Isabela Ribeiro

In vitro and in vivo experimental models for drug screening and development for Chagas disease

Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.

New, Improved Treatments for Chagas Disease: From the R&D Pipeline to the Patients

Endemic throughout Latin America with a prevalence rate of approximately 1.4%, Chagas disease (CD) is estimated to kill 14,000 people every year, which is more people in the region each year than any other parasite-born disease, including malaria [1],[2]. Brazilian physician Carlos Chagas first described CD exactly a century ago [3], and its socioeconomic impact makes it the most important parasitic disease in the Americas [4]. Estimated to infect somewhere between 8 to 14 million people, CD both afflicts the poor and, like other neglected tropical diseases, “promotes poverty” [2],[5]. Through its impact on worker productivity, and by causing premature disability and death, CD annually costs an estimated 667,000 disability-adjusted life years lost [1],[6]. In the case of Brazil alone, losses of over US

Towards a Paradigm Shift in the Treatment of Chronic Chagas Disease

1.3 billion in wages and industrial productivity were due to the disabilities of workers with CD [7]. CD is an important public health issue, both in Latin America and increasingly around the world: the infection rate in endemic areas is estimated to be 1.4% [8], with geographic variation from 0.1% to 45.2% [9]. Vectorial transmission has been significantly reduced due to control efforts like the Southern Cone Initiative [10],[11] and others [11],[12]. However, there are areas producing new cases such as regions untouched by vector control efforts [13], special areas with non-domiciliated triatomine [14], and the Amazon region with recent cases reported via oral transmission and by wild triatomine [15]. And still to this day, millions of patients remain without adequate treatment for this silently debilitating and potentially fatal disease. Although no official global figures exist, it is estimated that no more than 1% of those infected are believed to receive any treatment at all. An increasing number of CD patients are also seen in non-endemic, developed countries because of globalization and the movement of unknowingly infected people from Latin America to other parts of the world [16],[17],[18]. The appearance of Trypanosoma cruzi in blood banks in the United States has led the Food and Drug Administration (FDA) to recently issue a draft guidance on CD screening [19].

Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

ABSTRACT Treatment for Chagas disease with currently available medications is recommended universally only for acute cases (all ages) and for children up to 14 years old. The World Health Organization, however, also recommends specific antiparasite treatment for all chronic-phase Trypanosoma cruzi-infected individuals, even though in current medical practice this remains controversial, and most physicians only prescribe palliative treatment for adult Chagas patients with dilated cardiomyopathy. The present opinion, prepared by members of the NHEPACHA network (Nuevas Herramientas para el Diagnóstico y la Evaluación del Paciente con Enfermedad de Chagas/New Tools for the Diagnosis and Evaluation of Chagas Disease Patients), reviews the paradigm shift based on clinical and immunological evidence and argues in favor of antiparasitic treatment for all chronic patients. We review the tools needed to monitor therapeutic efficacy and the potential criteria for evaluation of treatment efficacy beyond parasitological cure. Etiological treatment should now be mandatory for all adult chronic Chagas disease patients.

Fexinidazole: A Potential New Drug Candidate for Chagas Disease

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.

An unfolding tragedy of Chagas disease in North America.

Background New safe and effective treatments for Chagas disease (CD) are urgently needed. Current chemotherapy options for CD have significant limitations, including failure to uniformly achieve parasitological cure or prevent the chronic phase of CD, and safety and tolerability concerns. Fexinidazole, a 2-subsituted 5-nitroimidazole drug candidate rediscovered following extensive compound mining by the Drugs for Neglected Diseases initiative and currently in Phase I clinical study for the treatment of human African trypanosomiasis, was evaluated in experimental models of acute and chronic CD caused by different strains of Trypanosoma cruzi. Methods and Findings We investigated the in vivo activity of fexinidazole against T. cruzi, using mice as hosts. The T. cruzi strains used in the study were previously characterized in murine models as susceptible (CL strain), partially resistant (Y strain), and resistant (Colombian and VL-10 strains) to the drugs currently in clinical use, benznidazole and nifurtimox. Our results demonstrated that fexinidazole was effective in suppressing parasitemia and preventing death in infected animals for all strains tested. In addition, assessment of definitive parasite clearance (cure) through parasitological, PCR, and serological methods showed cure rates of 80.0% against CL and Y strains, 88.9% against VL-10 strain, and 77.8% against Colombian strain among animals treated during acute phase, and 70% (VL-10 strain) in those treated in chronic phase. Benznidazole had a similar effect against susceptible and partially resistant T. cruzi strains. Fexinidazole treatment was also shown to reduce myocarditis in all animals infected with VL-10 or Colombian resistant T. cruzi strains, although parasite eradication was not achieved in all treated animals at the tested doses. Conclusions Fexinidazole is an effective oral treatment of acute and chronic experimental CD caused by benznidazole-susceptible, partially resistant, and resistant T. cruzi. These findings illustrate the potential of fexinidazole as a drug candidate for the treatment of human CD.

Benznidazole and Posaconazole in experimental Chagas disease: positive interaction in concomitant and sequential treatments.

In North America, Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) was first reported in Mexico in 1940 [1] and in the United States in Texas in 1955 [2]. However, based on ancient mummified remains discovered in the Rio Grande Valley, human T. cruzi infection has been present in North America since prehistoric times [3].

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

Background Current chemotherapy for Chagas disease is unsatisfactory due to its limited efficacy, particularly in the chronic phase, with frequent side effects that can lead to treatment discontinuation. Combined therapy is envisioned as an ideal approach since it may improve treatment efficacy whilst decreasing toxicity and the likelihood of resistance development. We evaluated the efficacy of posaconazole in combination with benznidazole on Trypanosoma cruzi infection in vivo. Methods and Findings Benznidazole and posaconazole were administered individually or in combination in an experimental acute murine infection model. Using a rapid treatment protocol for 7 days, the combined treatments were more efficacious in reducing parasitemia levels than the drugs given alone, with the effects most evident in combinations of sub-optimal doses of the drugs. Subsequently, the curative action of these drug combinations was investigated, using the same infection model and 25, 50, 75 or 100 mg/kg/day (mpk) of benznidazole in combination with 5, 10 or 20 mpk of posaconazole, given alone or concomitantly for 20 days. The effects of the combination treatments on parasitological cures were higher than the sum of such effects when the drugs were administered separately at the same doses, indicating synergistic activity. Finally, sequential therapy experiments were carried out with benznidazole or posaconazole over a short interval (10 days), followed by the second drug administered for the same period of time. It was found that the sequence of benznidazole (100 mpk) followed by posaconazole (20 mpk) provided cure rates comparable to those obtained with the full (20 days) treatments with either drug alone, and no cure was observed for the short treatments with drugs given alone. Conclusions Our data demonstrate the importance of investigating the potential beneficial effects of combination treatments with marketed compounds, and showed that combinations of benznidazole with posaconazole have a positive interaction in murine models of Chagas disease.

Drug discovery for Chagas disease should consider Trypanosoma cruzi strain diversity

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

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

This opinion piece presents an approach to standardisation of an important aspect of Chagas disease drug discovery and development: selecting Trypanosoma cruzi strains for in vitro screening. We discuss the rationale for strain selection representing T. cruzi diversity and provide recommendations on the preferred parasite stage for drug discovery, T. cruzi discrete typing units to include in the panel of strains and the number of strains/clones for primary screens and lead compounds. We also consider experimental approaches for in vitro drug assays. The Figure illustrates the current Chagas disease drug-discovery and development landscape.