni antigen (SEA) has been largely used in Venezuela. The sensitivity of ELISA-SEA in our hands is 90%, moreover its specificity reach 92% when populations from non-endemic areas but heavily infected with other intestinal parasites are analyzed. The Schistosomiasis Control Program is currently carrying out the surveillance of endemic areas using ELISA-SEA as the first screening method, followed by the Circumoval Precipitin test for validation assay. The results with these two serological techniques allowed us to defined the criteria of chemotherapy in populations of the endemic areas. On the search of better diagnostic technique, Alkaline Phosphatase Immunoenzyme Assay (APIA) is being evaluated in field surveys.

This technique is based on the sensitization of different antigens in a single nitrocellulose strip, which react when exposed to an immune serum and thereafter with the appropriate peroxidase conjugate and the corresponding substrate. Signals in those reactive spots are recorded as black squares in a negative photographic film, using a chemiluminiscent substrate or as blue spots when a precipitable colorimetric substrate is used. This technique allows the simultaneous demonstration of antigenicity of different antigens (peptides, recombinant molecules, and crude preparations), with a high sensitivity and specificity. Its major value is based on its versatility, since it is possible to rapidly evaluate and to compare various antigenic preparations and to use it for diagnosis of different infectious, allergic and autoimmune diseases, at a low cost.

To establish the confirmatory diagnosis of Trypanosoma cruzi infection, at least two immunoserological tests (ELISA, Indirect hamaglutination, IH, Complement Fixation Test, CFT) were carried out in 254 donors, from public and private blood banks of Venezuela, during 48 months between 1997-1998 and 2003-2004, referred to the Immunology Section of the Tropical Medicine Institute in Caracas. Antibodies anti-T. cruzi were detected in 129/254 (50,79%) by ELISA-IgG or IH and CFT. The "artificial xenodiagnosis" was positive in 10/118 persons with positive confirmed serology. Of 129 donors found positive by the serological tests, 68 were living in the capital region and 61 in the interior of the country. Likewise 113 were born in the interior of the country, 8 in Caracas and 8 in Colombia. Of them, 12 individuals serologically confirmed declared to have donated blood in a minimum of 4 occasions before diagnosis. The present study emphasizes the importance of detection of antibodies against T. cruzi in the integral evaluation of blood donors, since many of them with antibodies anti-T. cruzi, have donated blood several times previous to diagnosis.

Seccion de Biohelmintiasis, Instituto de MedicinaTropical, Universidad Central de Venezuela, Caracas,Venezuela *Laboratorio para Estudios sobre Malaria,Instituto Nacional de Higiene, Direccion deMalariologia y Saneamiento Ambiental, Caracas,VenezuelaKey words: schistosomiasis - immunodianosis -synthetic peptides

Chagas disease is an endemic zoonosis native to the Americas and is caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The parasite is also highly genetically diverse, with six discrete typing units (DTUs) reported TcI – TcVI. These DTUs broadly correlate with several epidemiogical, ecological and pathological features of Chagas disease. In this manuscript we report the most comprehensive evaluation to date of the genetic diversity of T. cruzi in Venezuela. The dataset includes 778 samples collected and genotyped over the last twelve years from multiple hosts and vectors, including nine wild and domestic mammalian host species, and seven species of triatomine bug, as well as from human sources. Most isolates (732) can be assigned to the TcI clade (94.1%); 24 to the TcIV group (3.1%) and 22 to TcIII (2.8%). Importantly, among the 95 isolates genotyped from human disease cases, 79% belonged to TcI - a DTU common in the Americas, however, 21% belonged to TcIV- a little known genotype previously thought to be rare in humans. Furthermore, were able to assign multiple oral Chagas diseases cases to TcI in the area around the capital, Caracas. We discuss our findings in the context of T. cruzi DTU distributions elsewhere in the Americas, and evaluate the impact they have on the future of Chagas disease control in Venezuela., Author Summary Chagas disease is caused by a protozoan parasite called Trypanosoma cruzi. T. cruzi infects a wide variety of mammal species in Latin America as well as man, and is spread by multiple species of blood sucking triatomine insect vectors. The presence of genetic diversity in T. cruzi in the Americas is well established, with six different major genetic types in circulation. The genetic diversity of T. cruzi in Venezuela is relatively poorly understood. In this work we present the results from the genotyping of over seven hundred isolates from 17 of the 24 states. Our dataset comprises strains isolated from wild and domestic animals, several species of triatomine vector, as well as from human Chagas disease cases, including those associated with oral transmission of T. cruzi. Amongst other findings, our data reveal a surprisingly high frequency of atypical genotypes in humans, particularly TcIV, which has rarely been reported. We evaluate our findings in the context of T. cruzi diversity elsewhere in the Americas, and assess the impact they have on the future of Chagas disease control in Venezuela.

A worm found in histopathologic sections of a lung piece of a young Venezuelan male was identified as a Paragonimus sp. Definitive identification of the species was not possible since only a deteriorated segment of the worm was recovered, nevertheless comparison with other known species is discussed. This is the first report of an indigenous case of human paragonimiasis in Venezuela.

Different enzymes of Schistosoma mansoni are recognized by IgG antibodies present in the sera of infected human patients. The antigenicity of these enzymes suggests their possible use in immunodiagnostic assays that would take advantage of their activities.

According to the World Health Organization (WHO), one of the milestones set by the London Declaration for Chagas disease the interruption of Chagas disease transmission through blood transfusion in Latin America was achieved in most countries (20 out of 21) in 2015. This is a crucial step towards reaching the goal of controlling Chagas disease by 2020. However, many challenges remain: less than 1% of the 6 to 7 million people infected with Trypanosoma cruzi are treated, and new infections still occu