The Drugs for Neglected Diseases initiative (DNDi) has defined and implemented an early discovery strategy over the last few years, in fitting with its virtual R&D business model. This strategy relies on a medium- to high-throughput phenotypic assay platform to expedite the screening of compound libraries accessed through its collaborations with partners from the pharmaceutical industry. We review the pragmatic approaches used to select compound libraries for screening against kinetoplastids, taking into account screening capacity. The advantages, limitations and current achievements in identifying new quality series for further development into preclinical candidates are critically discussed, together with attractive new approaches currently under investigation.

The rTSSA-II (recombinant Trypomastigote Small Surface II) antigen was evaluated by ELISA to detect anti-Trypanosoma cruzi antibodies in sera from naturally infected dogs and humans. For this evaluation ELISA-rTSSA-II was standardized and groups were classified according to the results obtained through xenodiagnosis, ELISA and PCR. Sensitivity (Se), Specificity (Sp), Kappa index (KI) and area under curve (AUC) were determined. The Se was determined by using 14 sera from dogs infected with T. cruzi VI (TcVI) whereas Sp was determined by using 95 non-chagasic sera by xenodiagnosis, ELISA-Homogenate and PCR. The performance of ELISA-rTSSA-II in dog sera was high (AUC=0·93 and KI=0·91). The Se was 92·85% (1 false negative) and Sp was 100%. Two sera from dogs infected with TcI and 1 with TcIII were negative. For patients infected with T. cruzi, reactivity was 87·8% (36/41), there was only 1 indeterminate, and Sp was 100%. Fifty-four sera from non-chagasic and 68 sera from patients with cutaneous leishmaniasis did not react with rTSS-II. ELISA-rTSSA-II showed a high performance when studying sera from naturally infected dogs and it also presented 100% Sp. This assay could be an important tool to carry out sero-epidemiological surveys on the prevalence of T. cruzi circulating lineages in the region.

Chagas' disease is an opportunistic infection in the setting of HIV/AIDS. The arrival of HIV-positive immigrants from endemic areas to non-endemic countries makes possible the detection of Chagas' disease in this group of patients. We describe the results of a screening programme conducted in the HIV-positive immigrant population arriving from endemic areas who attended the Tropical Medicine Unit of Hospital Universitario Central of Asturias during 2008. We determined anti-T. cruzi antibodies in all HIV patients arriving from endemic areas who were followed up. The ID-Chagas antibody test was used as a screening assay. The positive cases were confirmed with ELISA, IFAT and PCR. We analysed 19 HIV-positive immigrants, of which two (10·5%) had a positive antibody test for Chagas' disease confirmed. PCR was positive in both cases. There was no difference between the co-infected and the non-co-infected patients with respect to race, place of birth and residence, CD4+ cell count, and HIV viral load count. Direct microscopic examination of blood was negative in both positive cases. The positive patients were a man from Bolivia and woman from Paraguay. The overlap of HIV and T. cruzi infection occurs not only in endemic areas but also in non-endemic areas of North America and Europe where the diagnosis may be even more difficult due to low diagnostic suspicion. The implementation of screening programmes in this population group is needed for the early diagnostic of Chagas' disease.

The biodiversity of Trypanosoma cruzi is one of the main factors complicating the understanding of its molecular epidemiology. As an alternative to classical genetic methods, investigators have used DNA-mediated transformation techniques to study this diversity. Recently, transient expression data were shown to correlate with the genetic data. This led investigators to speculate on a potential speciation within clonal populations of Trypanosoma cruzi. To further test the phylogenetic significance of transient expression analysis in Trypanosoma cruzi, multiple plasmids were used to drive the expression of a reporter gene in different parasite populations. In our study, population specific expression of the reporter gene was observed but the variability revealed by these transient expression assays was very large and did not follow the grouping of Trypanosoma cruzi populations in 2 lineages. In this report, we discuss some of the limitations of transient assays on such a diverse parasite.

Phosphoarginine is a cell energy buffer molecule synthesized by the enzyme arginine kinase. In Trypanosoma cruzi, the aetiological agent of Chagas' disease, 2 different isoforms were identified by data mining, but only 1 was expressed during the parasite life cycle. The digitonin extraction pattern of arginine kinase differed from those obtained for reservosomes, glycosomes and mitochondrial markers, and similar to the cytosolic marker. Immunofluorescence analysis revealed that although arginine kinase is localized mainly in unknown punctuated structures and also in the cytosol, it did not co-localize with any of the subcelular markers. This punctuated pattern has previously been observed in many cytosolic proteins of trypanosomatids. The knowledge of the subcellular localization of phosphagen kinases is a crucial issue to understand their physiological role in protozoan parasites.

During invasion of multicellular organisms, protozoan parasites expose functional molecules that become targets for the host immune response. Recent research on Trypanosoma cruzi, the agent of Chagas' disease, suggests a new model of how the parasite might deal with this problem. Several antigens of T. cruzi have tandemly repeated amino acid motifs in molecules with as yet unknown functions. In two cases, these repeats are in molecules with a defined structure or function. Both proteins are implicated in the invasion of host-cells by the parasite. One of these is the core protein of a putative mucin-like glycoprotein that has Thr/Pro–rich repeats which, by themselves, might define the structure of a highly O-glycosylated molecule. The other protein is SAPA/trans-sialidase/neuraminidase, a molecule able to transfer sialic acid, that has so far only been described in trypanosomes. The amino acid repeats present in SAPA/trans-sialidase/neuraminidase are unrelated to the enzymic activity and constitute an immunodominant C–terminal domain. The N–terminal domain of SAPA/trans-sialidase/neuraminidase controls the enzymic activity since a recombinant molecule lacking the repeats conserves trans-sialidase activity. That both domains are functionally independent is also indicated by experiments that show that antibodies directed against the amino acid repeats are unable to inhibit trans-sialidase activity. A large number of proteins having trans-sialidase related sequences but lacking enzymic activity are also present in the surface membrane of the parasite. The immunodominant SAPA/trans-sialidase/neuraminidase repeats, together with the complex network of cross-reacting epitopes present in related but enzymically inactive proteins might contribute to the delay in mounting an effective antibody response. However, antibodies neutralizing trans-sialidase activity are generated later during the infection. These antibody specificities are directed to the enzymic domain of the molecule and might contribute to the control of parasite dissemination after the early period of the infection.

We previously reported that blood forms of Trypanosoma cruzi express alpha- and beta-adrenergic receptors and that binding of specific agonists to these receptors modifies the infective capacity of the parasite in vitro. The present study has revealed that the inhibitory effect of the beta-adrenergic agonist L-isoproterenol and the stimulatory effect of the alpha-adrenergic agonist L-phenylephrine are not produced when the parasite is subjected to prolonged exposure to otherwise effective doses of these agonists or when supraoptimal doses of these agonists are used. We refer to these phenomena as ‘desensitization’ because of their analogy with vertebrate cells becoming desensitized by prolonged exposure to, or relatively high concentrations of, adrenergic agonists. At a constant agonist concentration, T. cruzi desensitization was time-dependent and, when the time of parasite treatment with the agonists was not changed, the higher concentrations of the agonist tested were the most effective in producing desensitization. The reduced infectivity resulting from treatment with optimal doses of L-isoproterenol was accompanied by elevated levels of cyclic adenosine mono- phosphate (cAMP) which were not detectable when L-isoproterenol concentrations producing desensitization were used. This finding implicated cAMP as a likely second signal in the inhibitory mechanisms of this agonist. No significant change in cAMP was detectable in parasites treated with L-phenylephrine, leaving open the question about how optimal doses of this alpha-adrenergic agonist enhance T. cruzi infectivity. Parasite responsiveness to alpha- and beta-adrenergic agonists as well as the desensitization effects define a system which regulates infectivity and could be modified at the host tissue level by naturally occurring agonists.

Blastocrithidia triatomae parasitizes vectors of Chagas' disease and is very difficult to cultivate in conventional media. However, co-cultivation with a cell line of its host Triatoma infestans (TI-32; in Schneider's Drosophila medium supplemented with 20% foetal calf serum and 10% tryptose phosphate broth) led to vigorous growth at 24 or 28 °C without an adaptation phase. More than 60 primary cultures were initiated successfully without any failures. Subcultures could be started immediately at weekly intervals. The doubling time was similar in both the primary cultures (41 h) and the 33rd subculture (39 h). The importance of the reduviid cells for B. triatomae became clear after removal of the insect cells, when multiplication of epimastigotes stopped and mainly cysts were formed. Cysts produced in vitro were infective for reduviids. Scanning electron microscopy showed that B. triatomae attached to the host cells, inserted its flagellum into them and destroyed them.

Trypanosoma cruzi specific sequences were amplified by the polymerase chain reaction from total blood of human chagasic patients and normal individuals. A 330 bp fragment originating from kinetoplast DNA was specifically detected in most chagasic individuals. We tested the sensitivity and specificity of this method in normal and affected individuals attending the Evandro Chagas Hospital, Rio de Janeiro. The results of these tests were compared with serological diagnosis performed using standard techniques, and in some cases with xenodiagnosis. We found that none of the serologically negative individuals gave any specific amplification product, whereas 55 out of 61 patients previously serodiagnosed as chagasic were positive using the PCR method (sensitivity: 90%). Xenodiagnosis, which is currently considered to be the most sensitive parasitological technique for Chagas' disease diagnosis, detected only 12 out of 28 serologically positive patients (sensitivity: 43%). The usefulness of the PCR method was further investigated with chagasic patients who had received anti-parasite treatment with benznidazole. It has always been difficult to evaluate the incidence of cure in such cases by serology, since a humoral response against T. cruzi antigens may remain for years even in the absence of the parasite. We observed a positive amplification result in only 9 out of 32 treated patients who remained reactive when tested using classical serology. These observations suggest that PCR is the most sensitive technique available for direct detection of T. cruzi in chagasic patients and that it can be a very useful instrument for the follow-up of patients after specific treatment.

Chagas' disease is one of the few functional gastrointestinal disorders for which a causative agent has been identified. However, some pathological aspects of the chagasic megasyndromes are still incompletely understood. Chagasic megacolon is characterized by an inflammatory process, organ dilatation and neuronal reduction in both plexuses of the enteric nervous system (ENS). Although some studies on the ENS in Chagas' disease have been performed, the process of neuronal destruction and neuronal regeneration still remains unclear. Our hypothesis is that the regeneration process of the ENS may be involved with the mechanisms that prevent or retard organ dilatation and chagasic megacolon development. For that reason, we evaluated the neuronal regeneration with the marker GAP-43 in the colon's neuronal plexuses from chagasic patients with megacolon, and from non-infected individuals. Visual examination and quantitative analysis revealed an increased neuronal regeneration process in the dilated portion from chagasic patients when compared with the non-dilated portion and with non-infected individuals. We believe that this increased regeneration can be interpreted as an accentuated neuronal plasticity that may be a response of the ENS to avoid megacolon propagation to the entire organ and maintain the colon functional innervation.

We have previously reported that genetic immunization with Tc13Tul antigen of Trypanosoma cruzi, the aetiological agent of Chagas' disease, triggers harmful effects and non-protective immune responses. In order to confirm the role of Tc13 antigens during T. cruzi infection, herein we studied the humoral and cellular immune responses to the Tc13Tul molecule and its EPKSA C-terminal portion in BALB/c T. cruzi-infected mice or mice immunized with recombinant Tc13Tul. Analysis of the antibody response showed that B-cell epitopes that stimulate a sustained IgM production along the infection and high levels of IgG in the acute phase are mainly located at the Tc13 N- and C-terminal domains, respectively. DTH assays showed that T-cell epitopes are mainly at the Tc13 N-terminal segment and that they do not elicit an efficient memory response. Recombinant Tc13Tul did not induce IFN-γ secretion in either infected or immunized mice. However, a putative CD8+Tc13Tul-derived peptide was found to elicit IFN-γ production in chronically infected animals. Immunization with recombinant Tc13Tul did not induce pathology in tissues and neither did it protect against the infection. Our results show that in the outcome of T. cruzi infection the Tc13 family protein mainly triggers non-protective immune responses.

Circulating antibodies in chagasic patients interact with myocardial β adrenergic and muscarinic cholinergic receptors, triggering intracellular signals that alter cardiac function along the course of the disease. However, until now, experimental data in models of chronically infected chagasic mice linking the effects on myocardial β adrenergic and muscarinic receptors to cardiopulmonary dysfunction is lacking. Thus, we studied C57BL/6 mice 8 months after intraperitoneal injection of 100 trypomastigote forms of the Colombian strain of T. cruzi. Uninfected mice, matched in age, were used as controls. Histopathological analyses (inflammation and fibrosis) and radio-ligand binding assays for estimation of muscarinic and adrenergic receptor density were performed in myocardium tissue samples. When compared to controls, infected mice had electrical conduction disturbances, diastolic dysfunction, lower O2 consumption and anaerobic threshold. In addition, hearts of chronic chagasic mice had intense inflammation and fibrosis, and decreased β adrenergic and increased muscarinic receptor densities than normal controls. Our data suggest that chronic T. cruzi infection causes alterations in cardiac receptor density and fibrosis deposition which can be associated with cardiac conduction abnormalities, diastolic dysfunction and lower exercise capacity, associating for the first time all these functional and histopathological alterations in chagasic mice.

The application of genome-scale approaches to study Trypanosoma cruzi–host interactions at different stages of the infective process is becoming possible with sequencing and assembly of the T. cruzi genome nearing completion and sequence information available for both human and mouse genomes. Investigators have recently begun to exploit DNA microarray technology to analyze host transcriptional responses to T. cruzi infection and dissect developmental processes in the complex T. cruzi life-cycle. Collectively, information generated from these and future studies will provide valuable insights into the molecular requirements for establishment of T. cruzi infection in the host and highlight the molecular events coinciding with disease progression. While the field is in its infancy, the availability of genomic information and increased accessibility to relatively high-throughput technologies represents a significant advancement toward identification of novel drug targets and vaccine candidates for the treatment and prevention of Chagas' disease.

The aims of this study were to characterize human American tegumentary leishmaniasis, which includes cutaneous, mucocutaneous and mucosal leishmaniasis, in Northwest Argentina, to determine the prevalence of double infection with Trypanosoma cruzi and to identify the species of Leishmania in this area. Most of the 330 leishmaniasis patients presented cutaneous ulcers (96·1%), 2·4% mucocutaneous and 1·5% the mucosal form (‘espundia’). The aetiological agents, determined by isoenzyme electrophoresis, were identified as Leishmania (Viannia) braziliensis in 16 out of 20 isolates and in the remaining 4 as Leishmania (Leishmania) amazonensis, the first ever-documented in Argentina. Sera analysed by ELISA and IFA using complex antigen from both T. cruzi and L. braziliensis showed a very high percentage of positives (66·3–78·2%). When antigens for specific diagnosis of Chagas' disease were used, 40·9% of the leishmaniasis patients were also found to be infected by T. cruzi. These results indicate that the strong immune response against T. cruzi gave no protection to Leishmania, in spite of the serological cross-reaction between these parasites. In addition, we showed that more than 40% of the patients would be misdiagnosed as chagasic if complex antigens, as epimastigotes or soluble fraction from epimastigotes, were used in IFA or ELISA. This is of paramount importance not only because patients' treatment would be associated to misdiagnosis but the fact that in many countries in Central and South America, a positive test for Chagas' disease means a rejection for those seeking employment.

Isozyme analysis (12 enzymes: 14 loci) was conducted on 99 isolates of Trypanosoma cruzi: 77 from Guatemala, 5 from Mexico and 17 from South American countries. Analyses of 4 population-genetic indices were undertaken to assess the possibility of genetic exchange occurring among Guatemalan isolates. The results provide evidence for a degree of genetic exchange occurring among isolates from this relatively small geographical area. Previous studies of population genetics on T. cruzi might have failed to detect this phenomenon because they tended to use isolates originating far from one another, rendering gene exchange unlikely for geographical reasons. Phylogenetic data, presented here, show considerable differences in genetic structure between Central and South American isolates, suggesting that different biological and clinical properties might be expected. For example, there are differences in clinical syndromes between Central and South America, a situation discussed further here.

The crystal structure of tyrosine aminotransferase (TAT) from the parasitic protozoan Trypanosoma cruzi, which belongs to the aminotransferase subfamily Iγ, has been determined at 2.5 Å resolution with the R-value R = 15.1%. T. cruzi TAT shares less than 15% sequence identity with aminotransferases of subfamily Iα but shows only two larger topological differences to the aspartate aminotransferases (AspATs). First, TAT contains a loop protruding from the enzyme surface in the larger cofactor-binding domain, where the AspATs have a kinked α-helix. Second, in the smaller substrate-binding domain, TAT has a four-stranded antiparallel β-sheet instead of the two-stranded β-sheet in the AspATs. The position of the aromatic ring of the pyridoxal-5′-phosphate cofactor is very similar to the AspATs but the phosphate group, in contrast, is closer to the substrate-binding site with one of its oxygen atoms pointing toward the substrate. Differences in substrate specificities of T. cruzi TAT and subfamily Iα aminotransferases can be attributed by modeling of substrate complexes mainly to this different position of the cofactor-phosphate group. Absence of the arginine, which in the AspATs fixes the substrate side-chain carboxylate group by a salt bridge, contributes to the inability of T. cruzi TAT to transaminate acidic amino acids. The preference of TAT for tyrosine is probably related to the ability of Asn17 in TAT to form a hydrogen bond to the tyrosine side-chain hydroxyl group.

We have previously reported that the mechanisms of neuromyopathic damage induced by Trypanosoma cruzi are mediated by T cells and are parasite-strain dependent. In the present study our aim was to determine whether the humoral response against muscle and nervous system is also parasite-strain dependent. Of the sera from mice infected with RA and CA-I T. cruzi strains, 93% reacted against antigens of the nervous system (sciatic nerve, spinal cord and brain). No differences in the ability to recognize heart antigens were found between RA (48%) and CA-I (63%) antisera. Reactivity against skeletal muscle was only relevant in anti-CA-I sera at 270 days post-infection. Each of the antisera assayed in Western blots developed a particular antigenic pattern, but 3 antigens in the nervous system of molecular weight 48, 60 and 70 kDa were detected by 42, 28 and 23% of the sera, respectively. On the other hand, deposits of IgG were observed at the interstitial matrix in sciatic nerve and as endomisial and sarcolemmal patterns in skeletal muscle by IFAT for both RA and CA-I antisera. Absorption of sera with parasite antigens did not abolish the autoreactivity. Our results suggest that major serum autoreactivity from T. cruzi-infected mice is not parasite-strain dependent and does not arise from molecular mimicry.

A large field study has been performed in the Cochabamba region of Bolivia with the aim of comparing the polymerase chain reaction (PCR) with other diagnostic methods for Chagas' disease. The amplification of Trypanosoma cruzi-specific kinetoplast DNA sequences in blood samples was compared with classical serological methods, specific IgM detection and direct parasite visualization for 268 school children in a single village where Chagas' disease transmission is active. Of 113 children positive by classical serology or buffy coat examination, 106 were detected by PCR (sensitivity: 93·8%). We did not observe any significant difference of PCR sensitivity between initial (IgM and/or buffy coat positive) and indeterminate stage (only IgG positive) patients. Among the remaining 155 children unconfirmed as chagasic (who were either only IgM positive, IgG-, IgM-, and buffy coat-negative) only 1 case was PCR positive. This case may be due to DNA contamination, or to a very recent infection not detected otherwise, or to specific immune depression. These results show that PCR is a very sensitive parasitological test for Chagas' disease in active transmission regions. The future follow-up of the possibly infected patients who were only IgM-positive should clarify the interest of PCR and IgM tests in the detection of starting infections.

Trypanosoma cruzi, the agent of Chagas' disease, exhibits considerable biological variability. Moreover, it has been postulated that populations of this protozoan are subdivided into natural clones, which can be separated from each other by considerable levels of evolutionary divergence. The authors have proposed that this long-term clonal evolution may have a profound impact on Trypanosoma cruzi biological diversity. In order to test this hypothesis, 16 T. cruzi stocks representing 3 major clonal genotypes of the parasite were analysed for 8 different in vitro biological parameters. The overall results show a strong statistical linkage between genetic and biological differences. This is in agreement with the working hypothesis, although a notable biological variability is observable among the stocks of each of the 3 major clonal genotypes. The authors propose that T. cruzi genetic variability must be taken into account in any applied study dealing with this parasite.