A estruturação do Programa de Capacitação Profissional de Biossegurança no contexto do Projeto de Modernização da Gestão Científica do Instituto Oswaldo Cruz

In the present paper we report a Biosafety Training Program (BTP) developed at Oswaldo Cruz Institute (IOC/Fiocruz) aligned with the Scientific Management Modernization Project of IOC. The program, including the "Biosafety Course in Biomedical Research Laboratory", was structured according to the planning-development-evaluation cycle. Initially, for the diagnosis cycle, the IOC laboratories representatives answered a questionnaire that showed that both professional categories (middle and higher levels) were interested in participating in BTP and mentioned, as preferential themes, biosafety and good laboratory practices. In the planning phase it was defined that BTP would be divided into two projects (Good Laboratory Practices in Public Health for middle level professionals and Biosafety Course in Biomedical Research Laboratories for higher level professionals). During the development phase of the Biosafety Course, the following modules were chosen: introduction, chemical, physical and biological risks, quality management and animal experimentation. Thus, in the period from 2006 to 2008, 315 professionals were trained and the respective evaluations were performed according to David Kirkpatrick's model. The first level of evaluation, called reaction, showed that 54.03% of the professionals said that the course was excellent, 39.59% classified the course as good and 6.38% as regular or did not express any opinion. For learning evaluation, pre and post-tests were carried out in each module. All the modules showed an increase in the grades of the post-test when compared to the pre-test. The results pointed to strategies that should be followed in order to improve this biosafety continuing education model.O presente artigo apresenta as etapas de estruturação do Programa de Capacitação Profissional de Biossegurança (PCPB), em consonância com o Projeto de Modernização da Gestão Científica do Instituto Oswaldo Cruz (IOC), detalhando o ciclo planejamento-desenvolvimento-avaliação, em especial do Curso de Biossegurança em Laboratório de Pesquisa Biomédica. Inicialmente, para o ciclo diagnóstico foram aplicados questionários aos interlocutores dos laboratórios do IOC, os quais revelaram interesse de participação no PCPB para ambas as categorias profissionais (níveis médio e superior), indicando como temáticas preferenciais biossegurança e boas práticas de laboratório. Na fase de planejamento foi definido que o PCPB seria subdivido em dois projetos (Boas Práticas de Laboratório de Saúde Pública para os profissionais de nível médio e Curso de Biossegurança para Laboratórios de Pesquisa Biomédica para profissionais de nível superior). A seguir, na fase de estruturação do curso, os módulos contemplados incluíram: introdutório; riscos químico, físico e biológico; gestão da qualidade e experimentação animal. Assim, no período 2006-2008, foram capacitados 315 profissionais e realizadas avaliações segundo o modelo de David Kirkpatrick. O primeiro nível, chamado de reação, foi aferido e demonstrou que 54,03% dos profissionais declararam que o curso foi excelente; 39,59% classificaram como bom e os demais 6,38% acharam que foi regular ou não opinaram. Para a avaliação do aprendizado foram realizados, a cada módulo, pré e pós-testes. Foi verificado que todos os módulos tiveram acréscimos nas médias do pós-teste em relação ao pré-teste. Os resultados obtidos apontaram estratégias a serem seguidas no aperfeiçoamento desse modelo de educação continuada em biossegurança

Parasite, Compartments, and Molecules: Trick versus Treatment on Chagas Disease

Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic to Latin America, standing out as a socio-economic problem for low-income tropical populations. Such disease affects millions of people worldwide and emerges in nonendemic areas due to migration and climate changes. The current chemotherapy is restricted to two nitroderivatives (benznidazole and nifurtimox), which is unsatisfactory due to limited efficacy (particularly in chronic phase) and adverse side effects. T. cruzi life cycle is complex, including invertebrate and vertebrate hosts and three developmental forms (epimastigotes, trypomastigotes, and amastigotes). In this chapter, we will discuss promising cellular and molecular targets present in the vertebrate-dwelling forms of the parasite (trypomastigotes and amastigotes). Among the cellular targets, the mitochondrion is the most frequently studied; while among the molecular ones, we highlight squalene synthase, C14α-sterol demethylase, and cysteine proteases. In this scenario, proteomics becomes a valuable tool for the identification of other molecular targets, and some previously identified candidates will be also discussed. Multidisciplinary studies are needed to identify novel key molecules in T. cruzi in order to increase trypanocidal activity and reduce mammalian toxicity, ensuring the development of novel drugs for Chagas disease

Experimental Chemotherapy for Chagas Disease: A Morphological, Biochemical, and Proteomic Overview of Potential Trypanosoma cruzi Targets of Amidines Derivatives and Naphthoquinones

Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately eight million individuals in Latin America and is emerging in nonendemic areas due to the globalisation of immigration and nonvectorial transmission routes. Although CD represents an important public health problem, resulting in high morbidity and considerable mortality rates, few investments have been allocated towards developing novel anti-T. cruzi agents. The available therapy for CD is based on two nitro derivatives (benznidazole (Bz) and nifurtimox (Nf)) developed more than four decades ago. Both are far from ideal due to substantial secondary side effects, limited efficacy against different parasite isolates, long-term therapy, and their well-known poor activity in the late chronic phase. These drawbacks justify the urgent need to identify better drugs to treat chagasic patients. Although several classes of natural and synthetic compounds have been reported to act in vitro and in vivo on T. cruzi, since the introduction of Bz and Nf, only a few drugs, such as allopurinol and a few sterol inhibitors, have moved to clinical trials. This reflects, at least in part, the absence of well-established universal protocols to screen and compare drug activity. In addition, a large number of in vitro studies have been conducted using only epimastigotes and trypomastigotes instead of evaluating compounds' activities against intracellular amastigotes, which are the reproductive forms in the vertebrate host and are thus an important determinant in the selection and identification of effective compounds for further in vivo analysis. In addition, due to pharmacokinetics and absorption, distribution, metabolism, and excretion characteristics, several compounds that were promising in vitro have not been as effective as Nf or Bz in animal models of T. cruzi infection. In the last two decades, our team has collaborated with different medicinal chemistry groups to develop preclinical studies for CD and investigate the in vitro and in vivo efficacy, toxicity, selectivity, and parasite targets of different classes of natural and synthetic compounds. Some of these results will be briefly presented, focusing primarily on diamidines and related compounds and naphthoquinone derivatives that showed the most promising efficacy against T. cruzi

Phenotypic evaluation of nucleoside analogues against Trypanosoma cruzi infection: in vitro and in vivo approaches

Chagas disease, caused by Trypanosoma cruzi (T. cruzi), is a serious public health problem. Current treatment is restricted to two drugs, benznidazole and nifurtimox, displaying serious efficacy and safety drawbacks. Nucleoside analogues represent a promising alternative as protozoans do not biosynthesize purines and rely on purine salvage from the hosts. Protozoan transporters often present different substrate specificities from mammalian transporters, justifying the exploration of nucleoside analogues as therapeutic agents. Previous reports identified nucleosides with potent trypanocidal activity; therefore, two 7-derivatized tubercidins (FH11706, FH10714) and a 3′-deoxytubercidin (FH8513) were assayed against T. cruzi. They were highly potent and selective, and the uptake of the tubercidin analogues appeared to be mediated by the nucleoside transporter TcrNT2. At 10 μM, the analogues reduced parasitemia >90% in 2D and 3D cardiac cultures. The washout assays showed that FH10714 sterilized the infected cultures. Given orally, the compounds did not induce noticeable mouse toxicity (50 mg/kg), suppressed the parasitemia of T. cruzi-infected Swiss mice (25 mg/kg, 5 days) and presented DNA amplification below the limit of detection. These findings justify further studies with longer treatment regimens, as well as evaluations in combination with nitro drugs, aiming to identify more effective and safer therapies for Chagas disease

In Vitro and In Vivo Investigation of the Efficacy of Arylimidamide DB1831 and Its Mesylated Salt Form - DB1965 - against Trypanosoma cruzi Infection

Chagas disease is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi. At present, nifurtimox and benznidazole, both compounds developed empirically over four decades ago, represent the chemotherapeutic arsenal for treating this highly neglected disease. However, both drugs present variable efficacy depending on the geographical area and the occurrence of natural resistance, and are poorly effective against the later chronic stage. As a part of a search for new therapeutic opportunities to treat chagasic patients, pre-clinical studies were performed to characterize the activity of a novel arylimidamide (AIA - DB1831 (hydrochloride salt) and DB1965 (mesylate salt)) against T.cruzi. These AIAs displayed a high trypanocidal effect in vitro against both relevant forms in mammalian hosts, exhibiting a high selectivity index and a very high efficacy (IC50 value/48 h of 5–40 nM) against intracellular parasites. DB1965 shows high activity in vivo in acute experimental models (mouse) of T.cruzi, showing a similar effect to benznidazole (Bz) when compared under a scheme of 10 daily consecutive doses with 12.5 mg/kg. Although no parasitological cure was observed after treating with 20 daily consecutive doses, a combined dosage of DB1965 (5 mg/kg) with Bz (50 mg/kg) resulted in parasitaemia clearance and 100% animal survival. In summary, our present data confirmed that aryimidamides represent promising new chemical entities against T.cruzi in therapeutic schemes using the AIA alone or in combination with other drugs, like benznidazole

Combined Treatment of Heterocyclic Analogues and Benznidazole upon Trypanosoma cruzi In Vivo

Chagas disease caused by Trypanosoma cruzi is an important cause of mortality and morbidity in Latin America but no vaccines or safe chemotherapeutic agents are available. Combined therapy is envisioned as an ideal approach since it may enhance efficacy by acting upon different cellular targets, may reduce toxicity and minimize the risk of drug resistance. Therefore, we investigated the activity of benznidazole (Bz) in combination with the diamidine prodrug DB289 and in combination with the arylimidamide DB766 upon T. cruzi infection in vivo. The oral treatment of T.cruzi-infected mice with DB289 and Benznidazole (Bz) alone reduced the number of circulating parasites compared with untreated mice by about 70% and 90%, respectively. However, the combination of these two compounds decreased the parasitemia by 99% and protected against animal mortality by 100%, but without providing a parasitological cure. When Bz (p.o) was combined with DB766 (via ip route), at least a 99.5% decrease in parasitemia levels was observed. DB766+Bz also provided 100% protection against mice mortality while Bz alone provided about 87% protection. This combined therapy also reduced the tissular lesions induced by T. cruzi infection: Bz alone reduced GPT and CK plasma levels by about 12% and 78% compared to untreated mice group, the combination of Bz with DB766 resulted in a reduction of GPT and CK plasma levels of 56% and 91%. Cure assessment through hemocultive and PCR approaches showed that Bz did not provide a parasitological cure, however, DB766 alone or associated with Bz cured ≥13% of surviving animals

In Vitro and In Vivo Biological Effects of Novel Arylimidamide Derivatives against Trypanosoma cruzi

ABSTRACT Chagas disease (CD), a neglected tropical disease caused by Trypanosoma cruzi , remains a serious public health problem in several Latin American countries. The available chemotherapies for CD have limited efficacy and exhibit undesirable side effects. Aromatic diamidines and arylimidamides (AIAs) have shown broad-spectrum activity against intracellular parasites, including T. cruzi . Therefore, our aim was to evaluate the biological activity of eight novel AIAs (16DAP002, 16SAB079, 18SAB075, 23SMB022, 23SMB026, 23SMB054, 26SMB070, and 27SMB009) against experimental models of T. cruzi infection in vitro and in vivo . Our data show that none of the compounds induced a loss of cellular viability up to 32 μM. Two AIAs, 18SAB075 and 16DAP002, exhibited good in vitro activity against different parasite strains (Y and Tulahuen) and against the two relevant forms of the parasite for mammalian hosts. Due to the excellent selective indexes of 18SAB075, this AIA was moved to in vivo tests for acute toxicity and parasite efficacy; nontoxic doses (no-observed-adverse-effect level [NOAEL], 50 mg/kg) were employed in the tests for parasite efficacy. In experimental models of acute T. cruzi infection, 18SAB075 reduced parasitemia levels only up to 50% and led to 40% protection against mortality (at 5 mg/kg of body weight), being less effective than the reference drug, benznidazole