Mariano J. Levin

Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients.

Background This report describes a real-time PCR (Q-PCR) strategy to quantify Trypanosoma cruzi (T. cruzi) DNA in peripheral blood samples from Chagas disease patients targeted to conserved motifs within the repetitive satellite sequence. Methodology/Principal Findings The Q-PCR has a detection limit of 0.1 and 0.01 parasites/mL, with a dynamic range of 106 and 107 for Silvio X10 cl1 (T. cruzi I) and Cl Brener stocks (T. cruzi IIe), respectively, an efficiency of 99%, and a coefficient of determination (R 2) of 0.998. In order to express accurately the parasitic loads: (1) we adapted a commercial kit based on silica-membrane technology to enable efficient processing of Guanidine Hydrochloride-EDTA treated blood samples and minimize PCR inhibition; (2) results were normalized incorporating a linearized plasmid as an internal standard of the whole procedure; and (3) a correction factor according to the representativity of satellite sequences in each parasite lineage group was determined using a modified real-time PCR protocol (Lg-PCR). The Q-PCR strategy was applied (1) to estimate basal parasite loads in 43 pediatric Chagas disease patients, (2) to follow-up 38 of them receiving treatment with benznidazole, and (3) to monitor three chronic Chagas heart disease patients who underwent heart-transplantation and displayed events of clinical reactivation due to immunosupression. Conclusion/Significance All together, the high analytical sensitivity of the Q-PCR strategy, the low levels of intra- and inter-assay variations, as well as the accuracy provided by the Lg-PCR based correction factor support this methodology as a key laboratory tool for monitoring clinical reactivation and etiological treatment outcome in Chagas disease patients.

Molecular karyotype of clone CL Brener chosen for the Trypanosoma cruzi Genome Project

a Escola Paul&a de Medicina, Rua Botucatu, 862, CEP 04023-062, Stio Pa&o, Brazil b Institute de Quimica da USP, Sao Paula, Brazil ’ Institute de Inuestigaciones en Engenharia Genetica y Biologia Molecular, Buenos Aires. Argentina ’ Instituto de Parasitologia y Biomedicina, Granada, Spain e FIOCRUZ, Rio de Janeiro, Brazil f Instituto de Biofisica, UFRJ, Rio de Janeiro, Brazil g Centro de Biologia Celular, XV, Caracas, Venezuela h Centro de Biologia Molecular, IJAM_ Madrid, Spain

Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation.

BACKGROUND One hundred years after the discovery of Chagas disease, it remains a major neglected tropical disease. Chronic Chagas heart disease (cChHD) is the most severe manifestation. Heart transplantation is the proper treatment for end-stage heart failure, although reactivation of disease may result after receipt of immunosuppressive therapy. T. cruzi strains cluster into 6 discrete typing units (DTUs; I-VI) associated with different geographical distribution, transmission cycles and varying disease symptoms. In the southern cone of South America, T. cruzi II, V, and VI populations appear to be associated with Chagas disease and T. cruzi I with sylvatic cycles. METHODS Molecular characterization of DTUs, T. cruzi I genotypes (on the basis of spliced-leader gene polymorphisms), and minicircle signatures was conducted using cardiac explant specimens and blood samples obtained from a cohort of 16 Argentinean patients with cChHD who underwent heart transplantation and from lesion samples obtained from 6 of these patients who presented with clinical reactivation of Chagas disease. RESULTS Parasite persistence was associated with myocarditis progression, revealing T. cruzi I (genotype Id) in 3 explant samples and T. cruzi II, V, or VI in 5 explant samples. Post-heart transplantation follow-up examination of bloodstream DTUs identified T. cruzi I in 5 patients (genotypes Ia or Id) and T. cruzi II, V, or VI in 7 patients. T. cruzi I, V, and VI were detected in skin chagoma specimens, and T. cruzi V and VI were detected in samples obtained from patients with myocarditis reactivations. Multiple DTUs or genotypes at diverse body sites and polymorphic minicircle signatures at different cardiac regions revealed parasite histotropism. T. cruzi I infections clustered in northern Argentina (latitude, 23 degrees S-27 degrees S), whereas T. cruzi II, V, or VI DTUs were more ubiquitous. CONCLUSIONS Multiple DTUs coexist in patients with Chagas disease. The frequent finding of T. cruzi I associated with cardiac damage was astounding, revealing its pathogenic role in cChHD at the southern cone.

Functional analysis of the intergenic regions of TcP2β gene loci allowed the construction of an improved Trypanosoma cruzi expression vector

TcP2beta ribosomal protein genes in Trypanosoma cruzi are encoded by four different loci, H6.4, H1.8, H1.5 and H1.3. All loci have a similar organization, except for H1.8 that harbors two TcP2beta genes arranged in tandem and separated by a short repetitive sequence, named SIRE (short interspersed repetitive element), which is also found upstream of the first gene of the tandem and downstream of the second. In this locus the trans-splicing signal of TcP2beta is located within the SIRE element, while in the other loci it is positioned within the first 50bases upstream of the AUG with an AG acceptor site at position -12 respective to the initiation codon. Transient transfection experiments were used to evaluate the efficiency of these two different trans-splicing regions to drive CAT activity. The region named HX1 located upstream the TcP2beta H1. 8 gene was clearly more efficient than the SIRE sequence contained in the region named HX2. Therefore, we decided to use the HX1 region to ameliorate the performance of the cryptic trans-splicing signal present in the T. cruzi expression vector pRIBOTEX (Martinez-Calvillo, S., López, I., Hernandez, H., 1997. pRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants. Gene 199, 71-76). By insertion of the region HX1 downstream of the ribosomal promoter of pRIBOTEX, we constructed pRHX1CAT40 that, in stable transfected cells, was able to drive CAT activity 2760 times more efficiently than the control plasmids. Based on this, a novel plasmid vector was conceived, named pTREX-n, which retains the neo gene of pRIBOTEX as a positive selectable marker and replaces the CAT-SV40 cassette in pRHX1CAT40 by a multiple cloning site.

Molecular epidemiology of domestic and sylvatic Trypanosoma cruzi infection in rural northwestern Argentina

Genetic diversity of Trypanosoma cruzi populations and parasite transmission dynamics have been well documented throughout the Americas, but few studies have been conducted in the Gran Chaco ecoregion, one of the most highly endemic areas for Chagas disease, caused by T. cruzi. In this study, we assessed the distribution of T. cruzi lineages (identified by PCR strategies) in Triatoma infestans, domestic dogs, cats, humans and sylvatic mammals from two neighbouring rural areas with different histories of transmission and vector control in northern Argentina. Lineage II predominated amongst the 99 isolates characterised and lineage I amongst the six isolates obtained from sylvatic mammals. T. cruzi lineage IIe predominated in domestic habitats; it was found in 87% of 54 isolates from Tr. infestans, in 82% of 33 isolates from dogs, and in the four cats found infected. Domestic and sylvatic cycles overlapped in the study area in the late 1980s, when intense domestic transmission occurred, and still overlap marginally. The introduction of T. cruzi from sylvatic into domestic habitats is likely to occur very rarely in the current epidemiological context. The household distribution of T. cruzi lineages showed that Tr. infestans, dogs and cats from a given house compound shared the same parasite lineage in most cases. Based on molecular evidence, this result lends further support to the importance of dogs and cats as domestic reservoir hosts of T. cruzi. We believe that in Argentina, this is the first time that lineage IIc has been isolated from naturally infected domestic dogs and Tr. infestans.

Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes.

The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia+Tc Id, Tc Ia+Tc Ie and Tc Id+Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time.

Expression of the transferrin gene during development of non-hepatic tissues: High level of transferrin mRNA in fetal muscle and adult brain

Using a cloned rat transferrin cDNA probe, we looked for transferrin mRNA in the various rat tissues during development. In all the cases the mRNA detected seemed to be the same and to be product of a single gene. The transferrin gene is early expressed at a high level during liver differentiation. In the muscle and other non-hepatic and non-nervous tissues, the gene expression is maximal just before birth (19-20th day of gestational age), then markedly decreases during the postnatal development, the mRNA level being very low in the adult tissues. In brain, by contrast, transferrin mRNA level is very low before birth, then gradually increases during the postnatal development and reaches a plateau in the adult. Maximal mRNA concentration in fetal muscle (2 days before birth) and adult brain is about 1:7 to 1:10 of that obtained in adult liver. These results are analyzed in the light of the evidence that transferrin is not only an iron-binding protein, but also a factor involved in cell proliferation and differentiation, and particularly in nerve control of muscle differentiation.

Differential Profile and Biochemical Effects of Antiautonomic Membrane Receptor Antibodies in Ventricular Arrhythmias and Sinus Node Dysfunction

BackgroundThe relationship between anti-&bgr;-adrenergic (anti-&bgr;R) and anti-M2-cholinergic (anti-M2R) receptor antibodies (Abs) and cardiac arrhythmias and their biochemical effects have not been systematically investigated. Methods and ResultsWe studied 41 patients, 28 with ventricular arrhythmias (primary or due to Chagas’ heart disease or idiopathic dilated cardiomyopathy; group I), 13 with sinus node dysfunction (primary or caused by Chagas’ heart disease; group II), and 10 healthy controls (group III). The chronotropic effects of the IgG and immunopurified anti-&bgr;1RAbs or anti-M2RAbs were assessed on cultured cardiomyocytes before and after exposure to atropine and propranolol. The biochemical effects of the IgG from 9 patients from group I, 6 from group II, and 6 controls were evaluated on COS7 cells transfected with genes encoding for &bgr;1,&bgr;2-adrenergic receptors (cAMP increment) or M2-cholinergic receptors (phosphatidylinositol increment). The IgG from group I patients exerted a positive chronotropic action, with a high prevalence of anti-&bgr;RAbs (75%) and low prevalence of anti-M2RAbs (10.7%) and induced a clear-cut and long-lasting increment in cAMP. The IgG from group II patients depressed chronotropism, with a high prevalence of anti-M2RAbs (76.9%) and low prevalence of anti-&bgr;RAbs (15.4%) and evoked a marked augmentation of phosphatidylinositol. ConclusionsOur results demonstrate a strong correlation between anti-&bgr;RAbs and ventricular arrhythmias and anti-M2RAbs and sinus node dysfunction. Anti-&bgr;RAbs increase and anti-M2RAbs inhibit cAMP production. These findings offer new insight into the etiology and pathophysiology of cardiac arrhythmias, with therapeutic implications.

Chagas' disease diagnosis: a multicentric evaluation of Chagas Stat-Pak, a rapid immunochromatographic assay with recombinant proteins of Trypanosoma cruzi.

A rapid serologic test for diagnosis of T. cruzi infection (Chagas Stat Pak) was developed using recombinant proteins in an immunochromatographic assay. This cassette format test was evaluated first in blind with a panel of 393 coded serum samples. The Chagas Stat-Pak identified 197 infected (98.5% sensitivity) and 183 non-infected individuals (94.8% specificity). A second evaluation was performed with 352 sera from four Latin America countries tested independently in each country, showing a sensitivity of 100% and specificity of 98.6%. A third set of tests comparing sera with plasma and eluates from filter paper as well as serum preserved in 50% glycerol did show identical results as those obtained with serum. This rapid test (15 min) uses one device per sample, does not require refrigeration nor a laboratory structure or specialized skills to be performed, accepts different types of samples and may be stored for long periods of time for result checking and documentation. These attributes together with the high sensitivity and specificity demonstrated herein, make this test a suitable tool for field studies, small laboratories and emergencies at blood banks in the countryside of endemic areas.

Humoral autoimmune response to ribosomal P proteins in chronic Chagas heart disease.

The C terminal region of a Trypanosoma cruzi ribosomal P protein, encoded by the λgtll JL5 recombinant, defined a major antigenic determinant in chronic Chagas heart disease. Immunopurified anti‐JL5 antibodies were tested for anti‐human ribosome reactivity by immunoblotting. They recognized the parasite ribosomal P proteins and clearly reacted with the corresponding human P proteins. The peptide R‐13, that comprises the 13 C terminal residues of the JL5 recombinant and defines the specificity shared between chronic Chagas heart disease anti‐JL5 antibodies and the systemic lupus erythematosus (SLE) anti‐P antibodies, was used to study the specificity and the IgG subclass distribution of the anti‐R‐13 response by ELISA. The R‐13 autoepitope is recognized mainly by sera from chagasic patients, but not by sera from malaria patients. Moreover, there was a significant correlation between anti‐R‐13 antibody levels and anti‐T. cruzi antibody titres. The anti‐R‐13 response was mainly restricted to the IgG1 heavy chain isotype and correlated with the anti‐T. cruzi isotype distribution.