Jacqueline Bua

Biological markers for evaluating therapeutic efficacy in Chagas disease, a systematic review

The most neglected aspects of Chagas disease (CD) have been patient care and treatment. Despite recent progress in the development of potentially improved drugs, there is no consensus among different research groups on the lack of therapeutic response markers to evaluate efficacy of newly proposed drugs early after treatment. A systematic review of current evidence regarding molecules which are potential biomarkers for therapeutic response has been conducted using quality assessment and target responses as primary criteria. The review provides a panorama of the cumulative evidence and specific needs for development of a battery of complementary biomarkers which together fulfill ideal or acceptable criteria to evaluate early responses to treatment for chronic CD. There are several marker candidates which together may fulfill acceptable criteria to indicate the efficacy of a trypanocidal treatment. Data from ongoing studies are considered essential to improve assessment of existing markers and to identify those for early follow-up of treated patients.

How to Improve the Early Diagnosis of Trypanosoma cruzi Infection: Relationship between Validated Conventional Diagnosis and Quantitative DNA Amplification in Congenitally Infected Children

Background According to the Chagas congenital transmission guides, the diagnosis of infants, born to Trypanosoma cruzi infected mothers, relies on the detection of parasites by INP micromethod, and/or the persistence of T. cruzi specific antibody titers at 10–12 months of age. Methodology and Principal Findings Parasitemia levels were quantified by PCR in T. cruzi-infected children, grouped according to the results of one-year follow-up diagnosis: A) Neonates that were diagnosed in the first month after delivery by microscopic blood examination (INP micromethod) (n = 19) had a median parasitemia of 1,700 Pe/mL (equivalent amounts of parasite DNA per mL); B) Infants that required a second parasitological diagnosis at six months of age (n = 10) showed a median parasitemia of around 20 Pe/mL and 500 Pe/mL at 1 and 6 months old, respectively, and C) babies with undetectable parasitemia by three blood microscopic observations but diagnosed by specific anti - T. cruzi serology at around 1 year old, (n = 22), exhibited a parasitemia of around 5 Pe/mL, 800 Pe/mL and 20 Pe/mL 1, 6 and 12 month after delivery, respectively. T. cruzi parasites were isolated by hemoculture from 19 congenitally infected children, 18 of which were genotypified as DTU TcV, (former lineage TcIId) and only one as TcI. Significance This report is the first to quantify parasitemia levels in more than 50 children congenitally infected with T. cruzi, at three different diagnostic controls during one-year follow-up after delivery. Our results show that the parasite burden in some children (22 out of 51) is below the detection limit of the INP micromethod. As the current trypanocidal treatment proved to be very effective to cure T. cruzi - infected children, more sensitive parasitological methods should be developed to assure an early T. cruzi congenital diagnosis.

Galectin-1 Prevents Infection and Damage Induced by Trypanosoma cruzi on Cardiac Cells

Background Chronic Chagas cardiomyopathy caused by Trypanosoma cruzi is the result of a pathologic process starting during the acute phase of parasite infection. Among different factors, the specific recognition of glycan structures by glycan-binding proteins from the parasite or from the mammalian host cells may play a critical role in the evolution of the infection. Methodology and Principal Findings Here we investigated the contribution of galectin–1 (Gal–1), an endogenous glycan-binding protein abundantly expressed in human and mouse heart, to the pathophysiology of T. cruzi infection, particularly in the context of cardiac pathology. We found that exposure of HL–1 cardiac cells to Gal–1 reduced the percentage of infection by two different T. cruzi strains, Tulahuén (TcVI) and Brazil (TcI). In addition, Gal–1 prevented exposure of phosphatidylserine and early events in the apoptotic program by parasite infection on HL–1 cells. These effects were not mediated by direct interaction with the parasite surface, suggesting that Gal–1 may act through binding to host cells. Moreover, we also observed that T. cruzi infection altered the glycophenotype of cardiac cells, reducing binding of exogenous Gal–1 to the cell surface. Consistent with these data, Gal–1 deficient (Lgals1 -/-) mice showed increased parasitemia, reduced signs of inflammation in heart and skeletal muscle tissues, and lower survival rates as compared to wild-type (WT) mice in response to intraperitoneal infection with T. cruzi Tulahuén strain. Conclusion/Significance Our results indicate that Gal–1 modulates T. cruzi infection of cardiac cells, highlighting the relevance of galectins and their ligands as regulators of host-parasite interactions.

Improving access to Chagas disease diagnosis and etiologic treatment in remote rural communities of the Argentine Chaco through strengthened primary health care and broad social participation

Background Rural populations in the Gran Chaco region have large prevalence rates of Trypanosoma cruzi infection and very limited access to diagnosis and treatment. We implemented an innovative strategy to bridge these gaps in 13 rural villages of Pampa del Indio held under sustained vector surveillance and control. Methodology The non-randomized treatment program included participatory workshops, capacity strengthening of local health personnel, serodiagnosis, qualitative and quantitative PCRs, a 60-day treatment course with benznidazole and follow-up. Parents and healthcare agents were instructed on drug administration and early detection and notification of adverse drug-related reactions (ADR). Healthcare agents monitored medication adherence and ADRs at village level. Principal findings The seroprevalence of T. cruzi infection was 24.1% among 395 residents up to 18 years of age examined. Serodiagnostic (70%) and treatment coverage (82%) largely exceeded local historical levels. Sixty-six (85%) of 78 eligible patients completed treatment with 97% medication adherence. ADRs occurred in 32% of patients, but most were mild and manageable. Four patients showing severe or moderate ADRs required treatment withdrawal. T. cruzi DNA was detected by qPCR in 47 (76%) patients before treatment, and persistently occurred in only one patient over 20–180 days posttreatment. Conclusions and significance Our results demonstrate that diagnosis and treatment of T. cruzi infection in remote, impoverished rural areas can be effectively addressed through strengthened primary healthcare attention and broad social participation with adequate external support. This strategy secured high treatment coverage and adherence; effectively managed ADRs, and provided early evidence of positive therapeutic responses.

Oxidative stress damage in the protozoan parasite Trypanosoma cruzi is inhibited by Cyclosporin A.

Cyclosporin A (CsA) specifically inhibits the mitochondrial permeability transition pore (mPTP). Opening of the mPTP, which is triggered by high levels of matrix [Ca2+] and/or oxidative stress, leads to mitochondrial dysfunction and thus to cell death by either apoptosis or necrosis. In the present study, we analysed the response of Trypanosoma cruzi epimastigote parasites to oxidative stress with 5 mm H2O2, by studying several features related to programmed cell death and the effects of pre-incubation with 1 μ m of CsA. We evaluated TcPARP cleavage, DNA integrity, cytochrome c translocation, Annexin V/propidium iodide staining, reactive oxygen species production. CsA prevented parasite oxidative stress damage as it significantly inhibited DNA degradation, cytochrome c translocation to cytosol and TcPARP cleavage. The calcein-AM/CoCl2 assay, used as a selective indicator of mPTP opening in mammals, was also performed in T. cruzi parasites. H2O2 treatment decreased calcein fluorescence, but this decline was partially inhibited by pre-incubation with CsA. Our results encourage further studies to investigate if there is a mPTP-like pore and a mitochondrial cyclophilin involved in this protozoan parasite.

A homolog of cyclophilin D is expressed in Trypanosoma cruzi and is involved in the oxidative stress–damage response

Mitochondria have an important role in energy production, homeostasis and cell death. The opening of the mitochondrial permeability transition pore (mPTP) is considered one of the key events in apoptosis and necrosis, modulated by cyclophilin D (CyPD), a crucial component of this protein complex. In Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, we have previously described that mitochondrial permeability transition occurs after oxidative stress induction in a cyclosporin A-dependent manner, a well-known cyclophilin inhibitor. In the present work, a mitochondrial parasite cyclophilin, named TcCyP22, which is homolog to the mammalian CyPD was identified. TcCyP22-overexpressing parasites showed an enhanced loss of mitochondrial membrane potential and loss of cell viability when exposed to a hydrogen peroxide stimulus compared with control parasites. Our results describe for the first time in a protozoan parasite that a mitochondrial cyclophilin is a component of the permeability transition pore and is involved in regulated cell death induced by oxidative stress.

Some Limitations for Early Diagnosis of Congenital Chagas Infection by PCR

Case report of an infrequent diagnosis of congenital Trypanosoma cruzi infection, causative agent of Chagas disease, negative for parasite diagnosis until 7 months after delivery. Trypanosoma cruzi, the causing agent of Chagas disease, can be transmitted to the offspring of infected pregnant women, thus being an epidemiologically important way of parasite transmission in humans. In addition, the migration of infected women from endemic areas to nonendemic countries may export this parasite infection. The diagnosis of congenital Chagas disease relies on the detection of the parasite because maternal antibodies are passively transferred to infants during pregnancy. The diagnosis of congenital infection can also be confirmed by detection of infant-specific anti–T cruzi antibodies at 10 months after delivery. Because early detection of T cruzi infection in newborns allows an efficient trypanocidal treatment and cure, more sensitive molecular techniques such as DNA amplification are being used for a prompt parasitological diagnosis of children born to seropositive mothers. In this report, we describe a diagnosis case of a child congenitally infected with T cruzi who tested negative for parasite detection both by microscopic observation and DNA amplification at 20 days and 6 months after delivery. However, at 7 months of age, a hemoculture was made from the infant’s blood, and the infective parasite was finally isolated and classified as T cruzi discrete typing unit I. In a retrospective study, real-time polymerase chain reaction also allowed detecting the parasite but failed to detect any parasite load in earlier control samples. This case report stresses that even when molecular techniques are negative, a long-term follow-up is necessary for the diagnosis of infants congenitally infected with T cruzi.

Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates

Trypanosoma cruzi is a genetically heterogeneous group of organisms that cause Chagas disease. It has been long suspected that the clinical outcome of the disease and response to therapeutic agents are, at least in part, related to the genetic characteristics of the parasite. Herein, we sought to validate the significance of the genotype of T. cruzi isolates recovered from patients with different clinical forms of Chagas disease living in Argentina on their biological behaviour and susceptibility to drugs. Genotype identification of the newly established isolates confirmed the reported predominance of TcV, with a minor frequency of TcI. Epimastigote sensitivity assays demonstrated marked dissimilar responses to benznidazole, nifurtimox, pentamidine and dihydroartemisinin in vitro. Two TcV isolates exhibiting divergent response to benznidazole in epimastigote assays were further tested for the expression of anti-oxidant proteins. Benznidazole-resistant BOL-FC10A epimastigotes had decreased expression of Old Yellow Enzyme and cytosolic superoxide dismutase, and overexpression of mitochondrial superoxide dismutase and tryparedoxin- 1, compared to benznidazole-susceptible AR-SE23C parasites. Drug sensitivity assays on intracellular amastigotes and trypomastigotes reproduced the higher susceptibility of AR-SE23C over BOL-FC10A parasites to benznidazole observed in epimastigotes assays. However, the susceptibility/resistance profile of amastigotes and trypomastigotes to nifurtimox, pentamidine and dihydroartemisinin varied markedly with respect to that of epimastigotes. C3H/He mice infected with AR-SE23C trypomastigotes had higher levels of parasitemia and mortality rate during the acute phase of infection compared to mice infected with BOL-FC10A trypomastigotes. Treatment of infected mice with benznidazole or nifurtimox was efficient to reduce patent parasitemia induced by either isolate. Nevertheless, qPCR performed at 70 dpi revealed parasite DNA in the blood of mice infected with AR-SE23C but not in BOL-FC10A infected mice. These results demonstrate high level of intra-type diversity which may represent an important obstacle for the testing of chemotherapeutic agents.

Distinct Treatment Outcomes of Antiparasitic Therapy in Trypanosoma cruzi-Infected Children Is Associated With Early Changes in Cytokines, Chemokines, and T-Cell Phenotypes

Background: In contrast to adults, Trypanosoma cruzi-infected children have more broadly functional Trypanosoma cruzi-specific T cells, and the total T-cell compartment exhibits fewer signs of immune exhaustion. However, not much is known about the link between immunocompetence and the treatment efficacy for human Chagas disease. Methods: Using cytokine enzyme-linked immunosorbent spot (ELISPOT) polychromatic flow cytometry, cytometric bead assay, multiplex serological assays and quantitative PCR, we evaluated T. cruzi-specific T-cell and antibody immune responses, T-cell phenotypes and parasitemia in children in the early chronic phase of Chagas disease undergoing anti-Trypanosoma cruzi treatment. Results: Treatment with benznidazole or nifurtimox induced a decline in T. cruzi-specific IFN-γ- and IL-2-producing cells and proinflammatory cytokines and chemokines. T-cell responses became detectable after therapy in children bearing T-cell responses under background levels prior to treatment. The total frequencies of effector, activated and antigen-experienced T cells also decreased following anti-T. cruzi therapy, along with an increase in T cells expressing the receptor of the homeostatic cytokine IL-7. Posttreatment changes in several of these markers distinguished children with a declining serologic response suggestive of successful treatment from those with sustained serological responses in a 5-year follow-up study. A multivariate analysis demonstrated that lower frequency of CD4+CD45RA−CCR7−CD62L− T cells prior to drug therapy was an independent indicator of successful treatment. Conclusions: These findings further validate the usefulness of alternative metrics to monitor treatment outcomes. Distinct qualitative and quantitative characteristics of T cells prior to drug therapy may be linked to treatment efficacy.

Improved Immuno-Detection of a Low-Abundance Cyclophilin Allows the Confirmation of its Expression in a Protozoan Parasite

Protein samples can be challenging to analyze due to the presence of high-abundance proteins masking lowabundance proteins of interest, such as biomarkers and novel physiological mediators. Cyclophilins are chaperones involved in the cis/trans isomerization of peptidyl-prolyl bonds in peptides or proteins and have been found in every organism sequenced to date. Although considerable progress has been made in the characterization of some cyclophilins expressed in diverse parasites invading humans, the main aspects of low-abundance members of this family remain unknown. In the present work, we present that the combined strategy of using more specific antibodies and increasing the presence of subcellular proteins in the sample, allowed us to confirm the expression of a 21.1 kDa cyclophilin for the first time in Trypanosoma cruzi.