Trypanosoma cruzi: allopurinol in the treatment of mice with experimental acute Chagas disease

The therapeutic effect of allopurinol was studied in an experimental Trypanosoma cruzi infection (Chagas disease) in outbred IVIC-NMRI and inbred C57B1/6J mice intraperitoneally inoculated with the parasites 2–6 days before drug treatment. Allopurinol protected against T. cruzi infection. This effect was evidenced by highly significant reductions in both parasitemias and mortality rates and increased survival time in allopurinol-treated animals compared with untreated infected mice. Allopurinol protected effectively when administered in 10 daily doses of 32–64 mg/kg body wt/day injected intraperitoneally. Using direct methods, parasitemia remained undetectable for at least 310 days. An indirect method, subinoculation to susceptible mice, showed a few circulating trypanosomes which decreased greatly in number after a second schedule of allopurinol treatment; finally no trypanosomes were detectable 275 days after treatment initiation. Allopurinol also induced a strong trypanostatic effect when tested in vitro on five different Trypanosoma cruzi strains (optimal inhibitory concentration: 3 μg/ml). These results suggest that allopurinol protects mice with acute Chagas infection by a direct trypanostatic effect. The low toxicity of this drug suggests its use in more chronic experimental Chagas infections.     

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients

Background

A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation.

Methodology/Findings

An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10⁻³ par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories.

Conclusion/Significance

This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.     

Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillos hosts of Trypanosoma cruzi II, including hybrids

Trypanosoma cruzi, the causative agent of Chagas disease, has at least two principal intraspecific subdivisions, T. cruzi I (TCI) and T. cruzi II (TCII), the latter containing up to five subgroups (a-e). Whilst it is known that TCI predominates from the Amazon basin northwards and TCII to the South, where the disease is considered to be clinically more severe, the precise clinical and evolutionary significance of these divisions remains enigmatic. Here, we present compelling evidence of an association between TCI and opossums (Didelphis), and TCII and armadillos, on the basis of key new findings from the Paraguayan Chaco region, together with a comprehensive analysis of historical data. We suggest that the distinct arboreal and terrestrial ecologies, respectively, of these mammal hosts provide a persuasive explanation for the extant T. cruzi intraspecific diversity in South America, and for separate origins of Chagas disease in northern South America and in the southern cone countries.     

Aspirin treatment of mice infected with Trypanosoma cruzi and implications for the pathogenesis of Chagas disease

Chagas disease, caused by infection with Trypanosoma cruzi, is an important cause of cardiovascular disease. It is increasingly clear that parasite-derived prostaglandins potently modulate host response and disease progression. Here, we report that treatment of experimental T. cruzi infection (Brazil strain) beginning 5 days post infection (dpi) with aspirin (ASA) increased mortality (2-fold) and parasitemia (12-fold). However, there were no differences regarding histopathology or cardiac structure or function. Delayed treatment with ASA (20 mg/kg) beginning 60 dpi did not increase parasitemia or mortality but improved ejection fraction. ASA treatment diminished the profile of parasite- and host-derived circulating prostaglandins in infected mice. To distinguish the effects of ASA on the parasite and host bio-synthetic pathways we infected cyclooxygenase-1 (COX-1) null mice with the Brazil-strain of T. cruzi. Infected COX-1 null mice displayed a reduction in circulating levels of thromboxane (TX)A(2) and prostaglandin (PG)F(2α). Parasitemia was increased in COX-1 null mice compared with parasitemia and mortality in ASA-treated infected mice indicating the effects of ASA on mortality potentially had little to do with inhibition of prostaglandin metabolism. Expression of SOCS-2 was enhanced, and TRAF6 and TNFα reduced, in the spleens of infected ASA-treated mice. Ablation of the initial innate response to infection may cause the increased mortality in ASA-treated mice as the host likely succumbs more quickly without the initiation of the "cytokine storm" during acute infection. We conclude that ASA, through both COX inhibition and other "off-target" effects, modulates the progression of acute and chronic Chagas disease. Thus, eicosanoids present during acute infection may act as immunomodulators aiding the transition to and maintenance of the chronic phase of the disease. A deeper understanding of the mechanism of ASA action may provide clues to the differences between host response in the acute and chronic T. cruzi infection.     

Trypanosoma cruzi ubiquitin as an antigen in the differential diagnosis of Chagas disease and leishmaniasis

In the present report we describe Trypanosoma cruzi ubiquitin as an antigen to be utilized in the differential diagnosis of Chagas disease and leishmaniasis. Initially, recombinant T. cruzi ubiquitin was evaluated against a panel of sera by phage dot immunoassay, showing a good performance against chagasic sera. However, the presence of a carboxy-terminal tail region encoding a ribosomal protein homologous to a related protein present in the genome of Leishmania sp. gave significant cross-reactivity with leishmanial sera. Therefore, ubiquitin was purified by a simple biochemical protocol and its immunoreactivity was studied by enzyme-linked immunosorbent assay. Analysis of 104 sera indicates that the response to ubiquitin is very sensitive towards chronic chagasic sera (98%) and, more important, highly species-specific, presenting better performance compared to the use of the recombinant protein or the total epimastigote extracts when tested against a panel of leishmanial sera, where out of a total of 70 sera tested, only five sera from the mucocutaneous form of the disease reacted with T. cruzi ubiquitin. On the other hand, Leishmania ubiquitin was not recognized by chagasic sera, but was recognized by sera from different forms of leishmaniasis. These results make ubiquitin an excellent candidate to be used in the differential diagnosis of these two parasitic diseases. The molecular basis for this highly species-specific response is discussed.     

Trypanosoma cruzi discrete typing units in Chagas disease patients from endemic and non-endemic regions of Argentina

Genetic diversity of Trypanosoma cruzi may play a role in pathogenesis of Chagas disease forms. Natural populations are classified into 6 Discrete Typing Units (DTUs) Tc I-VI with taxonomical status. This study aimed to identify T. cruzi DTUs in bloodstream and tissue samples of Argentinean patients with Chagas disease. PCR-based strategies allowed DTU identification in 256 clinical samples from 239 Argentinean patients. Tc V prevailed in blood from both asymptomatic and symptomatic cases and Tc I was more frequent in bloodstream, cardiac tissues and chagoma samples from immunosuppressed patients. Tc II and VI were identified in a minority of cases, while Tc III and Tc IV were not detected in the studied population. Interestingly, Tc I and Tc II/VI sequences were amplified from the same skin biopsy slice from a kidney transplant patient suffering Chagas disease reactivation. Further data also revealed the occurrence of mixed DTU populations in the human chronic infection. In conclusion, our findings provide evidence of the complexity of the dynamics of T. cruzi diversity in the natural history of human Chagas disease and allege the pathogenic role of DTUs I, II, V and VI in the studied population.     

Chronic Chagas disease with cardiodigestive involvement and the TcVI infective form of Trypanosoma cruzi. A case report

We report a patient with megacolon associated with TcVI infective lineage form of Trypanosoma cruzi. Although this megacolon was considered idiopathic, Chagas disease was suspected and diagnosed because of the concomitant cardiovascular involvement. Based on this case, we discuss the suitability of Chagas diagnosis in patients with tract motility involvement.     

Trypanosoma cruzi: 4-aminopyrazolopyrimidine in the treatment of experimental Chagas' disease

An allopurinol metabolite, 4-aminopyrazolopyrimidine, was tested on two different strains of mice (NMRI-IVIC and C57Bl/6J) that had been infected 4 days earlier with the virulent Ya strain of Trypanosoma cruzi. Low doses of 4-aminopyrazolopyrimidine (0.125-0.500 mg/kg body wt/day) for 10 days induced a significant reduction in parasitemia (direct counts and subinoculation experiments) and increased survival time (without any evidence of toxicity) compared with untreated animals. When tested in vitro, 4-aminopyrazolopyrimidine was sixfold more active than allopurinol as a trypanostatic drug. The low therapeutic doses of 4-aminopyrazolopyrimidine suggest that this drug may be useful in the treatment of acute Chagas' disease.     

Search for a platelet-activating factor receptor in the Trypanosoma cruzi proteome: a potential target for Chagas disease chemotherapy

Chagas disease (CD) causes the highest burden of parasitic diseases in the Western Hemisphere and is therefore a priority for drug research and development. Platelet-activating factor (PAF) causes the CD parasite Trypanosoma cruzi to differentiate, which suggests that the parasite may express PAF receptors. Here, we explored the T. cruzi proteome for PAF receptor-like proteins. From a total of 23,000 protein sequences, we identified 29 hypothetical proteins that are predicted to have seven transmembrane domains (TMDs), which is the main characteristic of the G protein-coupled receptors (GPCRs), including the PAF receptor. The TMDs of these sequences were independently aligned with domains from 25 animal PAF receptors and the sequences were analysed for conserved residues. The conservation score mean values for the TMDs of the hypothetical proteins ranged from 31.7-44.1%, which suggests that if the putative T. cruzi PAF receptor is among the sequences identified, the TMDs are not highly conserved. These results suggest that T. cruzi contains several GPCR-like proteins and that one of these GPCRs may be a PAF receptor. Future studies may further validate the PAF receptor as a target for CD chemotherapy.     

Stimulatory effects of leukotriene B4 on macrophage association with and intracellular destruction of Trypanosoma cruzi

The effects of leukotriene B4 (LTB4) on mouse peritoneal macrophage (MPH) association with and destruction of Trypanosoma cruzi were studied. The presence of 10(-8) to 10(-6) M LTB4 in co-cultures of MPH and T. cruzi enhanced their association (a term meaning surface binding and internalization), as evidenced by increases in the percentage of MPH associating with trypanosomes and the number of parasites per 100 MPH. Pretreatment of either parasites or MPH with LTB4 increased their association with the untreated counterpart, suggesting that the enhancement was a composite of effects on both cells. The effect of LTB4 on MPH was reversible, because normal levels of MPH-parasite association were recorded 60 min after the LTB4 pretreatment. However, the enhancement was demonstrable after the MPH were incubated with LTB4 for up to 24 hr, indicating that the effect lasted if LTB4 was present. Pretreatment with LTB4 also increased the capacity of MPH to take up glutaraldehyde-killed T. cruzi or latex beads, suggesting that LTB4 stimulated phagocytosis. Pretreatment of rat heart myoblasts--which are not phagocytic--with LTB4 also increased parasite association, suggesting that phagocytic ability was not an absolute requirement for production of the enhancement and that LTB4-induced alterations of the cell membrane facilitating parasite invasion may also be involved. An effect of LTB4 on MPH cytotoxicity was denoted by an increased rate of intracellular parasite killing. Two inhibitors of guanylate cyclase abrogated the enhancing effect of LTB4, suggesting that increased MPH levels of cyclic GMP--known to be increased by LTB4--mediated the effect. Because inflammatory cells, such as are found in acute chagasic lesions, are known to produce increased amounts of leukotrienes, the stimulatory effects of LTB4 could contribute to host defense against T. cruzi infection.     

Analysis of total human immunodeficiency virus (HIV)-specific CD4(+) and CD8(+) T-cell responses: relationship to viral load in untreated HIV infection

Human immunodeficiency virus (HIV)-specific T-cell responses are thought to play a key role in viral load decline during primary infection and in determining the subsequent viral load set point. The requirements for this effect are unknown, partly because comprehensive analysis of total HIV-specific CD4(+) and CD8(+) T-cell responses to all HIV-encoded epitopes has not been accomplished. To assess these responses, we used cytokine flow cytometry and overlapping peptide pools encompassing all products of the HIV-1 genome to study total HIV-specific T-cell responses in 23 highly active antiretroviral therapy na?ve HIV-infected patients. HIV-specific CD8(+) T-cell responses were detectable in all patients, ranging between 1.6 and 18.4% of total CD8(+) T cells. HIV-specific CD4(+) T-cell responses were present in 21 of 23 patients, although the responses were lower (0.2 to 2.94%). Contrary to previous reports, a positive correlation was identified between the plasma viral load and the total HIV-, Env-, and Nef-specific CD8(+) T-cell frequency. No correlation was found either between viral load and total or Gag-specific CD4(+) T-cell response or between the frequency of HIV-specific CD4(+) and CD8(+) T cells. These results suggest that overall frequencies of HIV-specific T cells are not the sole determinant of immune-mediated protection in HIV-infection.     

Extracellular ATP induces cell death in CD4+/CD8+ double-positive thymocytes in mice infected with Trypanosoma cruzi

In the acute phase of Trypanosoma cruzi infection, there is dramatic atrophy of the thymus. However, the pathways involved in this change have not yet been identified. This event is mainly characterized by a massive loss of cortical CD4+/CD8+ double-positive cells, but also by other structural and functional alterations in the organ. A number of molecules, including extracellular ATP, have been suggested to play a role in the selective processes that take place in the thymus. ATP and analogues trigger many different cellular responses in thymocytes and other cell types, such as the opening of plasma membrane cation channels and a pore that may induce cell death. Herein, we investigated the possible involvement of extracellular ATP in thymus atrophy induced by infection with T. cruzi. We observed that ATP induces an increase in plasma membrane permeabilization and cellular death in CD4+/CD8+ double-positive thymocytes collected from infected mice during the atrophy phase. No differences were observed prior to the atrophy phase or during the chronic phase. Our results indicate that P2Z/P2X7 receptors may play a central role in thymus atrophy during T. cruzi infection.     

Differential expression of Trypanosoma cruzi I associated with clinical forms of Chagas disease: overexpression of oxidative stress proteins in acute patient isolate

Chagas disease has a variable clinical course with different manifestations and heterogenous geographical distribution. Some studies suggest that this clinical variability could be influenced by the genetic variability of T. cruzi. Here we present the differential protein expression among trypomastigotes and amastigotes of T. cruzi group I isolates from patients with acute and chronic form of Chagas disease from Santander, Colombia. A total of 29 proteins were identified by MALDI-TOF and LC-MS/MS; twenty in trypomastigote and nine in amastigote stage. The 29 proteins identified were grouped in 7 functional categories: 1) metabolism 31%, 2) assembly of cytoskeleton 13.7%, 3) protein destination 13.7%, 4) defenses antioxidants 20.6%, 5) protein synthesis and cellular cycle 13.7%, 6) catabolism 6.8%, and 7) adhesion 3.4%. Tryparedoxin peroxidase, lipoamide dehydrogenase, tyrosine amino transferase and HSP70 were overexpressed in the acute Chagas isolate. Tryparedoxin peroxidase overexpression in the acute isolate was confirmed by Western blot analysis. Most of these proteins are associated with resistance to oxidative stress facilitating their survival within host cells. Therefore, these proteins may represent virulence factors associated with the development of the acute form of the disease and could be used as biomarkers of the clinical course of disease and as drug targets.     

Integration of Trypanosoma cruzi kDNA minicircle sequence in the host genome may be associated with autoimmune serum factors in Chagas disease patients

Integration of kDNA sequences within the genome of the host cell shown by PCR amplification with primers to the conserved Trypanosoma cruzi kDNA minicircle sequence was confirmed by Southern hybridization with specific probes. The cells containing the integrated kDNA sequences were then perpetuated as transfected macrophage subclonal lines. The kDNA transfected macrophages expressed membrane antigens that were recognized by antibodies in a panel of sera from ten patients with chronic Chagas disease. These antigens barely expressed in the membrane of uninfected, control macrophage clonal lines were recognized neither by factors in the control, non-chagasic subjects nor in the chagasic sera. This finding suggests the presence of an autoimmune antibody in the chagasic sera that recognizes auto-antigens in the membrane of T. cruzi kDNA transfected macrophage subclonal lines.     

Ecological Connectivity of Trypanosoma cruzi Reservoirs and Triatoma pallidipennis Hosts in an Anthropogenic Landscape with Endemic Chagas Disease

Traditional methods for Chagas disease prevention are targeted at domestic vector reduction, as well as control of transfusion and maternal-fetal transmission. Population connectivity of Trypanosoma cruzi-infected vectors and hosts, among sylvatic, ecotone and domestic habitats could jeopardize targeted efforts to reduce human exposure. This connectivity was evaluated in a Mexican community with reports of high vector infestation, human infection, and Chagas disease, surrounded by agricultural and natural areas. We surveyed bats, rodents, and triatomines in dry and rainy seasons in three adjacent habitats (domestic, ecotone, sylvatic), and measured T. cruzi prevalence, and host feeding sources of triatomines. Of 12 bat and 7 rodent species, no bat tested positive for T. cruzi, but all rodent species tested positive in at least one season or habitat. Highest T. cruzi infection prevalence was found in the rodents, Baiomys musculus and Neotoma mexicana. In general, parasite prevalence was not related to habitat or season, although the sylvatic habitat had higher infection prevalence than by chance, during the dry season. Wild and domestic mammals were identified as bloodmeals of T. pallidipennis, with 9% of individuals having mixed human (4.8% single human) and other mammal species in bloodmeals, especially in the dry season; these vectors tested >50% positive for T. cruzi. Overall, ecological connectivity is broad across this matrix, based on high rodent community similarity, vector and T. cruzi presence. Cost-effective T. cruzi, vector control strategies and Chagas disease transmission prevention will need to consider continuous potential for parasite movement over the entire landscape. This study provides clear evidence that these strategies will need to include reservoir/host species in at least ecotones, in addition to domestic habitats.     

Lactobacillus-dominated cervicovaginal microbiota associated with reduced HIV/STI prevalence and genital HIV viral load in African women

Cervicovaginal microbiota not dominated by lactobacilli may facilitate transmission of HIV and other sexually transmitted infections (STIs), as well as miscarriages, preterm births and sepsis in pregnant women. However, little is known about the exact nature of the microbiological changes that cause these adverse outcomes. In this study, cervical samples of 174 Rwandan female sex workers were analyzed cross-sectionally using a phylogenetic microarray. Furthermore, HIV-1 RNA concentrations were measured in cervicovaginal lavages of 58 HIV-positive women among them. We identified six microbiome clusters, representing a gradient from low semi-quantitative abundance and diversity dominated by Lactobacillus crispatus (cluster R-I, with R denoting ‘Rwanda'') and L. iners (R-II) to intermediate (R-V) and high abundance and diversity (R-III, R-IV and R-VI) dominated by a mixture of anaerobes, including Gardnerella, Atopobium and Prevotella species. Women in cluster R-I were less likely to have HIV (P=0.03), herpes simplex virus type 2 (HSV-2; P<0.01), and high-risk human papillomavirus (HPV; P<0.01) and had no bacterial STIs (P=0.15). Statistically significant trends in prevalence of viral STIs were found from low prevalence in cluster R-I, to higher prevalence in clusters R-II and R-V, and highest prevalence in clusters R-III/R-IV/R-VI. Furthermore, only 10% of HIV-positive women in clusters R-I/R-II, compared with 40% in cluster R-V, and 42% in clusters R-III/R-IV/R-VI had detectable cervicovaginal HIV-1 RNA (P_trend=0.03). We conclude that L. crispatus-dominated, and to a lesser extent L. iners-dominated, cervicovaginal microbiota are associated with a lower prevalence of HIV/STIs and a lower likelihood of genital HIV-1 RNA shedding.