INTRODUCTION
American and African trypanosomiasis constitute the two main human systemic trypanosomiases, which are neglected tropical diseases worldwide (Kennedy, Reference Kennedy2013; Bern, Reference Bern2015). American trypanosomiasis or Chagas disease is caused by the intracellular parasite Trypanosoma cruzi, which is mainly transmitted through contact with the feces of hematophagous Triatomine insects. About 6 to 7 million people are estimated to be infected worldwide, mostly in Latin America (WHO, 2016a ). Owing to population migration from Central and South American endemic countries, Chagas disease has also become a health problem in non-endemic areas, especially the USA and European countries, in which non-vectorial transmission routes related to blood and organ donation predominate (Bern, Reference Bern2015). In North America, this disease accounts for over 300 000 reported cases, while in Europe about 108 000 cases are estimated (Andrade et al. Reference Andrade, Gollob and Dutra2014). Chagas disease is characterized as the most common cause of non-ischaemic cardiomyopathy in South America (Bocchi, Reference Bocchi2013), leading to the death of many patients every year, mainly due to dilated cardiomyopathy, congestive heart failure, dysrhythmias and thromboembolic events occurring in approximately 30% of infected individuals (Marin-Neto et al. Reference Marin-Neto, Cunha-Neto, Maciel and Simões2007; Bern, Reference Bern2015). There is no effective vaccine for Chagas disease, and although drugs such as nifurtimox and benznidazole are effective in acute infections (about 60% cure), these drugs exhibit high toxicity and do not guarantee a cure (about 10–20%) in chronic infections (Cançado, Reference Cançado1999, Reference Cançado2002).
African trypanosomiasis or sleeping sickness is caused by the extracellular protozoa Trypanosoma brucei gambiense as well as Trypanosoma brucei rhodesiense in 36 sub-Saharan Africa countries (Giordani et al. Reference Giordani, Morrison, Rowan, de Koning and Barrett2016; WHO, 2016b ). Although the tsetse fly (genus Glossina) is responsible for the transmission of both parasite species, T. brucei gambiense accounts for more than 98% of reported cases (Kennedy, Reference Kennedy2013; Sudarshi and Brown, Reference Sudarshi and Brown2015). About 20 000 cases/year and 65 million people are at risk of infection (WHO, 2016b ). Due to infection of the central nervous system, neurological disorders (i.e. changes in behaviour, mental confusion, sensory disturbances and poor motor coordination) associated with meningoencephalitis are the most serious clinical manifestations of sleeping sickness, which has a high mortality rate (Kennedy, Reference Kennedy2013; WHO, 2016b ). Currently, there is no vaccine and no prospects that one will be developed in the near future (Avery, Reference Avery2013; Goupil and McKerrow, Reference Goupil and McKerrow2014). The only available treatment is based on single or combined chemotherapy with pentamidine, suramine, merlasoprol, eflornithine and nifurtimox. The therapeutic schemes based on these drugs are complicated to administer, present high toxicity and do not guarantee a cure, especially in advanced disease stages (Kennedy, Reference Kennedy2013; Giordani et al. Reference Giordani, Morrison, Rowan, de Koning and Barrett2016; WHO, 2016b ).
American and African trypanosomiasis are diseases closely related to poverty, and people suffering from these neglected diseases constitute an unattractive market to investments in research and drugs development by the private sector (Boelaert et al. Reference Boelaert, Meheus, Robays and Lutumba2010; Ekins et al. Reference Ekins, Williams, Krasowski and Freundlich2011). Considering that access to allopathic medicine is restricted in many endemic regions in Africa and South America, there is a growing demand for low cost and effective treatment strategies (Urbina, Reference Urbina2010; Ekins et al. Reference Ekins, Williams, Krasowski and Freundlich2011; Goupil and McKerrow, Reference Goupil and McKerrow2014). For trypanosomiasis, the prospect of discovering new antiparasitic molecules and developing new drugs has motivated research centers to investigate a large variety of plant species (Ndjonka et al. Reference Ndjonka, Rapado, Silber, Liebau and Wrenger2013; Hertweck, Reference Hertweck2015). However, screening has been based on isolated efforts directed by research groups worldwide, and currently there is no overview on the plant species investigated and their relevance in the therapeutic management of human systemic trypanosomiasis (Salem and Werbovetz, Reference Salem and Werbovetz2006; Olliaro et al. Reference Olliaro, Kuesel and Reeder2015).
Admittedly, studies on experimental animal models have provided the empirical basis for determining the applicability, efficacy and safety of plant derivatives in the treatment of parasitic diseases, parameters strictly necessary to support clinical studies in humans (Hooijmans and Ritskes-Hoitinga, Reference Hooijmans and Ritskes-Hoitinga2013). However, as the findings of preclinical studies often originate from relatively small experiments and are quite heterogeneous, they may not always be applicable in a translational context to enhance human health and well-being (Hooijmans and Ritskes-Hoitinga, Reference Hooijmans and Ritskes-Hoitinga2013; Van Luijk et al. Reference Van Luijk, Bakker, Rovers, Ritskes-Hoitinga, de Vries and Leenaars2014). Systematically reviewing the preclinical evidence in an objective manner (unlike the widely used narrative reviews) has never been carried out before and might provide us with reliable and solid evidence on whether or not plant extracts and its derivatives could be beneficial in the treatment of American and African trypanosomiasis. Thus, beyond determining the overall picture of all available experimental evidence on the plant species applied in the treatment of two neglected tropical diseases, their usage characteristics and effects, this systematic review was designed to determine if there is a rational basis for the selection of the plant species investigated, especially considering the geographic distribution of each species (native or exotic) as well as any evidence of ethnodirected bioprospecting.
METHODS
Search strategy and selection of the papers
The PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) statement was adopted for conducting this systematic review (Moher et al. Reference Moher, Liberati, Tetzlaff and Altman2009). Four researchers (RMP, GMZG, AMM and PFC) independently searched PubMed, Scopus and Web of Science databases for all original articles that investigated the applicability of plant extracts and its derivatives in the treatment of animal models of Chagas disease and sleeping sickness published up to August 28, 2016. The search strategy was based on three components: (i) animals, (ii) diseases and (iii) plant extracts. A search filter was initially developed for PubMed according to the platform's thesaurus – MeSH terms (Medical Subject Headings). To expand the recovery of relevant indexed studies and those in the indexing process, the commands [MeSH Terms] and [TIAB] were combined. To detect all animal studies in PubMed, a standardized animal filter was applied (Hooijmans et al. Reference Hooijmans, Tillema, Leenaars and Ritskes-Hoitinga2010). The same search filter used to diseases and plant extracts were adapted to Scopus and Web of Science. Another animal filter was created for Web of Science search and the Scopus animal filter was used in this database. The full search strategy is described in Supplementary File S1. No language or chronological restriction was applied in the article search.
The initial selection was independently performed by the investigators (RMP, GMZG, AMM and PFC), who screened the abstract of all recovered papers. Duplicate studies were removed by comparing the authors, title, year and journal of publication. In case of doubt, the entire publication was recovered and evaluated. Only studies investigating plant extracts, its fractions and isolated compounds were considered for potential inclusion in the systematic review. Studies using T. brucei brucei in experimental models of sleeping sickness were also included in the review, since there is evidence of human infection by this parasite strain (Deborggraeve et al. Reference Deborggraeve, Koffi, Jamonneau, Bonsu, Queyson, Simarro, Herdewijn and Büscher2008), which was also adopted as a classical model of human African trypanosomiasis in mice (Keita et al. Reference Keita, Bouteille, Enanga, Vallat and Dumas1997).
After initial screening, all potentially relevant studies were recovered in full-text and evaluated for eligibility. Study exclusion was based on well-defined criteria as follows: (i) studies exclusively investigating in vitro and human systems without the inclusion of experimental groups with other animal species, (ii) studies testing only synthetic or commercial substances and (iii) no full-text available and secondary studies (i.e. literature reviews, editorials, commentaries and letters to the editor). Eligibility was independently analysed by researchers and disagreements were resolved by consensus. The reference lists of the selected relevant papers were screened for potentially relevant papers.
Study characteristics and data extraction
Qualitative data were extracted from all included articles. Data extraction was categorized as follows: (i) publication characteristics: authors, years, journals and countries; (ii) characteristics of the animal model: species, sex, age, weight; (iii) characteristics of the disease model: parasite species and strain; (iv) characteristics of the administered treatment: plant family and species, part of the plant used, therapeutic formulation (i.e. crude extract, extract fractions, and isolated substances), dosimetry (i.e. dose, route, frequency and duration of the treatment); (v) treatment safety: host toxicity assays (i.e. LD50, liver enzymes, genotoxicity, mutagenicity); and (vi) main outcome measures.
Rational basis for plant research
Two criteria were considered to investigate the rational basis that supports the selection of the specific plant species investigated: (i) the geographical distribution of each plant species and (ii) any evidence of ethnodirected bioprospecting. The geographical distribution is directly related to the availability and ease of access plant species considering that native species present more regional relevance compared to more difficult to obtain exotic species.
As non-random screening proved to be a more efficient strategy for the discovery new potentially applicable plant resources with antimicrobial activities than random selection (Harvey, Reference Harvey2008; Pan et al. Reference Pan, Zhou, Gao, Yu, Zhang, Tang, Sun, Ma, Han, Fong and Ko2013; Silva et al. Reference Silva, Albuquerque, Costa Júnior, Lima, do Nascimento and Monteiro2014), ethnodirected bioprospecting was also admitted as an indicator of the rational selection of all plant species investigated. This selection strategy is coherent with the use of natural resources by local communities in an ethnomedical context, which takes into account the regional culture in an integrated system of beliefs and values (Staub et al. Reference Staub, Geck, Weckerle, Casu and Leonti2015). The popular indication was admitted when the papers included in the review presented referenced reports confirming the traditional use of the plant species investigated and its specific application to treat trypanosomiasis.
Bias analysis
We assessed the methodological quality (report bias) of all included papers by using the criteria described in the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (McGrath and Lilley, Reference McGrath and Lilley2015). These criteria are based on short descriptions of essential study characteristics such as ethnical statement, experimental procedures, sample size, animal allocation, statistical methods, baseline data, generalizability and funding. Considering the proposition of this systematic review and the specificity of the research subject, we constructed a table summarizing all relevant and applicable aspects described in the ARRIVE guidelines. The researchers independently assessed the reporting bias of all studies and discrepancies were resolved by consensus. Negative quality assessment does not necessarily indicate that the experiment has been carried out improperly; it rather indicates inadequate reporting quality (Hooijmans et al. Reference Hooijmans, Tillema, Leenaars and Ritskes-Hoitinga2010).
RESULTS
Included studies
Using our search strategy, 46 studies were recovered in all databases and included in the systematic review. The reference lists were screened and 20 studies were additionally identified. Thus, 66 relevant preclinical studies with animal models of human systemic trypanosomiasis from 1990 until 2016 were included in the review and used for data extraction. Fig. 1 shows the flowchart and each step performed in the selection process to recover relevant studies. The research filters presented here allowed us recover a similar number of registers in PubMed and Scopus, but a lower number in Web of Sciences.
Flow diagram the systematic review literature search results. Based on ‘Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement’. http://www.prisma-statement.org
Considering all papers investigating the applicability of plants and their derivatives against trypanosomatide parasites with the potential to induce human systemic trypanosomiasis (n = 258), most excluded studies were based on in vitro parasite viability assays (45·74%, n = 118), followed by duplicated studies (17·05%, n = 44), those investigating synthetic compounds (8·14%, n = 21) and others reasons such as non-primary studies and conference abstracts (11·24%, n = 29).
The general characteristics of all included studies are shown in Supplementary Table S1. Only 17 papers (25·76%) focused on American trypanosomiasis, while 49 studies (74·24%) investigated African trypanosomiasis. Publications originated from eight different countries for each disease (Fig. 2 and Supplementary Table S1).
Qualitative synthesis of all studies, diseases, parasites and plants investigated. Detailed data described in Supplementary Tables S1 and S2.
Animal models of American trypanosomiasis
For Chagas disease, most studies were produced in South/Central America (88·24%, n = 15), primarily from Mexico (29·41%, n = 5), Brazil and Argentina (17·65%, n = 3, each). All studies used mice (100·00%, n = 17) as the animal model. BALB/c (64·70%, n = 11) was the main mouse lineage used, followed by two studies (11·76%) using Swiss or C3H/HeN mice. In one study (5·88%) using C3H/HeN mice, CF1 animals were also investigated. The proportion of animal sex was 35·29% female (n = 6) or both 23·53% (n = 4); 41·18% (n = 7) of studies did not describe this information. The age of the animals ranged from 4 to 8 weeks, but this variable was neglected in five studies (29·41%). The animals’ weight ranged from 18–30 g. This parameter was not reported in 47·06% (n = 8) of studies. Studies of T. cruzi mainly used the Y (29·41%, n = 5), H4 (23·53%, n = 4), MHOM/GT/94/SMI-04 H4 or CL (11·75%, n = 2 each), RA, SN3, Tulahuen or Bolivian (5·88%, n = 1 each) strains (Supplementary Table S1).
Animal models of African trypanosomiasis
For sleeping sickness, Africa studies (89·80%, n = 44) were more frequent, especially those from Nigeria (81·63%, n = 40). As the animal model, studies used rats (53·06%, n = 26), mice (34·69%, n = 17), both (6·12%, n = 3), rabbits or dogs (2·04%, n = 1 each), and only one paper did not report the species investigated. Most studies did not describe the animal lineage (59·18%, n = 29), and most those reporting this parameter used Wistar rats (22·44%, n = 11) or Swiss mice (12·24%, n = 6). The proportion of animal sex was 26·53% male (n = 13), 2·04% female (n = 1) or both 40·82% (n = 20); 30·61% (n = 15) did not report this parameter. The age ranged from 1 to 16 weeks; however, this variable was neglected in most studies (79·59%, n = 39). The animals’ weight ranged from 100–250 g in rats, 15–50 g in mice, 1·05–1·6 kg in rabbits and 3–8 kg in dogs. This parameter was not reported in 18·37% (n = 9) of studies. Only three (6·12%) studies used the parasite species T. b. rhodesiense, while the other studies (93·88%, n = 63) used T. b. brucei. Most studies (51·02%, n = 25) did not describe the strain of T. brucei, or used the Federe strain (22·45%, n = 11) to induce infection (Supplementary Table S1).
Plants species and selection
There the botanical species investigated are described in Fig. 2, Supplementary Tables S1 and S2. Forty-four plant families were identified in this review, especially Fabaceae, Asteraceae, Meliaceae, Combretaceae, Rubiaceae and Euphorbiaceae, comprising 49·14% of all families described. Fabaceae and Asteraceae families were most frequently investigated, 12·07 and 9·48%, respectively. A total of 91 different plant species were investigated. Azadirachta indica, Khaya senegalensis, Psidium guajava, Terminalia avicennioides and Ocimum gratissimum were the most frequently investigated plant species (18·10%, n = 21). For American trypanosomiasis, 29 different species were tested, with mainly Carica papaya, Neurolaena lobata and Senna villosa investigated in 6·25% (n = 2, each) of the studies. For African trypanosomiasis, 62 different species were analysed, especially Azadirachta indica and Khaya senegalensis, which were tested in 5·95% (n = 5, each) of the studies, followed by Psidium guajava and Terminalia avicennioides (4·76%, n = 4, each), and also Ocimum gratissimum (3·57%, n = 3). Native and exotic plant species were investigated by 54·54% (n = 36) and 45·46% (n = 30) of the studies, respectively. Ethnodirected indications for trypanosomiasis were not reported in 68·18% (n = 45) studies (Supplementary Table S2).
Treatments, phytochemical screening and toxicity tests
As shown in Supplementary Table S1, 75·44% of studies (n = 43) investigated crude extracts, especially methanol (26·32%, n = 15) and ethanol (22·81%, n = 13) extracts, followed by fractions (12·28%, n = 7) or both (5·26%, n = 3), isolated molecules (1·75%, n = 1) or oil (3·51%, n = 2). The dose of the administered treatments ranged from 1 mg/kg to 80 g/kg of body mass. The main administration route was intraperitoneal (42·42%, n = 28), oral (33·33%, n = 22) or both (4·54%, n = 3), followed by intramuscular (3·03%, n = 2) and intravenous (1·51%, n = 1). Ten studies (15·15%) did not report the administration route. The treatment period ranged from 72 h to 120 days.
From the 15 (22·73%) studies that presented a phytochemical analysis and characterized the secondary metabolites tested, 11 investigated (16·67%) Chagas disease. The molecules identified with antitrypanosomal activity are summarized in Table 1. Toxicity to the host was reported in 32 studies (48·48%); the LD50 test (16·66%) was most frequently used.
Plant species and isolated molecules with in vivo anti-trypanosomal activity identified by phytochemical screening from chromatography and/or mass spectrometry
Main experimental evidence
There was considerable variability in the effects of plant derivatives in both diseases investigated. From all studies reviewed, plant species with positive and negative results that controlled key outcomes in Chagas disease and sleeping sickness (i.e. parasitaemia, parasite load and mortality), as well as those with evidence of host toxicity are extracted and summarized in Fig. 3. Detailed positive and negative outcomes, including haematological and serum biochemical parameters, inflammation, redox metabolism and organ damage can be found in Supplementary Table S3.
Synthesis of the plant species with positive and negative effects in the treatment of African and American trypanosomiasis. Details of what were considered as positive and negative effects are listed in Supplementary Table S3.
Although well-defined effects have been frequently observed in the treatment of African trypanosomiasis, plant species such as Ocimum gratissimum (Adamu et al. Reference Adamu, Nwosu and Agbede2009; Olukunle et al. Reference Olukunle, Abatan, Soniran, Takeet, Idowu, Akande, Biobaku and Jacobs2010; Adelodun et al. Reference Adelodun, Elusiyan, Olorunmola, Adewoyin, Omisore, Adepiti, Agbedahunsi and Adewunmi2013), Guiera senegalensis (Youan et al. Reference Youan, Coulibaly, Miezan, Doua and Bamba1997; Aderbauer et al. Reference Aderbauer, Clausen, Kershaw and Melzig2008), Securidaca longepedunculata (Youan et al. Reference Youan, Coulibaly, Miezan, Doua and Bamba1997; Aderbauer et al. Reference Aderbauer, Clausen, Kershaw and Melzig2008) and Khaya senegalensis (Youan et al. Reference Youan, Coulibaly, Miezan, Doua and Bamba1997; Ibrahim et al. Reference Ibrahim, Njoku and Sallau2008, Reference Ibrahim, Musa, Aliyu, Mayaki, Gideon and Islam2013a ; Antia et al. Reference Antia, Olayemi, Aina and Ajaiyeoba2009; Adelodun et al. Reference Adelodun, Elusiyan, Olorunmola, Adewoyin, Omisore, Adepiti, Agbedahunsi and Adewunmi2013) presented divergent results.
In the treatment of sleeping sickness, the species Afzelia africana, Khaya senegalensis (Ibrahim et al. Reference Ibrahim, Njoku and Sallau2008; Antia et al. Reference Antia, Olayemi, Aina and Ajaiyeoba2009), Terminalia superba (Antia et al. Reference Antia, Olayemi, Aina and Ajaiyeoba2009), Moringa peregrina (Ayyari et al. Reference Ayyari, Salehi, Ebrahimi, Zimmermann, Portmann, Krauth-Siegel, Kaiser, Brun, Rezadoost, Rezazadeh and Hamburger2014), Zanthoxylium zanthoxyloides (Mann et al. Reference Mann, Ifarajimi, Adewoye, Ukam, Udeme, Okorie, Sakpe, Ibrahim, Yahaya, Kabir and Ogbadoyi2011) and Butyrospermun paradoxun (Mbaya et al. Reference Mbaya, Nwosu and Onyeyili2007) presented some indications of host toxicity. In the treatment of Chagas disease, only the species Solanum americanum (Cáceres et al. Reference Cáceres, López, González, Berger, Tada and Maki1998) induced host toxicity.
Reporting bias
Using the criteria proposed by the ARRIVE guidelines, 37 items related to study bias were analysed to determine possible inadequate reporting quality (Supplementary Table S4). None of the studies fulfilled all ARRIVE criteria, and only a mean of 19·74 ± 4 items were attended by the included studies. When the ARRIVE items were individually investigated by disease, none of the studies on Chagas disease reported the route of administration, experimental replicates, motivation for animal exclusion or study limitations. Three or fewer studies reported the duration of treatment (n = 2, 11·8%), statistical design of experimental groups (n = 3, 17·6%) and randomization of animals and groups (n = 3, 17·6%). Maintaining proportionality in relation to the total number of studies on sleeping sickness, nine or fewer studies reported information such as the rational basis for the choice of animal model (n = 3, 6·12%) and administration route (n = 3, 6·12%), duration of treatment (n = 4, 8·16%), previous health check of the animals (n = 8, 16·33%), statistical design of experimental groups (n = 2, 4·08%), experimental replicates (n = 2, 4·08%), motivation for animal exclusion (n = 3, 6·12%) and study limitations (n = 8, 16·33%).
DISCUSSION
Our findings indicate that although there have been research initiatives in developed countries, the search for new treatment options for sleeping sickness and Chagas disease from plant-derived natural resources is a concern of developing countries. Although both diseases have been investigated in a similar number of countries, studies on sleeping sickness were more frequent. Well-defined geographic patterns were expected, in which research initiatives on a particular disease are concentrated in their respective endemic areas (i.e. sleeping sickness in African countries, Chagas disease in South and Central American countries). While most studies on sleeping sickness were performed in Nigeria, research efforts on Chagas disease were more homogeneously distributed among the South American countries.
Although the research motivation was not always explicit, it is possible that differences in the frequency of studies on Chagas disease and sleeping sickness are related to the epidemiological profile and natural history of each disease. Over the past decades, Chagas disease has manifested epidemiological compression in endemic countries, especially in response to investments in vectorial control and parasite screening in organ and tissue banks (Bern, Reference Bern2015; Dias et al. Reference Dias2016). Furthermore, few patients are diagnosed in the acute phase, and most (almost 70%) develop the indeterminate chronic form of Chagas disease, which can remain stable for 20 years or more (Marin-Neto et al. Reference Marin-Neto, Cunha-Neto, Maciel and Simões2007; Dias et al. Reference Dias2016). However, beyond the diversity of African countries affected by sleeping sickness, the most recent epidemic lasted until the late 1990s. In this period, this disease was the first or second greatest cause of mortality, even ahead of HIV/AIDS, in communities in the Democratic Republic of the Congo, South Sudan and Angola (WHO, 2016b ). Only recently (2009) the efforts to control the disease have been effective, reducing the new cases (below 10 000/year) for the first time in 50 years (WHO, 2016b ). As the disease (especially when caused by T. b. rhodesiense) develops weeks or months after infection, with more than 80% of deaths occurring within 6 months of the onset of illness (Welburn et al. Reference Welburn, Fèvre, Coleman, Odiit and Maudlin2001), immediate efforts are required to control infection and prevent progression to the neurological stage, in which treatment is more risky and less effective (Enanga et al. Reference Enanga, Burchmore, Stewart and Barrett2002; Hedley et al. Reference Hedley, Fink, Sparkes and Chiodini2016; WHO, 2016b ). Limitations in diagnostics and lower socioeconomic status in developing countries also represent important stimuli for continued efforts in the search of new options to treat both of these neglected tropical diseases. Thus, plant-based products seem to be an attractive alternative against parasitic diseases, especially in poor endemic rural communities, in which allopathic drugs are expensive and frequently not available (Ndjonka et al. Reference Ndjonka, Rapado, Silber, Liebau and Wrenger2013; Hertweck, Reference Hertweck2015).
Although the studies included in this review show wide methodological variability, some points of convergence were observed for each disease investigated. For sleeping sickness and Chagas disease, rats and mice were used as the main animal model. While animal lineage was frequently neglected in studies on sleeping sickness, BALB/c mice were consistently used as model of Chagas disease. Animal species and genetic background are crucial elements in preclinical investigations of parasitic diseases, since these factors are directly related to host resistance and susceptibility to the pathogen (Andrade et al. Reference Andrade, Machado, Chiari, Pena and Macedo2002; León et al. Reference León, Montilla, Vanegas, Castillo, Parra and Ramírez2017). Rats are resistant and constitute a limited model of T. cruzi infection, requiring high parasite inocula to develop low parasitaemia, with minor or absent classical morphofunctional organ injuries (i.e., myocarditis, heart electrical disturbances and polyneuropathies) that characterize Chagas disease (Rivera-Vanderpas et al. Reference Rivera-Vanderpas, Rodriguez, Afchain, Bazin and Capron1983; Melo and Machado, Reference Melo and Machado2001). Conversely, albino mice (BALB/c and Swiss) are highly susceptible to T. cruzi infection. However, mouse models are far from the human condition, since these models presents a high mortality rate in the acute phase of the infection and frequently does not develop chronic disease, especially chronic Chagas heart disease and mega syndromes (Andrade et al. Reference Andrade, Machado, Chiari, Pena and Macedo2002; León et al. Reference León, Montilla, Vanegas, Castillo, Parra and Ramírez2017). Although canine and primate models are considered to be the most closely related to both human acute and chronic Chagas disease (Guedes et al. Reference Guedes, Veloso, Tafuri, Galvão, Carneiro, Lana, Chiari, Ataide Soares and Bahia2002; Carvalho et al. Reference Carvalho, Andrade, Xavier, Mangia, Britto, Jansen, Fernandes, Lannes-Vieira and Bonecini-Almeida2003), the limited availability of dogs and monkeys, the high cost of maintenance in large animal facilities and ethical issues related to animal welfare limit the use of these models (Hasiwa et al. Reference Hasiwa, Bailey, Clausing, Daneshian, Eileraas, Farkas, Gyertyán, Hubrecht, Kobel, Krummenacher, Leist, Lohi, Miklósi, Ohl, Olejniczak, Schmitt, Sinnett-Smith, Smith, Wagner, Yager, Zurlo and Hartung2011).
For sleeping sickness, rats were mainly used. Rats have been consistently used since they develop the morphological and functional (including behavioural) disturbances typically observed in the human disease (Darsaud et al. Reference Darsaud, Bourdon, Chevrier, Keita, Bouteille, Queyroy, Canini, Cespuglio, Dumas and Buguet2003; Amrouni et al. Reference Amrouni, Meiller, Gautier-Sauvigné, Piraud, Bouteille, Vincendeau, Buguet and Cespuglio2011), especially neurological symptoms such as hypo-activity, anorexia and circadian rhythm disruption (Darsaud et al. Reference Darsaud, Bourdon, Chevrier, Keita, Bouteille, Queyroy, Canini, Cespuglio, Dumas and Buguet2003; Chevrier et al. Reference Chevrier, Canini, Darsaud, Cespuglio, Buguet and Bourdon2005). Mice are also described as relevant animal models, especially due to the high infectivity by all T. brucei strains (de Menezes et al. Reference de Menezes, Queiroz, Gomes, Marques and Jansen2004; Antoine-Moussiaux et al. Reference Antoine-Moussiaux, Magez and Desmecht2008). Although inbred mice (i.e. BALB/c, C57Bl/6) are advantageous as they provide controlled, homogeneous and reproducible pathological manifestations, especially immunological responses; these models do not represent the genetic variability of humans. This aspect is overcome by using outbred rodents (i.e. Swiss mice, Wistar rats); this, however, leads to variable phenotypes and unstable pathological outcomes that impair results interpretation, reproducibility and generalizability (Festing, Reference Festing1999, Reference Festing2016). Considering the investigations of antiparasitic products, the use of susceptible animal lineages could be more relevant, since resistant strains could make it difficult to assess the extent to which parasite control is associated with a direct trypanocidal effect of the tested product or only rather the natural response of host defence mechanisms.
Animals of both sexes were used in both disease models; however, this information was under-reported, hampering studies reproducibility. Proportionally, while female animals were frequently used in Chagas disease models, in sleeping sickness female animals were mainly used in mixed groups, and rarely alone. There are studies suggesting that male animals are more susceptible to both T. brucei (Murray et al. Reference Murray, Morrison and Whitelaw1982) and T. cruzi (Schuster and Schaub, Reference Schuster and Schaub2001) infections. While the relationship between sex and disease evolution is not objectively clarified, both sexes seem to be potentially useful in sleeping sickness or Chagas disease models. However, from a methodological point of view, sex standardization has been a traditional strategy to control the impact of biological variability on outcome measures, with a relevant influence on the comparability and generalizability of experimental findings (Clayton and Collins, Reference Clayton and Collins2014). Conversely, the age and weight of animals are two related parameters that exerts a direct impact on disease evolution, since parasite control is immune mediated (Vincendeau and Bouteille, Reference Vincendeau and Bouteille2006; Machado et al. Reference Machado, Dutra, Esper, Gollob, Teixeira, Factor, Weiss, Nagajyothi, Tanowitz and Garg2012). These parameters were more homogeneous but less reported in studies on Chagas disease, and more variable for sleeping sickness. These characteristics appear to be directly influenced by the use of rabbits and dogs in studies on sleeping sickness. However, even studies using only rats or mice showed greater age and weight variability, indicating reduced homogeneity in T. brucei-infected groups. Taking into account that the immune response has a curvilinear activation profile and effectiveness through the host life cycle, discrepant animal ages can be associated with divergent innate and acquired immune responses and variable infection patterns (Shearer, Reference Shearer1997; Boldizsar et al. Reference Boldizsar, Mikecz and Glant2010). Thus, heterogeneous ages and the absence of this information impair the comparison between studies and understanding to what extent the immune response may have influenced the results of the therapies investigated.
Although the parasite strains used to induce both diseases have also presented wide variability, the antiparasitic assays were coherently based on acute experimental models. There is evidence that each parasite strain presents different virulence and pathogenicity patterns. Thus, by using virulent strains such as H4 (Guzman-Marin et al. Reference Guzman-Marin, Jimenez-Coello, Puerto-Solis, Ortega-Pacheco and Acosta-Viana2012) and pathogenic strains such as Y (Martínez-Díaz et al. Reference Martínez-Díaz, Escario, Nogal-Ruiz and Gómez-Barrio2001; Santos et al. Reference Santos, Novaes, Cupertino, Bastos, Klein, Silva, Fietto, Talvani, Bahia and Oliveira2015) in mouse models of Chagas disease, and the virulent Federe strain in the rat model of sleeping sickness (Adamu et al. Reference Adamu, Nwosu and Agbede2009; Ibrahim et al. Reference Ibrahim, Musa, Aliyu, Mayaki, Gideon and Islam2013a , Reference Ibrahim, Aliyu, Abdullahi, Solomon, Toko, Garba, Bashir and Habila b ), authors have aligned experimental design with important outcome measures such as mortality, parasitaemia and parasite load, which are essential indicators of the antiparasitic potential of natural or synthetic drugs (Aderbauer et al. Reference Aderbauer, Clausen, Kershaw and Melzig2008; Novaes et al. Reference Novaes, Gonçalves, Penitente, Bozi, Neves, Maldonado, Natali and Talvani2016). Since parasitaemia can drop to undetectable levels in chronic stages (Marin-Neto et al. Reference Marin-Neto, Cunha-Neto, Maciel and Simões2007; Bern, Reference Bern2015), it is difficult to accurately determine if the beneficial outcomes are determined by a direct parasitic control of the products tested or by a secondary effect of the product on the metabolism and structure of the target organs in both diseases. This aspect could partially explain the rarity of studies investigating the chronic phase of T. cruzi (Jiménez-Coello et al. Reference Jiménez-Coello, Acosta-Viana, Ortega-Pacheco, Perez-Gutierrez and Guzman-Marin2014) and T. brucei (Nasimolo et al. Reference Nasimolo, Kiama, Gathumbi, Makanya and Kagira2014) infections.
Beyond all differences in animal models and parasite strains, plant families and species were also quite variable in studies on Chagas disease and sleeping sickness. While the investigation of native and exotic plant species was relatively similar, most studies reported the use plants with ethnodirected indications for trypanosomiasis treatment. Taking into account the fact that random screening of plants in the search for new biomolecules or drugs presents poor efficiency, and is time-consuming and very expensive (Ramesha et al. Reference Ramesha, Gertsch, Ravikanth, Priti, Ganeshaiah and Uma Shaanker2011; Pan et al. Reference Pan, Zhou, Gao, Yu, Zhang, Tang, Sun, Ma, Han, Fong and Ko2013), appropriating popular knowledge and traditional usage represents a rational approach in natural products research (Heinrich and Gibbons, Reference Heinrich and Gibbons2001; Silva et al. Reference Silva, Albuquerque, Costa Júnior, Lima, do Nascimento and Monteiro2014). In fact, most investigated plants presented beneficial results in the treatment of Chagas disease and sleeping sickness, indicating a convergence between the ethnomedical system and an effective pharmacological trypanocidal effect. Although ethnomedicine often indicates a history of safe usage of several plant species, there is the misconception that in cases of traditional use by human populations, the biological safety of plant species is assured (Marcus and Snodgrass, Reference Marcus and Snodgrass2005; Ekor, Reference Ekor2014). This aspect was clearly identified in the present review, since seven plant species (A. africana, K. senegalensis, T. superba, M. peregrina, Z. zanthoxyloides, B. paradoxun and S. americanum) presented some indication of host toxicity. Although poorly understood, host toxicity could be related to the highly concentrated extracts used in the treatments, a different aspect compared to the ethnomedical practice, which is mainly based on less concentrated plant-based products (Calixto, Reference Calixto2000; Silva et al. Reference Silva, Albuquerque, Costa Júnior, Lima, do Nascimento and Monteiro2014). As more than half of the studies did not perform host toxicity tests, the safety of the plant products tested cannot be proven. This is a serious issue, since positive results cannot be considered in isolation to determine the biological/pharmacological relevance of the tested product, which should be strictly based on a risk-benefit analysis (Calixto, Reference Calixto2000; Muller and Milton, Reference Muller and Milton2012). Nowadays, toxicological tests are indicated much earlier and play a central role in determining the safety of candidate drugs (Pritchard et al. Reference Pritchard, Jurima-Romet, Reimer, Mortimer, Rolfe and Cayen2003; Santos et al. Reference Santos, Novaes, Cupertino, Bastos, Klein, Silva, Fietto, Talvani, Bahia and Oliveira2015). Thus, LD50 and dose–response genotoxicity tests, behavioural and toxicokinetics assays in one or more rodent (especially mice and rats) or non-rodent (especially dogs and non-human primates) species have been strongly indicated. Such investigations are useful in providing data on the biological safety of a new chemical substance, guiding the refinement of pharmacological studies (Pritchard et al. Reference Pritchard, Jurima-Romet, Reimer, Mortimer, Rolfe and Cayen2003; Parasuraman, Reference Parasuraman2011).
In addition to this limited analytical rigour, the plant species O. gratissimum, G. senegalensis, S. longepedunculata and K. senegalensis presented contradictory results in different studies, an aspect potentially related to the profound differences in animal models (mice vs rats), parasite strains (i.e. pathogenicity vs. virulence) and experimental protocols (i.e. extract dose, time and route of administration) applied in sleeping sickness treatment. Unconventional routes of administration were another important limiting factor observed in half of the studies reviewed. Since most studies were based on ethnodirected plant indications and tested crude extracts, it is not reasonable to adopt a route other than oral (i.e. intraperitoneal, intramuscular, etc.), which is traditionally used when non-fractioned extracts are used (Ajazuddin and Saraf, Reference Ajazuddin and Saraf2010; Kesarwani et al. Reference Kesarwani, Gupta and Mukerjee2013). This is not a trivial matter since it directly interferes with the pharmacokinetics and pharmacodynamics of the secondary metabolites present in the administered plant preparations (De Smet and Brouwers, Reference De Smet and Brouwers1997; Bhattaram et al. Reference Bhattaram, Graefe, Kohlert, Veit and Derendorf2002). In addition, since more than two-thirds of the studies did not present a complete phytochemical screening of the investigated plant products, it was rarely possible to establish, which secondary metabolites are associated with the results. This is a central limitation that hampers the discovery of specific molecules with biotechnological potential to be used in the development of new drugs (Atanasov et al. Reference Atanasov, Waltenberger, Pferschy-Wenzig, Linder, Wawrosch, Uhrin, Temml, Wang, Schwaiger, Heiss, Rollinger, Schuster, Breuss, Bochkov, Mihovilovic, Kopp, Bauer, Dirsch and Stuppner2015). This limitation was overcome only in those studies using extract fractions and isolated molecules, in which glycosides (Ayyari et al. Reference Ayyari, Salehi, Ebrahimi, Zimmermann, Portmann, Krauth-Siegel, Kaiser, Brun, Rezadoost, Rezazadeh and Hamburger2014), flavonoids (Marín et al. Reference Marín, Ramírez-Macías, López-Céspedes, Olmo, Villegas, Díaz, Rosales, Gutiérrez-Sánchez and Sánchez-Moreno2011; da Rocha et al. Reference da Rocha, Queiroz, Meira, Moreira, Soares, Marcourt, Vilegas and Wolfender2014), benzenetriols (Ibrahim et al. Reference Ibrahim, Musa, Aliyu, Mayaki, Gideon and Islam2013a ), lactones (Sülsen et al. Reference Sülsen, Frank, Cazorla, Anesini, Malchiodi, Freixa, Vila, Muschietti and Martino2008, Reference Sülsen, Frank, Cazorla, Barrera, Freixa, Vila, Sosa, Malchiodi, Muschietti and Martino2011; Ibrahim et al. Reference Ibrahim, Aliyu, Abdullahi, Solomon, Toko, Garba, Bashir and Habila2013b ), lignans (Bastos et al. Reference Bastos, Albuquerque and Silva1999), proanthocyanidins (Kubata et al. Reference Kubata, Nagamune, Murakami, Merkel, Kabututu, Martin, Kalulu, Huq, Yoshida, Ohnishi-Kameyama, Kinoshita, Duszenko and Urade2005), alkaloids (Ferreira et al. Reference Ferreira, Cebrián-Torrejón, Corrales, Vera de Bilbao, Rolón, Gomez, Leblanc, Yaluf, Schinini, Torres, Serna, Rojas de Arias, Poupon and Fournet2011), steroids (Meira et al. Reference Meira, Guimarães, Dos Santos, Moreira, Nogueira, Tomassini, Ribeiro, de Souza, Ribeiro Dos Santos and Soares2015) and terpenoids (Ramírez-Macías et al. Reference Ramírez-Macías, Marín, Chahboun, Messouri, Olmo, Rosales, Gutierrez-Sánchez, Alvarez-Manzaneda and Sánchez-Moreno2012; Lozano et al. Reference Lozano, Strauss, Spina, Cifuente, Tonn, Rivarola and Sosa2016) were the main classes of secondary metabolites associated with antitrypanosomal activity in both diseases. The relevance of phytochemical screening was clearly demonstrated by the identification of quinine more than 190 years ago, the first antimalarial drug isolated from the bark of the Cinchona tree (Rubiaceae) (Schmidt et al. Reference Schmidt, Khalid, Romanha, Alves, Biavatti, Brun, Da Costa, de Castro, Ferreira, de Lacerda, Lago, Leon, Lopes, das Neves Amorim, Niehues, Ogungbe, Pohlit, Scotti, Setzer, de N C Soeiro, Steindel and Tempone2012). In addition, artemisinin, a sesquiterpene lactone isolated from Artemisia annua, showed high effectiveness against malaria, providing important contributions in the clinical management of chloroquine-resistant infections (van Agtmael et al. Reference van Agtmael, Eggelte and van Boxtel1999).
Methodological limitations were objectively identified from the analysis of reporting quality. Surprisingly, almost half of the essential criteria to be reported in in vivo animal studies were neglected. In general, underreported aspects such as the duration of treatment, administration route, experimental replicates, randomization of animals and groups are serious limitations to the internal and external validity of the included studies, impairing experimental reproducibility and the reliability of results. A potential strategy for a long-term solution to minimizing the risks of publication bias may be introducing basic requirements that must be incorporated into preclinical studies of animal models. For this, there are several guidelines regarding experimental design and the key aspects that should be reported when animal research data are publicly disclosed, such as SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE; http://www.SYRCLE.nl) and Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES; http://www.camarades.info).
Although our systematic review represents a proposal to group and critically analyse the evidence on the applicability of plant derivatives in the treatment of two neglected tropical diseases, the interpretation of the results should consider some limitations. Our sampling frame was based on specific databases. Thus, some articles may have been not recovered due to the boundaries applied in the search strategy, as well as limitations in the algorithms adopted in the search interfaces of each database. These aspects directly affect the sensitivity and specificity of the search strategy, which may have contributed to identifying key articles. We attempted to mitigate these limitations by screening the reference lists of all articles, which are not limited to databases or any keyword-based search model. The relevant number of additionally recovered papers indicates the utility of this approach in a heterogeneous area such as the evaluation of plant extracts in the treatment of parasitic diseases.
Based on this systematic review, it was possible to conclude that there is an important geographic and ethnodirected component involved in the research initiatives on the relevance of plant derivatives in the treatment of Chagas disease and sleeping sickness. Furthermore, there is a mismatch between the quantity and quality of the evidence. Although most studies adopt coherent animal models of infection and report beneficial trypanocidal results in vivo, the analysis of reporting quality suggested that the current evidence is at a high risk of bias. The absence or incomplete characterization of the animal models, experimental groups, treatment protocols, phytochemical screening and toxicity analysis of the plant products, impair the internal validity of the individual animal studies. Together with these limitations, contradictory results based on heterogeneous studies of the same plant species compromise the external validity of the evidence, making it difficult to translate animal data into clinical practice, as well as the relevance of the plant species as potential biotechnological targets in the development of new drugs to treat Chagas disease and sleeping sickness. Taking into account that poor reporting quality does not always reflect the quality of the research actually carried out, we hope that our critical analysis may help to streamline preclinical research to reduce methodological bias, thus improving data reliability and generalizability.
FINANCIAL SUPPORT
This work was supported by the Brazilian agencies ‘Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)’ and ‘Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)’.
SUPPLEMENTARY MATERIAL
To view supplementary material for this article, please visit https://doi.org/10.1017/S0031182017000634.
