Trypanosomatids are flagellated protozoan parasites present in tropical and subtropical areas that cause a wide range of diseases in humans and animals, such as Chagas Disease (CD), Cutaneous and Visceral Leishmaniasis (CL and VL, respectively), and African sleeping sickness (1), which are mainly transmitted by vector insects (2). Most of the diseases caused by trypanosomatids can result in morbidity and mortality in adults, children, and animals. About half a billion people live in areas at risk for these diseases, and more than 20 million have infections caused by at least one of them, accounting for over 100,000 deaths a year (1, 3).

Leishmaniasis and CD are in the list of most relevant Neglected Tropical Diseases (NTD) according to the World Health Organization (WHO) caused by infection with Leishmania spp. and Trypanosoma cruzi, respectively (4). CD, which is transmitted by triatomine bugs, is endemic in Latin America (5, 6). CL and VL, on the other hand, are transmitted by infected phlebotomine sandflies mainly in poor and underprivileged populations (7), and are considered the two main distinct manifestations of Leishmaniasis (8). VL, the most severe form, is caused by Leishmania infantum and Leishmania donovani in the Americas and Eastern Hemisphere, respectively. It affects people every year and can present high death rates if not properly treated (9). CL is caused by many Leishmania species of subgenus Leishmania and Viannia in Africa, Asia, and Latin America (9, 10).

During the infection process, the parasites can invade several nucleated cells of the host through a broad variety of receptors (11). One of the first defense mechanisms of the hosts is complement activation, which recognizes and controls parasite invasion and presents important biological responses such as opsonization, phagocytosis and lysis of microorganisms and cells (12, 13), acting through classical, alternative and lectin pathways (12). The classical pathway is initiated by immune complexes, depending mainly on the interaction of C1 with antigen-antibody complexes, or occasionally by pathogen-associated molecular patterns (PAMPs) (12, 14, 15). The lectin pathway starts the activation process, and is triggered by the binding of pattern-recognition molecules (PRMs), such as mannose-binding lectin (MBL), ficolins and collectins, to PAMPs on parasite surface (15–17). It prompts the activation of the alternative pathway, which occurs when the bond between the C3 α-chain and thiol-ester hydrolyzes spontaneously, exhibiting a reactive site (12). Nevertheless, trypanosomatids have mechanisms to break recognition by the host immune system and escape complement attack; therefore, the infection depends on parasite persistence and host immune response (15, 18).

Capacity of the parasites to invade the host cells and proceed with the infection process depends on their ability to escape the host protection mechanisms and its genetic background (12, 15). Consequently, genetic modifications to the components of the three complement pathways, such as single nucleotide polymorphisms (SNPs), can have an impact on immunocompetence and influence susceptibility to infectious diseases (19, 20). In this context, human complement receptor 1 (CR1) levels were shown to be modulated by genetic variants in exon 29, which led to decreased expression in CD patients, indicating that low levels of this protein may influence T. cruzi infection (21, 22). Furthermore, previous studies have shown that gene polymorphisms are associated with mannose-binding lectin-associated serine protease 2 (MASP-2) and MBL expression levels, and are able to cause immunodeficiency, promote infection progression, and influence several infections in VL (23–25), since those genes are related to the lectin pathway activation (26). An illustration of common MBL2 polymorphisms are mutations in exon 1, which may lead to translational modifications and diminish serum protein levels (27–29). Still referring to the lectin pathway, ficolin-2 (FCN2) is known to be a highly polymorphic encoding gene, and elevated serum levels may facilitate the development of VL (30–32). Moreover, FCN2 polymorphisms can also influence the development of the cardiodigestive clinical form in chronic CD (20), delaying complement activation when the genetic modifications interfere with the binding capacity of the variant pathogen molecules toward their ligands (30, 33).

Additionally, studies in this area are scarce, and the reason why some individuals develop diseases more easily than others is still not fully understood. Therefore, this systematic review aims to report polymorphisms and expression levels of complement genes and understand how they are involved in the susceptibility or resistance to infectious diseases caused by trypanosomatids.

The complement system participates actively in first-line immune defense against pathogens (43). Proteins act on pathogen opsonization in a cascade reaction and induce inflammation, which activates immune cells against the invading agent and assist in maintaining host homeostasis (44). Studies have confirmed that genetic mutations in complement genes, as well as modifications in their expression levels, are associated with development of several human disorders, such as schizophrenia (45), age-related macular degeneration (46), Alzheimer’s disease (47), myocardial infarction (48), and stroke (49). In addition, these events were also interrelated with susceptibility to several bacterial and viral infectious diseases, such as meningitis (50, 51), leprosy (52), hepatitis B (53), and diseases caused by trypanosomatids: CD (22), VL (36), and CL (8).

In most cases, the expression levels of these proteins are directly affected by mutations in their respective genes, as a cause-and-effect relationship (54); therefore, nine of the 12 selected papers included both gene and protein conditions. Exons are composed of the coding region as well as of the 3’ and 5’ untranslated regions of the RNA, thus they were considered the main targets of almost all papers. However, previous studies have elucidated how genetic alterations in the promoter region are capable of impacting on protein concentration (30, 55), activity (56), and transcriptional efficiency (57) in the organism, and nine of the selected papers also included an analysis of this regulatory region.

The lectin complement pathway is, by far, the most explored in the context of progression of infectious diseases. This is due to the fact this pathway is activated by PRMs, providing first-line immunological defense against pathogens (58) and contributing to clearance of microbes (59). Moreover, it has already been described that several protozoan species are able to activate this pathway with high efficiency, since depletion of MBL and ficolins in serum reduced the complement deposition and lysis by approximately 70% in T. cruzi (60). This explains why ten of the 12 selected papers included in this systematic review focused on this complement pathway.

Population genetics studies demand the inclusion of several methodological criteria to ensure their reliability. One of the fundamental principles is the assessment of HWE, which is used to estimate the number of homozygous and heterozygous variant carriers based on their allele frequency in populations that are not evolving (61). Fortunately, almost all included papers used this criterion in their methodology, which sustained the findings of association of SNPs with disease progression (22). Interestingly, while some studies analyzed the SNPs out of HWE (22, 40), others chose to remove them from the analysis (32, 36). This methodological divergence could influence the conclusion from the data obtained; therefore, it is important to adopt a standard protocol to ensure the quality of the presented evidence in future studies.

In the genetic-based context, the study of SNPs enables the formation of haplotypes, which are sets of SNPs found in the same chromosome (42). The distance between the SNPs in a haplotype allows the LD analysis, which is also important in this field since it can be associated with disease progression among its multiple applications (62). In this context, the lectin pathway, once again, plays an important role, since FCN2 haplotypes have been associated with protection against VL (32) and susceptibility to CL (8); the MBL2 haplotype was interrelated with protection against VL (36); the COLEC11 haplotype was associated with susceptibility to CD (18), and the MASP2 haplotype was related to both resistance and susceptibility to CD (25), sustaining the importance of performing this analysis.

ELISA has been the most used technique in the analysis of expression levels of complement proteins (22, 36, 38). Since this tool is antibody-based, it provides high accuracy for identification of proteins and peptides (63). However, this assay presents limitation in large-scale studies, as most ELISA kits are antigen-specific (64). Currently, there are tools that can optimize this analysis and increase the number of targets and samples, such as mass spectrometry-based proteomics (65). It enables large-scale quantitative analysis of these serum proteins by using a small sample volume, and generates considerable information regarding patient physiology; however, it should be considered that the large abundance of other proteins and their interactions could be a challenge for this strategy (66). In contrast, several techniques have been used to perform identification of SNPs, such as conventional PCR (25), real-time PCR (8), and SNaPshot Multiplex kit (39). However, most of the studies used the Sanger sequencing method (18, 32, 36), which was the first approach to be developed (67). Although this method is still used to validate more modern techniques (68), it has a high cost per run, which hinders the investigation of many targets at once (69). New Generation Sequencing (NGS) could strengthen these studies, considering that it is able to generate a large amount of data faster and cheaper (67), which broadens the research field since it allows the investigation of more genes in the same study.

Another important aspect in the context of methodology planning in a research proposal is sample size determination, which was considered only by three papers in this systematic review. It can be calculated in several ways (70) and sustains the representativity of the sample with the original population, since a small number could not generate statistical significance, and a large number could be a waste of resources (71). However, it could be unapproachable, especially in developing countries, considering that it requires funding to include several places in the study area, and there is a risk of not reaching the calculated number due to the lack of interest or availability of volunteers (72). The inclusion of diversity in experimental groups is also of vital importance, and was considered in nine of the papers. It reduces potential bias, since gender and ethnic origin directly impact on the results of a research in its various topics, including pain response (73) and autoimmune (74) and infectious diseases (75). One paper did not describe any information regarding the experimental group characteristics; therefore, it is not possible to analyze these results for bias (41). The definition of the correct control groups also sustains the truthfulness of the research results (76), and should be described clearly in the methodology section. In this context, three papers did not specify the origin of the control group (18, 20, 38), which could not represent the experimental group location if collected in a different place.

Chagas Disease was more explored than Leishmaniasis in the specific literature. The most used diagnostic tool for CD among the selected papers was based on serological methods such as immunofluorescence and antigen detection (18, 40). For VL, detection of the rk39 parasite antigen was the most used technique (32, 36), while Giemsa staining of lesion samples was the most used for CL diagnosis (8). Interestingly, none of the papers chose molecular tests, which are more accurate and can identify co-infections and recognize low antibody producing patients (77). However, some studies suggested a combination of serological and molecular assays to maximize the chances of performing a correct diagnosis (77, 78). These tests have different specificity and sensitivity rates, which should be considered in the interpretation of results (77). T. cruzi parasites can be classified into six different types due to propagation of polymorphism events; therefore, the targets for molecular tests should be considered carefully to include as many variants as possible (79). The correct treatment for VL depends on accurate and early diagnosis. Although the rk39 antigen-based test provides a quick and selective result, it is not able to differentiate precisely between an active and a past diagnosis. Therefore, molecular methods such as PCR should be used in parallel to ensure the correct diagnosis and monitor the treatment efficacy (80). Giemsa staining of skin lesion is widely used in CL diagnosis; however, it is not the most sensitive technique and demands experienced professionals to be performed safely. PCR is the most sensitive method, providing fast results and offering additional analysis of species identification (81). The choice of the diagnostic method is associated with the selection of the appropriate sample for the test. In this context, understanding the infection dynamics of each parasite is vital, since molecular tests detect the parasite genetic material, which is found more abundantly in their target organ. Fortunately, nearly all papers included in this systematic review were careful about this matter.

The complement system plays an important role in the initial phase of infectious processes, and it was proved that genetic alteration in its genes considerably affects the progression of several conditions, including diseases caused by trypanosomatids. The lectin pathway is the most explored topic of complement in the literature, and its protein levels are associated with both susceptibility and resistance to diseases.

Chagas Disease is more studied than Leishmaniasis in the specific literature, and most of the papers assessed were careful about experimental planning. However, current studies should focus on including more accurate and modern diagnosis and analysis techniques, since several complement proteins have not been investigated in this field of research yet.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

TT, LM, PS and FF: Study conception and design. TT and LM: Data collection. TT, LM, and PS: Data analysis. TT, LM, and PS: Data interpretation. TT and LM: Drafted the manuscript. PS and FF: Revised the manuscript. PS and FF supervised the study. All authors contributed to the article and approved the submitted version.

This study was supported by Fundação Oswaldo Cruz (FIOCRUZ) and Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) (PROEP) (442055/2019-6). FF holds a grant from CNPq for productivity in research (309862/2015-9).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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