Loop-Mediated Isothermal Amplification of Trypanosoma cruzi DNA for Point-of-Care Follow-Up of Anti-Parasitic Treatment of Chagas Disease.

A loop-mediated isothermal amplification assay was evaluated as a surrogate marker of treatment failure in Chagas disease (CD). A convenience series of 18 acute or reactivated CD patients who received anti-parasitic treatment with benznidazole was selected-namely, nine orally infected patients: three people living with HIV and CD reactivation, five chronic CD recipients with reactivation after organ transplantation and one seronegative recipient of a kidney and liver transplant from a CD donor. Fifty-four archival samples (venous blood treated with EDTA or guanidinium hydrochloride-EDTA buffer and cerebrospinal fluid) were extracted using a Spin-column manual kit and tested by T. cruzi Loopamp kit (Tc-LAMP, index test) and standardized real-time PCR (qPCR, comparator test). Of them, 23 samples were also extracted using a novel repurposed 3D printer designed for point-of-care DNA extraction (PrintrLab). The agreement between methods was estimated by Cohen's kappa index and Bland-Altman plot analysis. The T. cruzi Loopamp kit was as sensitive as qPCR for detecting parasite DNA in samples with parasite loads higher than 0.5 parasite equivalents/mL and infected with different discrete typing units. The agreement between qPCR and Tc-LAMP (Spin-column) or Tc-LAMP (PrintrLab) was excellent, with a mean difference of 0.02 [CI = -0.58-0.62] and -0.04 [CI = -0.45-0.37] and a Cohen's kappa coefficient of 0.78 [CI = 0.60-0.96] and 0.90 [CI = 0.71 to 1.00], respectively. These findings encourage prospective field studies to validate the use of LAMP as a surrogate marker of treatment failure in CD.

Polymorphisms of the TLR4 gene: Risk factor for chronicity and severity in oral vectorial Chagas disease.

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.

Ten-year follow-up of the largest oral Chagas disease outbreak. Laboratory biomarkers of infection as indicators of therapeutic failure.

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.

Genome of the fatal tapeworm Sparganum proliferum uncovers mechanisms for cryptic life cycle and aberrant larval proliferation.

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.

Purification of Glycosylphosphatidylinositol-Anchored Mucins from Trypanosoma cruzi Trypomastigotes and Synthesis of α-Gal-Containing Neoglycoproteins: Application as Biomarkers for Reliable Diagnosis and Early Assessment of Chemotherapeutic Outcomes of Chagas Disease.

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.

Nifurtimox response of Trypanosoma cruzi isolates from an outbreak of Chagas disease in Caracas, Venezuela.

BACKGROUND & OBJECTIVES: 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. METHODS: 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 (IC50) values. RESULTS: Parasite 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 IC50 values ranging from 4.07 ± 1.82 to 94.92 ± 7.24 µM. INTERPRETATION & CONCLUSION: The high heterogeneity in nifurtimox IC50 values in the isolates and clones from the OChD patients, suggests that the therapeutic failure to nifurtimox could be due in part to a phenotypic variability that existed in the wild parasite population at the original source of contamination. Though, further pharmacological studies are needed to confirm the existence of natural nifurtimox resistance in the parasite.

An ecological overview on the factors that drives to Trypanosoma cruzi oral transmission.

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.

Improvements and Variants of the Multiple Antigen Blot Assay-MABA: An Immunoenzymatic Technique for Simultaneous Antigen and Antibody Screening.

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.

Analytical performance of a multiplex Real-Time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.

BACKGROUND: The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. METHODS/PRINCIPAL FINDINGS: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. CONCLUSIONS/SIGNIFICANCE: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.

Rapid detection of Trypanosoma cruzi in human serum by use of an immunochromatographic dipstick test.

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%).

[A confirmatory diagnosis of antibodies anti-Trypanosoma cruzi in donors referred by blood banks in Venezuela]. / Diagnóstico confirmatorio de anticuerpos anti-Trypanosoma cruzi en donantes referidos por bancos de sangre en Venezuela.

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.

Clinical and ultrasound findings before and after praziquantel treatment among Venezuelan schistosomiasis patients.

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.