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Genomic surveillance: a potential shortcut for effective Chagas disease management

Abstract

Chagas disease is an enduring public health issue in many Latin American countries, receiving insufficient investment in research and development. Strategies for disease control and management currently lack efficient pharmaceuticals, commercial diagnostic kits with improved sensitivity, and vaccines. Genetic heterogeneity of Trypanosoma cruzi is a key aspect for novel drug design since pharmacological technologies rely on the degree of conservation of parasite target proteins. Therefore, there is a need to expand the knowledge regarding parasite genetics which, if fulfilled, could leverage Chagas disease research and development, and improve disease control strategies. The growing capacity of whole-genome sequencing technology and its adoption as disease surveillance routine may be key for solving this long-lasting problem.

Key words:
Chagas disease; Trypanosoma cruzi; whole genome sequencing; genetic variation; trypanocidal agents; computational biology


Approximately 7 million people worldwide are infected by Trypanosoma cruzi, the causative agent of Chagas disease (CD).11. WHO - World Health Organization. Chagas disease (also known as American trypanosomiasis) [Internet]. 2022 [cited 2022 Jun 10]. Available from: https://www.who.int/news-room/fact-sheets/detail/chagas-disease-(american-trypanosomiasis).
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The infection is endemic in poor rural regions of Latin American countries, habitats to both the vector and the parasite of CD, though recent studies have shown the emergence of triatomine vectors in urban settings suggesting an expansion of risk areas.22. Parente CC, Bezerra FSM, Parente PI, Dias-Neto RV, Xavier SCC, Ramos AN, et al. Community-based entomological surveillance reveals urban foci of chagas disease vectors in Sobral, State of Ceara, Northeastern Brazil. PLoS One. 2017; 12(1): 1-11.,33. Provecho YM, Fernández MP, Salvá L, Meli S, Cano F, Sartor P, et al. Urban infestation by Triatoma infestans (Hemiptera: Reduviidae), an overlooked phenomena for Chagas disease in Argentina. Mem Inst Oswaldo Cruz. 2021; 116(1): 1-6 In such regions, the disease is primarily transmitted orally or through vector exposure, sustaining the chance of outbreaks.44. Franco-Paredes C, Villamil-Gómez WE, Schultz J, Henao-Martínez AF, Parra-Henao G, Rassi A, et al. A deadly feast: elucidating the burden of orally acquired acute Chagas disease in Latin America - Public health and travel medicine importance. Travel Med Infect Dis. 2020; 36: 101565.,55. Noya B, Díaz-Bello Z, Colmenares C, Ruiz-Guevara R, Mauriello L, Zavala-Jaspe R, et al. Large urban outbreak of orally acquired acute Chagas disease at a school in Caracas, Venezuela. J Infect Dis. 2010; 201(9): 1308-15.,66. Alarcón de Noya B, Díaz-Bello Z, Colmenares C, Ruiz-Guevara R, Mauriello L, Muñoz-Calderón A, et al. Update on oral Chagas disease outbreaks in Venezuela: epidemiological, clinical and diagnostic approaches. Mem Inst Oswaldo Cruz. 2015; 110(3): 377-86.,77. Ramírez JD, Montilla M, Cucunubá ZM, Floréz AC, Zambrano P, Guhl F. Molecular epidemiology of human oral Chagas disease outbreaks in Colombia. PLoS Negl Trop Dis. 2013; 7(2): e2041.,88. Souza-Lima RC, Barbosa MGV, Coura JR, Arcanjo ARL, Nascimento AS, Ferreira JMBB, et al. Outbreak of acute Chagas disease associated with oral transmission in the Rio Negro region, Brazilian Amazon. Rev Soc Bras Med Trop. 2013; 46(4): 510-4. Adding to that, CD has also become a concern in non-endemic settings (e.g., North American, European, and Asian countries) due to population movement,99. Lidani KCF, Andrade FA, Bavia L, Damasceno FS, Beltrame MH, Messias-Reason IJ, et al. Chagas disease: from discovery to a worldwide health problem. Front Public Health. 2019; 7(6): 1-13.,1010. Meymandi SK, Forsyth CJ, Soverow J, Hernandez S, Sanchez D, Montgomery SP, et al. Prevalence of Chagas disease in the Latin American-born population of Los Angeles. Clin Infect Dis. 2017; 64(9): 1182-8.,1111. Ramos-Sesma V, Navarro M, Llenas-García J, Gil-Anguita C, Torrus-Tendero D, Wikman-Jorgensen P, et al. Community-based screening of Chagas disease among Latin American migrants in a non-endemic country: an observational study. Infect Dis Poverty. 2021; 10(1): 117.,1212. Velasco M, Gimeno-Feliú LA, Molina I, Salas-Coronas J, Solà I, Monge-Maillo B, et al. Screening for Trypanosoma cruzi infection in immigrants and refugees: systematic review and recommendations from the Spanish Society of Infectious Diseases and Clinical Microbiology. Euro Surveill. 2020; 25(8): 1900393. and approximately 100,000 infected individuals were estimated to live in nine European countries in 2009.1313. Basile L, Jansá JM, Carlier Y, Salamanca DD, Angheben A, Bartoloni A, et al. Chagas disease in European countries: the challenge of a surveillance system. Euro Surveill. 2011; 16(37): 3. This represents a risk as these patients may vertically transmit CD to their offspring1414. Francisco-González L, Rubio-San-Simón A, González-Tomé MI, Manzanares Á, Epalza C, Santos MM, et al. Congenital transmission of Chagas disease in a non-endemic area, is an early diagnosis possible? PLoS One. 2019; 14(7): e0218491.,1515. Howard E, Xiong X, Carlier Y, Sosa-Estani S, Buekens P. Frequency of the congenital transmission of Trypanosoma cruzi : a systematic review and meta-analysis. BJOG. 2014; 121(1): 22-33. or through blood transfusion to other people1616. Angheben A, Boix L, Buonfrate D, Gobbi F, Bisoffi Z, Pupella S, et al. Chagas disease and transfusion medicine: a perspective from non-endemic countries. Blood Transfus. 2015; 13(4): 540-50.,1717. Flores-Chavez M, Fernandez B, Puente S, Torres P, Rodriguez M, Monedero C, et al. Transfusional Chagas disease: parasitological and serological monitoring of an infected recipient and blood donor. Clin Infect Dis. 2008; 46(5): e44-7. demanding both individual and community health care assistance.

Brazil, one of the endemic countries for CD, has a universal health care system that covers treatment regimens and national vector control programs that have significantly decreased disease incidence in the last decades.1818. Dias JCP, Ramos AN, Gontijo ED, Luquetti A, Shikanai-Yasuda MA, Coura JR, et al. II Consenso Brasileiro em Doença de Chagas, 2015. Epidemiol Serv Saúde. 2016; 25(núm. esp.): 7-86.,1919. Rojas de Arias A, Monroy C, Guhl F, Sosa-Estani S, Santos WS, Abad-Franch F. Chagas disease control-surveillance in the Americas: the multinational initiatives and the practical impossibility of interrupting vector-borne Trypanosoma cruzi transmission. Mem Inst Oswaldo Cruz. 2022; 117: 1-15. However, since most acute cases are asymptomatic or present nonspecific symptoms, and chronic cases do not receive a mandatory notification, CD national surveillance and control strategies are currently insufficient, which is reflected in the number of CD-associated deaths: ~ 4,500/year between 2010 and 2019.2020. SVS - Secretaria de Vigilância em Saúde. Boletim Epidemiológico Doença de Chagas. Brasília; 2021. Available from: https://www.gov.br/saude/pt-br/media/pdf/2021/abril/14/boletim_especial_chagas_14abr21_b.pdf.
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Between 2000 and 2011, CD was the leading cause of death by a single pathogen causing a neglected tropical disease in Brazil.2121. Martins-Melo FR, Ramos AN, Alencar CH, Heukelbach J. Mortality from neglected tropical diseases in Brazil, 2000-2011. Bull World Health Organ. 2016; 94(2): 103-10. On the other hand, there were only 5,184 acute cases reported between 2001 and 2018, comprising 288/year on average,2222. Santos EF, Silva ÂAO, Leony LM, Freitas NEM, Daltro RT, Regis-Silva CG, et al. Acute Chagas disease in Brazil from 2001 to 2018: a nationwide spatiotemporal analysis. PLoS Negl Trop Dis. 2020; 14(8): e0008445. suggesting huge underreporting. In other words, in the absence of systematic diagnosis and effective treatment, patients progress to the chronic phase, in which the most severe manifestations and a significantly reduced chance of treatment success prevail.2323. Urbina JA. Etiologic treatment of Chagas disease: old drugs, new insights, challenges, and perspectives. In: Pinazo Delgado MJ, Gascón J, editors. Chagas Disease Springer, Cham. 2020. p. 123-44. Available from: http://link.springer.com/10.1007/978-3-030-44054-1_8.
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,2424. Urbina JA. Recent clinical trials for the etiological treatment of chronic chagas disease: advances, challenges and perspectives. J Eukaryot Microbiol. 2015; 62(1): 149-56. The available chemotherapeutics against parasites that cause CD induce severe adverse effects in patients and have low efficacy in the late stage of infection, hampering treatment adhesion and leading to uncertain prognostic.2525. Coura JR, de Castro SL. A critical review on Chagas disease chemotherapy. Mem Inst Oswaldo Cruz. 2002; 97(1): 3-24.,2626. Jackson Y, Wyssa B, Chappuis F. Tolerance to nifurtimox and benznidazole in adult patients with chronic Chagas' disease. J Antimicrob Chemother. 2020; 75(3): 690-6.

Insufficient investments in treatment and vaccine development are an important issue that contributes to the challenge of overcoming the disease. Between 2009 and 2018, US$ 236.31 million was invested in research and development (R&D) for CD, representing only 0.67% of the investment in neglected diseases in the reference period.2727. Sangenito LS, Branquinha MH, Santos ALS. Funding for Chagas disease: a 10-year (2009-2018) survey. Trop Med Infect Dis. 2020; 5(2): 88.,2828. WHO - World Health Organization. R&D funding flows for neglected diseases by disease, year and funding category. 2021. Available from: https://www.who.int/observatories/global-observatory-on-health-research-and-development/monitoring/r-d-funding-flows-for-neglected-diseases-by-disease-year-and-funding-category#scope-and-limitations.
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Almost half of this budget (47%) was applied in basic research, 42.5% in drug development and roughly 10% in vaccines and diagnostics. Importantly, as a disease that affects primarily poor populations, pharmaceutical companies historically have shown a lack of interest in R&D for CD, in which funding is mainly provided by the public sector.2727. Sangenito LS, Branquinha MH, Santos ALS. Funding for Chagas disease: a 10-year (2009-2018) survey. Trop Med Infect Dis. 2020; 5(2): 88. Although having a lower death incidence in comparison to other neglected diseases (e.g., HIV/AIDS, malaria, and tuberculosis), which therefore receive proportionally more investments, CD is a relevant cause of premature death and life-long disability.2929. Martins-Melo FR, Carneiro M, Ribeiro ALP, Bezerra JMT, Werneck GL. Burden of Chagas disease in Brazil, 1990-2016: findings from the Global Burden of Disease Study 2016. Int J Parasitol. 2019; 49(3-4): 301-10. This “misconception of mildness” may partially account for the lack of urgency of disease management and the persistence of this harmful and silent medical condition.

Another critical aspect of CD control is that T. cruzi shows intense genetic diversity, which may contribute to the observed variability in disease outcome, clinical manifestations and treatment efficacy.2525. Coura JR, de Castro SL. A critical review on Chagas disease chemotherapy. Mem Inst Oswaldo Cruz. 2002; 97(1): 3-24.,3030. Nielebock MAP, Moreira OC, Xavier SCC, Miranda LFC, Lima ACB de, Pereira TO de JS, et al. Association between Trypanosoma cruzi DTU TcII and chronic Chagas disease clinical presentation and outcome in an urban cohort in Brazil. PLoS One. 2020; 15(12): e0243008. This genetic diversity has also been hampering the search for new treatments, the development of vaccines, and limiting the efficacy of commercial serological diagnosis kits, since all these pharmaceutical technologies rely on the conservation of parasite target proteins. Hence, a more rational approach to disease control should be taken. In this opinion article, we present some of the challenges in CD management imposed by T. cruzi genetic diversity, and hypothesise how whole-genome sequencing (WGS) of clinical samples coupled with national disease surveillance could not only enable optimised control strategies in endemic regions but also fuel scientific research projects that focus on developing pharmaceuticals technologies.

The treatment problem

Pharmacological treatment is essential in CD control and management considering disease endemicity and the constant risk of infection. The therapeutic guidelines currently adopted prescribe the only two drugs that have been available for CD treatment since the 1960s: Benznidazole (Bz) and Nifurtimox (first and second-line drugs, respectively). Their exact mechanism of action is not fully understood, but it is known so far that in T. cruzi these nitroheterocyclic prodrugs are activated by a type I nitroreductase enzyme (TcNTR) through the reduction of a nitro group.3131. Hall BS, Wilkinson SR. Activation of benznidazole by trypanosomal type I nitroreductases results in glyoxal formation. Antimicrob Agents Chemother. 2012; 56(1): 115-23. This reaction produces molecules capable of binding and causing damage to parasite nucleic acids and essential proteins resulting in a trypanocidal effect. Although effective in the initial phase of infection, Bz and Nifurtimox have a long list of intricate problems that may result from their administration.2424. Urbina JA. Recent clinical trials for the etiological treatment of chronic chagas disease: advances, challenges and perspectives. J Eukaryot Microbiol. 2015; 62(1): 149-56. First, treatment can last up to 90 days and is often indistinctly associated with severe adverse effects which contribute to reduced treatment adhesion. Also, their efficacy is not fully established in the chronic phase and, for this reason, these drugs are frequently not prescribed in these cases.2424. Urbina JA. Recent clinical trials for the etiological treatment of chronic chagas disease: advances, challenges and perspectives. J Eukaryot Microbiol. 2015; 62(1): 149-56.,2525. Coura JR, de Castro SL. A critical review on Chagas disease chemotherapy. Mem Inst Oswaldo Cruz. 2002; 97(1): 3-24.,2626. Jackson Y, Wyssa B, Chappuis F. Tolerance to nifurtimox and benznidazole in adult patients with chronic Chagas' disease. J Antimicrob Chemother. 2020; 75(3): 690-6.,3232. Campos M, Leon LL, Taylor MC, Kelly JM. Benznidazole-resistance in Trypanosoma cruzi: evidence that distinct mechanisms can act in concert. Mol Biochem Parasitol. 2014; 193(1): 17-9. Therefore, even 113 years after CD’s first description, we cannot assure cure for patients in the late stage of infection and this represents one of the major flaws in CD control as the current epidemiological profile is primarily composed of patients in such condition.

Drug resistance is another remarkable characteristic of the treatment and different mechanisms are suggested to play a role, including efflux pumps, mutations in the TcNTR gene and others that have not been elucidated so far.3232. Campos M, Leon LL, Taylor MC, Kelly JM. Benznidazole-resistance in Trypanosoma cruzi: evidence that distinct mechanisms can act in concert. Mol Biochem Parasitol. 2014; 193(1): 17-9.,3333. Zingales B, Araujo RGA, Moreno M, Franco J, Aguiar PHN, Nunes SL, et al. A novel ABCG-like transporter of Trypanosoma cruzi is involved in natural resistance to benznidazole. Mem Inst Oswaldo Cruz. 2015; 110(3): 433-44.,3434. Sayé M, Miranda MR, di Girolamo F, de los Milagros Cámara M, Pereira CA. Proline modulates the Trypanosoma cruzi resistance to reactive oxygen species and drugs through a novel D, L-proline transporter. PLoS One. 2014; 9(3): 1-9.,3535. Campos M, Castro-Pinto D, Ribeiro G, Berredo-Pinho M, Gomes LHF, da Silva Bellieny MS, et al. P-glycoprotein efflux pump plays an important role in Trypanosoma cruzi drug resistance. Parasitol Res. 2013; 112(6): 2341-51.,3636. Townson SM, Boreham PFL, Upcroft P, Upcroft JA. Resistance to the nitroheterocyclic drugs. Acta Trop. 1994; 56(2-3): 173-94. Some strains of T. cruzi naturally differ in their susceptibility to Bz and might even be entirely refractory to it.3737. Filardi LS, Brener Z. Susceptibility and natural resistance of Trypanosoma cruzi strains to drugs used clinically in Chagas disease. Trans R Soc Trop Med Hyg. 1987; 81(5): 755-9. Resistant clones are readily obtained by drug selection in the laboratory, usually showing cross-resistance to different nitroheterocyclic molecules.3838. Campos MC, Phelan J, Francisco AF, Taylor MC, Lewis MD, Pain A, et al. Genome-wide mutagenesis and multi-drug resistance in American trypanosomes induced by the front-line drug benznidazole. Sci Rep. 2017; 7(1): 14407.,3939. Mejia AM, Hall BS, Taylor MC, Gómez-Palacio A, Wilkinson SR, Triana-Chávez O, et al. Benznidazole-resistance in Trypanosoma cruzi is a readily acquired trait that can arise independently in a single population. J Infect Dis. 2012; 206(2): 220-8.,4040. Wilkinson SR, Taylor MC, Horn D, Kelly JM, Cheeseman I. A mechanism for cross-resistance to nifurtimox and benznidazole in trypanosomes. Proc Natl Acad Sci. 2008; 105(13): 5022-7. As an example, Mejia et al.3939. Mejia AM, Hall BS, Taylor MC, Gómez-Palacio A, Wilkinson SR, Triana-Chávez O, et al. Benznidazole-resistance in Trypanosoma cruzi is a readily acquired trait that can arise independently in a single population. J Infect Dis. 2012; 206(2): 220-8. observed that T. cruzi GAL61 grown in the laboratory in the presence of Bz lost one of the chromosomes containing the TcNTR I gene and that the second copy of the gene acquired mutations that led to non-synonymous amino acid variations in the protein sequence, including conserved positions. Recombinant proteins expressed from the TcNTR I mutant gene were unable to reduce both Bz and Nifurtimox. The authors suggested that the natural range of Bz susceptibility does not result exclusively from TcNTR I intrinsic sequence diversity. Instead, this variability may have different, so far unknown, underlying mechanisms. Finally, they propose that resistance caused by TcNTR I mutations may be a trait that emerges only under selective pressure.

Similarly, Campos et al.3838. Campos MC, Phelan J, Francisco AF, Taylor MC, Lewis MD, Pain A, et al. Genome-wide mutagenesis and multi-drug resistance in American trypanosomes induced by the front-line drug benznidazole. Sci Rep. 2017; 7(1): 14407. generated Bz-resistant clones of the T. cruzi Y strain after four months under selective drug pressure. They observed that the clones also displayed nonsense mutations in the TcNTR I gene and a genome-wide accumulation of single nucleotide polymorphisms (SNP), both emerging during the selection process. Additionally, these clones exhibited cross-resistance against Nifurtimox, impairment of the DNA repair system, and reduced fitness in mice infections. Further investigations are certainly required to explore whether Bz activity can produce new T. cruzi variants during a human infection under a treatment regime, but the in vitro results demonstrate different mechanisms through which the parasite could survive despite Bz exposure, highlighting its intrinsic genomic plasticity.

Genomic plasticity: a major obstacle to CD treatment, prevention, and diagnosis

The identification of new drug targets is an intrinsically difficult task as many aspects of this process need to be fine-tuned. An ideal target must be essential for parasite survival, structurally unrelated to proteins from the host, expressed in the infecting forms, and present well-documented information about their biochemical characteristics.4141. Osorio-Méndez JF, Cevallos AM. Discovery and genetic validation of chemotherapeutic targets for Chagas' disease. Front Cell Infect Microbiol. 2019; 8: 1-16. Besides, in CD this challenge is broadened by the uncertainty of choosing targets displaying intraspecific or intrastrain sequence diversity, which can lead to treatment failure. For instance, the T. cruzi phylogenetic tree is currently based on seven discrete typing units (DTU, TcI-VI, and TcBat), each comprising strains that share a set of well-defined molecular markers but are not genetically identical.4242. Zingales B, Miles MA, Campbell DA, Tibayrenc M, Macedo AM, Teixeira MMG, et al. The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect Genet Evol. 2012; 12(2): 240-53.

In addition to this constitutive diversity, the parasite also exhibits genomic plasticity, often presenting high SNP density and structural variations (gene and chromosome copy number variations, insertions, and deletions).4343. Reis-Cunha JL, Rodrigues-Luiz GF, Valdivia HO, Baptista RP, Mendes TAO, de Morais GL, et al. Chromosomal copy number variation reveals differential levels of genomic plasticity in distinct Trypanosoma cruzi strains. BMC Genomics. 2015; 16(1): 499.,4444. Herreros-Cabello A, Callejas-Hernández F, Gironès N, Fresno M. Trypanosoma Cruzi genome: organization, multi-gene families, transcription, and biological implications. Genes (Basel). 2020; 11(10): 1196.,4545. Reis-Cunha JL, Baptista RP, Rodrigues-Luiz GF, Coqueiro-dos-Santos A, Valdivia HO, de Almeida LV, et al. Whole genome sequencing of Trypanosoma cruzi field isolates reveals extensive genomic variability and complex aneuploidy patterns within TcII DTU. BMC Genomics. 2018; 19(1): 816.,4646. Schwabl P, Maiguashca Sánchez J, Costales JA, Ocaña-Mayorga S, Segovia M, Carrasco HJ, et al. Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity. Sirugo G, editor. PLoS Genet. 2020; 16(12): e1009170.,4747. Wang W, Peng D, Baptista RP, Li Y, Kissinger JC, Tarleton RL. Strain-specific genome evolution in Trypanosoma cruzi, the agent of Chagas disease. PLoS Pathog. 2021; 17(1): e1009254.,4848. Weatherly DB, Boehlke C, Tarleton RL. Chromosome level assembly of the hybrid Trypanosoma cruzi genome. BMC Genomics. 2009; 10(1): 255. Its chromosomal DNA is composed of four main types of sequences. The first one is the conserved syntenic coding regions with a single locus that forms the parasite’s core genome. The second type is comprised by gene families encoded in multiple loci, displaying low sequence conservation such as mucins, trans-sialidases, TcGP63, amastin, TcTASV, mucin-associated surface proteins, and cruzipain.4949. Pech-Canul AC, Monteón V, Solís-Oviedo R-L. A brief view of the surface membrane proteins from Trypanosoma cruzi. J Parasitol Res. 2017; 2017: 1-13. The T. cruzi genome also presents non-coding repetitive sequences that participate in different cellular processes composing roughly half of the entire parasite genome.4444. Herreros-Cabello A, Callejas-Hernández F, Gironès N, Fresno M. Trypanosoma Cruzi genome: organization, multi-gene families, transcription, and biological implications. Genes (Basel). 2020; 11(10): 1196. Finally, thousands of transcriptionally active pseudogenes are also observed. These are intergenic regions displaying protein-coding features, resembling known functional protein(s), bearing substitution(s) and/or insertion(s)/deletion(s) disrupting their open reading frames, which are remnants of the processes of acquisition and gene loss throughout the parasite’s evolutionary history.5050. Abrahim M, Machado E, Alvarez-Valín F, de Miranda AB, Catanho M. GBE uncovering pseudogenes and intergenic protein-coding sequences in TriTryps' genomes. Genome Biol Evol. 2022; 14(10): 1-14. Surface protein sequence diversity enables immune evasion during infection and represent a major challenge in the development of vaccines.5151. Dumonteil E, Herrera C. The case for the development of a Chagas disease vaccine: Why? How? When? Trop Med Infect Dis. 2021; 6(1): 16. Also, variations in gene copy number and in the size of chromosomes have already been reported,4343. Reis-Cunha JL, Rodrigues-Luiz GF, Valdivia HO, Baptista RP, Mendes TAO, de Morais GL, et al. Chromosomal copy number variation reveals differential levels of genomic plasticity in distinct Trypanosoma cruzi strains. BMC Genomics. 2015; 16(1): 499.,5252. Baptista RP, Reis-Cunha JL, DeBarry JD, Chiari E, Kissinger JC, Bartholomeu DC, et al. Assembly of highly repetitive genomes using short reads: the genome of discrete typing unit III Trypanosoma cruzi strain 231. Microb Genom. 2018; 4(4): e000156. including in cells that presumably had a clonal relationship, as they were artificially cultured in mammalian epithelial cells.5353. Lima FM, Souza RT, Santori FR, Santos MF, Cortez DR, Barros RM, et al. Interclonal variations in the molecular karyotype of Trypanosoma cruzi: chromosome rearrangements in a single cell-derived clone of the G strain. PLoS One. 2013; 8(5): e63738. Moreover, T. cruzi can also engage in sexual reproduction promoting genetic exchange between different genotypes even though natural populations exhibit a clonal structure with intense homozygosity and linkage disequilibrium.5454. Berry ASF, Salazar-Sánchez R, Castillo-Neyra R, Borrini-Mayorí K, Chipana-Ramos C, Vargas-Maquera M, et al. Sexual reproduction in a natural Trypanosoma cruzi population. PLoS Negl Trop Dis. 2019; 13(5): 1-17.,5555. Schwabl P, Imamura H, Van den Broeck F, Costales JA, Maiguashca-Sánchez J, Miles MA, et al. Meiotic sex in Chagas disease parasite Trypanosoma cruzi. Nat Commun. 2019; 10(1): 3972.

In this sense, some studies have focused on investigating the sequence diversity of T. cruzi proteins relevant for CD management. As an example, the ergosterol biosynthesis pathway, a validated drug target in T. cruzi, had 20 of its encoding genes displaying extensive inter-strain polymorphisms.5656. Cosentino RO, Agüero F. Genetic Pprofiling of the isoprenoid and sterol biosynthesis pathway genes of Trypanosoma cruzi. PLoS One. 2014; 9(5): e96762. A total of 975 polymorphic sites were identified in all sequences, 28% of them leading to non-synonymous substitutions, with SNP density between genes varying from 2.68 to 11.39 SNPs for every 100 bp. Insights into potential structural impacts generated by non-synonymous variations indicated that the sterol 14-alpha demethylase (TcCYP51), an enzyme also validated as a drug target in T. cruzi, presents a crucial substitution (A117S) adjacent to the conserved residue Y116, which has been proposed to play a role in resistance to azole compounds in other species of microorganisms.5757. Kelly SL, Lamb DC, Kelly DE. Y132H substitution in Candida albicans sterol 14α-demethylase confers fluconazole resistance by preventing binding to haem. FEMS Microbiol Lett. 1999; 180(2): 171-5.,5858. Délye C, Laigret F, Corio-Costet MF. A mutation in the 14 alpha-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor. Appl Environ Microbiol. 1997; 63(8): 2966-70.

Likewise, ribose-5-phosphate isomerase (Rpi) has been suggested as a new drug target in T. cruzi, and molecules that may work as selective inhibitors were identified by virtual screening.5959. Sinatti V, Baptista L, Alves-Ferreira M, Dardenne L, da Silva JHM, Guimarães A. In silico identification of inhibitors of ribose 5-phosphate isomerase from Trypanosoma cruzi using ligand and structure based approaches. J Mol Graph Model. 2017; 77: 168-80.,6060. Faria J, Loureiro I, Santarém N, Cecílio P, Macedo-Ribeiro S, Tavares J, et al. Disclosing the essentiality of ribose-5-phosphate isomerase B in Trypanosomatids. Sci Rep. 2016; 6(1): 1-16.,6161. Gonzalez SN, Mills JJ, Maugeri D, Olaya C, Laguera BL, Enders JR, et al. Design, synthesis, and evaluation of substrate - analogue inhibitors of Trypanosoma cruzi ribose 5-phosphate isomerase type B. Bioorg Med Chem Lett. 2021; 32: 1-6. An investigation of the sequence diversity among TcRpi protein sequences available in public databases showed 36 distinct clusters among 277 T. cruzi genomes (Azevedo et al., unpublished observations). Although most of the 160 amino acids in TcRpi are conserved, some polymorphic positions were observed immediately adjacent to the catalytic residues suggesting functional impact and the need of further investigation with experimental and computational approaches to address this issue.

Even the efficacy of CD serological diagnosis seems to be affected by sequence diversity, as evaluation of antigens used in commercial kits revealed that two out of seven display limited conservation (less than 80% identity) across 52 strains of different DTUs and countries.6262. Majeau A, Murphy L, Herrera C, Dumonteil E. Assessing Trypanosoma cruzi parasite diversity through comparative genomics: implications for disease epidemiology and diagnostics. Pathogens. 2021; 10(2): 212. This likely contributes to limited performance, causing false-negative results especially when screening samples from Central and North America, given that the sequences used are mostly of antigens from South America strains. The G-FINDER report on R&D for DC, also points out the need for improved pan-geographic accuracy in diagnostics kits and the capacity to perform an early assessment of treatment response.6363. An unmet need for Chagas' disease diagnostics. G-finder snapshots. 2021. Available from: https://policy-cures-website-assets.s3.ap-southeast-2.amazonaws.com/wp-content/uploads/2022/01/07004545/GFINDER_Snapshot_Chagas_Disease_Diagnostics.pdf.
https://policy-cures-website-assets.s3.a...

Beyond treatment and diagnostics, preventing T. cruzi infection through vaccination of vulnerable populations would be the state-of-the-art in disease prevention. Unfortunately, we still lack a vaccine and the parasite’s genetic diversity should also be carefully analysed in its development process as protective immunity may be strain-specific.5151. Dumonteil E, Herrera C. The case for the development of a Chagas disease vaccine: Why? How? When? Trop Med Infect Dis. 2021; 6(1): 16.,6464. Haolla FA, Claser C, de Alencar BCG, Tzelepis F, de Vasconcelos JR, de Oliveira G, et al. Strain-specific protective immunity following vaccination against experimental Trypanosoma cruzi infection. Vaccine. 2009; 27(41): 5644-53. Several studies suggest that the T. cruzi Tc24 protein could be used as a vaccine antigen candidate with great potential to treat and prevent cardiac fibrosis due to chronic CD. More recently, the possible impact of polymorphisms in Tc24 on immunogenicity was explored, with encouraging results.6565. Barry MA, Versteeg L, Wang Q, Pollet J, Zhan B, Gusovsky F, et al. A therapeutic vaccine prototype induces protective immunity and reduces cardiac fibrosis in a mouse model of chronic Trypanosoma cruzi infection. PLoS Negl Trop Dis. 2019; 13(5): e0007413.,6666. Versteeg L, Adhikari R, Poveda C, Villar-Mondragon MJ, Jones KM, Hotez PJ, et al. Location and expression kinetics of Tc24 in different life stages of Trypanosoma cruzi. PLoS Negl Trop Dis. 2021; 15(9): e0009689. Using WGS reads from samples of DTU TcI to TcVI, Arnal et al.6767. Arnal A, Villanueva-Lizama L, Teh-Poot C, Herrera C, Dumonteil E. Extent of polymorphism and selection pressure on the Trypanosoma cruzi vaccine candidate antigen Tc24. Evol Appl. 2020; 13(10): 2663-72. observed that the Tc24 sequence presents low levels of polymorphisms, loosely associated with the different lineages and not associated with the geographical location of sampling. Only 35 out of 211 codons were under selective pressure, most of them under purifying selection (deleterious variants that are being eliminated) and a small part under diversifying selection (sequence variants that are increasing in frequency). More importantly, the protein regions predicted as epitopes were conserved, supporting the use of Tc24 as a vaccine antigen. Hence, this example suggests that, besides the significant diversity observed in T. cruzi, there is still a chance of finding a well-suited protein for vaccine development.

In conclusion, genome plasticity being a remarkable feature in T. cruzi biology, partially accounts for the complexity of CD treatment, as well as many of the hurdles faced in effective diagnosis, drug and vaccine development. The above examples highlight the fact that, through evaluating sequence diversity and its functional impact, it is possible to significantly expand the set of information regarding a protein, enabling the choice of the most suitable pharmaceutical targets, and, most importantly, optimising the R&D process. By investigating the type and extent of selective pressure on the target genes it may also be possible to understand the rate and patterns of emerging mutations even before experimental essays, which could save investments and time. This whole evaluation process can be specially performed in a large-scale approach through in silico methods, as sequencing technologies are continually evolving as well as computational robustness.

Whole-genome sequencing: an essential tool on CD control strategies

A coordinated effort on exploring sequence diversity might be crucial to understand its extent and to accumulate the knowledge required to improved disease response, either on the individual or populational level. The incorporation of WGS of clinical samples in national protocols for epidemiological surveillance of CD would provide information not only to enable public health measures but also to plan an optimised treatment design strategy; some authors already proposed the incorporation of WGS based molecular genotyping specifically in TriTryps diseases (Leishmaniasis, CD, human African trypanosomiasis) control programs.6868. Domagalska MA, Dujardin J-C. Next-generation molecular surveillance of TriTryp diseases. Trends Parasitol. 2020; 36(4): 356-67.

It is suggested that T. cruzi WGS of clinical samples would provide crucial data to follow the evolution of an epidemic in time and space, characterise new transmission cycles, find the origin of outbreaks and their profile, detect new variants and sexual recombination events, and find genetic markers for traits of clinical and epidemiological relevance.6868. Domagalska MA, Dujardin J-C. Next-generation molecular surveillance of TriTryp diseases. Trends Parasitol. 2020; 36(4): 356-67. Through landscape genomics approaches, the provided data could also help understand how the parasite genome changes due to its interaction with different environments, uncovering gene fluxes dynamics and the emergence of new variants, supporting disease control public health measures.6969. Schwabl P, Llewellyn MS, Landguth EL, Andersson B, Kitron U, Costales JA, et al. Prediction and prevention of parasitic diseases using a landscape genomics framework. Trends Parasitol. 2017; 33(4): 264-75. In the R&D field, WGS of genotypes found in a given geographic region would enable the investigation of selective pressure on genes encoding proteins suggested as potential pharmacological targets, making it possible to select a target with a greater chance of success in a population. Also, it would be feasible to identify essential T. cruzi genes encoding functional but not structural analogues to human enzymes, which is crucial in drug design pipelines. The impact of T. cruzi genetic diversity on drug development strategies has been pointed out by Zingales et al.,7070. Zingales B, Miles MA, Moraes CB, Luquetti A, Guhl F, Schijman AG, et al. Drug discovery for Chagas disease should consider Trypanosoma cruzi strain diversity. Mem Inst Oswaldo Cruz. 2014; 109(6): 828-33. who emphasises that the choice of parasitic strains for in vitro testing can determine the outcome of the screening process, but there is still a lack of possible approaches to broadly overcome such an impact.

The Centre for Genomic Pathogen Surveillance has been conducting WGS of clinical samples for surveillance of human pathogens, such as carbapenem-resistant Klebsiella pneumoniae,7171. David S, Reuter S, Harris SR, Glasner C, Feltwell T, Argimon S, et al. Epidemic of carbapenem-resistant Klebsiella pneumoniae in Europe is driven by nosocomial spread. Nat Microbiol. 2019; 4(11): 1919-29.Staphylococcus aureus7272. Aanensen DM, Feil EJ, Holden MTG, Dordel J, Yeats CA, Fedosejev A, et al. Whole-genome sequencing for routine pathogen surveillance in public health: a population snapshot of invasive Staphylococcus aureus in Europe. mBio. 2016; 7(3): 1-15. and, more recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), specially to track antibiotic resistant strains. In a similar manner, the PulseNet International, a network of research laboratories in 88 countries on different continents, has been performing epidemiological surveillance of food-borne pathogens based on molecular methods. The project has recently implemented WGS into their pipelines and is currently aiming to expand its work to low and middle income countries.7373. Nadon C, Van Walle I, Gerner-Smidt P, Campos J, Chinen I, Concepcion-Acevedo J. PulseNet International: vision for the implementation of whole genome sequencing (WGS) for global food- borne disease surveillance. Euro Surveill. 2017; 22(23): 1-12.,7474. Davedow T, Carleton H, Kubota K, Palm D, Schroeder M, Gerner-Smidt P, et al. PulseNet International Survey on the implementation of whole genome sequencing in low and middle-income countries for foodborne disease surveillance. Foodborne Pathog Dis. 2022; 19(5): 332-40. These initiatives highlight that routinely performed large-scale WGS genotyping for infectious disease samples is a feasible approach and its use has been growing due to advances in sequencing technologies.

However, initiatives focused on T. cruzi sequencing and expanding the knowledge on its genome have been receiving insufficient investment and many research projects are unfinished. The T. cruzi genome project started in 1994 and the complete sequence of the hybrid lineage CL Brener was finished in 2005, alongside the genomic sequences of Leishmania major and Trypanosoma brucei.7575. Ash C, Jasny BR. Trypanosomatid genomes. Science. 2005; 309(5733): 399-399. In 1997, a database for T. cruzi genomic and biological information was made available centralising information from different biological databases.7676. Degrave W, Miranda AB, Amorim A, Brandão A, Aslett M, Vandeyar M. TcruziDB, an integrated database, and the WWW Information Server for the Trypanosoma cruzi genome project. Mem Inst Oswaldo Cruz. 1997; 92(6): 805-9. In 2009, the TcSNP database was created to integrate information on genetic variation of different T. cruzi genotypes.7777. Ackermann AA, Carmona SJ, Aguero F. TcSNP: a database of genetic variation in Trypanosoma cruzi. Nucleic Acids Res. 2009; 37: D544-9. In 2007, Andersson and colleagues built a database for T. cruzi repeated genes which could help understand the complexity of parasite genome.7878. Arner E, Kindlund E, Nilsson D, Farzana F, Ferella M, Tammi MT, et al. Database of Trypanosoma cruzi repeated genes: 20 000 additional gene variants. BMC Genomics. 2007; 8(1): 391. Unfortunately, besides their relevance and usefulness for the research community, all these initiatives were discontinued, with the TriTrypDB7979. Aslett M, Aurrecoechea C, Berriman M, Brestelli J, Brunk BP, Carrington M, et al. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 2010; 38(Suppl. 1): D457-62. and the NCBI databases8080. Agarwala R, Barrett T, Beck J, Benson DA, Bollin C, Bolton E, et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2018; 46(D1): D8-13. currently being the main sources of T. cruzi genetic data.

Whole-genome surveillance based on next-generation sequencing might be a potential answer to the impact of genetic variation in CD treatment development. Is estimated that half of the T. cruzi genome is composed of repetitive sequences diffusely distributed. In order to overcome this challenge, genomes have been sequenced using hybrid strategies combining a third-generation technology (usually from Pacific Biosciences) and a second-generation technology (such as Illumina), which have shown significant improvement in the number of contigs and the assembly result.4545. Reis-Cunha JL, Baptista RP, Rodrigues-Luiz GF, Coqueiro-dos-Santos A, Valdivia HO, de Almeida LV, et al. Whole genome sequencing of Trypanosoma cruzi field isolates reveals extensive genomic variability and complex aneuploidy patterns within TcII DTU. BMC Genomics. 2018; 19(1): 816.,8181. Berná L, Rodriguez M, Chiribao ML, Parodi-Talice A, Pita S, Rijo G, et al. Expanding an expanded genome: long-read sequencing of Trypanosoma cruzi. Microb Genomics. 2018; 4(5): 1-19.,8282. Koren S, Schatz MC, Walenz BP, Martin J, Howard JT, Ganapathy G, et al. Hybrid error correction and de novo assembly of single-molecule sequencing reads. Nat Biotechnol. 2012; 30(7): 693-700. The long-reads provided by PacBio’s Single-Molecule Real-Time sequencing (SMRT) technology allow sequencing of long tandem repeats and repeating sequences found at different loci in the genome, overcoming the risk of merging reads from different regions into a single sequence. However, this technology has low coverage and has higher error rate than first- and second-generation sequencing technologies, reducing its ability to identify SNPs and indels that are especially relevant for drug target selection. Therefore, SMRT technology reads need to be corrected by short reads generated by second-generation technologies that features short reads with high coverage or through other approaches. Short-reads coverage ensures proper identification of SNPs and indels, while long-reads increase the likelihood that genomic structure is reliable.

For instance, T. cruzi’s NGS raw sequences and their associated metadata could be deposited on a national server for genomic surveillance information of T. cruzi. Based on that data, bioinformaticians could work on target discovery workflows. Initially, data quality analysis should be performed, and short reads mapped against long reads. Both should be concatenated to allow the correction of erroneous base-calling and gaps and the resulting long-reads (which became more accurate) can be assembled. The de novo assembly becomes more feasible by using long reads allowing the identification of structural variations that are important markers of recombination events. Finally, genome annotations could be performed using basic local alignment search tool (BLAST) algorithms, since many genes and genomes of T. cruzi and other trypanosomatids have already been characterised and are publicly available in biological databases.5252. Baptista RP, Reis-Cunha JL, DeBarry JD, Chiari E, Kissinger JC, Bartholomeu DC, et al. Assembly of highly repetitive genomes using short reads: the genome of discrete typing unit III Trypanosoma cruzi strain 231. Microb Genom. 2018; 4(4): e000156.,5656. Cosentino RO, Agüero F. Genetic Pprofiling of the isoprenoid and sterol biosynthesis pathway genes of Trypanosoma cruzi. PLoS One. 2014; 9(5): e96762.,8383. Callejas-Hernández F, Rastrojo A, Poveda C, Gironès N, Fresno M. Genomic assemblies of newly sequenced Trypanosoma cruzi strains reveal new genomic expansion and greater complexity. Sci Rep. 2018; 8(1): 1-13.,8484. Callejas-Hernández F, Gironès N, Fresno M. Genome sequence of Trypanosoma cruzi strain Bug2148. Genome Announc. 2018; 6(3): 1-2.

WGS provides a wealth of information that can be applied for various purposes. In drug design, it would allow the identification of structurally unrelated functional analogues of human enzymes in T. cruzi. These are possible candidates for drug targeting, since the minimising potential off-target effects on the human protein. In addition, WGS data allows the investigation of selective pressure on genes that encode proteins suggested as potential pharmacological targets. Many new targets have already been proposed in the literature, but research on their diversity is still scarce.

To investigate sequence diversity, gene sequences from proteins of pharmacological interest could be retrieved from the samples by mapping their reference sequences against the whole genome data. Next, once the sequences from different samples were retrieved, they could be aligned in a multiple sequence alignment to group the sequences based on their identity and similarity creating gene clusters containing homologous sequences originating from the same genome (paralogous) and or homologous sequences shared between two or more genomes (orthologous), as well as groups containing taxonomically restricted sequences (singletons) (Figure). The final stage of the workflow should aim to explore the sequence diversity within the orthologous groups, in which, more conserved positions are expected between sequences of genes shared between representatives of the same DTU. Finally, the most suitable pharmacological targets should have the highest degree of conservation among the largest number of different DTUs comprising the same orthologous group. By analysing clinical T. cruzi samples from different DTUs in a specific geographic region, it is possible to evaluate the gene/protein with the highest chance of assuring treatment success when compared to all the potential targets.


Proposed workflow for investigation of sequence diversity among genes of pharmacological interest to Chagas disease (CD) treatment. In the first stage, gene sequences should be retrieved from whole-genome sequencing (WGS) data by mapping their reference sequences against the genomes. After that, all sequences should be aligned to determine similarity thresholds. Finally, the sequence diversity within each gene cluster could be investigated also using alignment tools but with a more restrictive sequence similarity cut-off.

Although sequencing T. cruzi genomes is crucial for CD control, it is important to consider that it still faces technical limitations that must be resolved or improved before its implementation on a large scale. First, reduced parasitemia during chronic infections limits the sensibility of molecular methods due to lower parasitic DNA concentration in blood.8585. Schijman AG, Bisio M, Orellana L, Sued M, Duffy T, Mejia Jaramillo AM, et al. International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLoS Negl Trop Dis. 2011; 5(1): e931.,8686. Ramírez JC, Cura CI, da Cruz Moreira O, Lages-Silva E, Juiz N, Velázquez E, et al. Analytical validation of quantitative real-time PCR methods for quantification of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. J Mol Diagnostics. 2015; 17(5): 605-15. Having in mind that most patients are asymptomatic and progress to the chronic stage of the disease, this characteristic of the infection may currently represent a limit to the number and types of samples to be sequenced. Another important aspect deals with the maintenance of samples in the laboratory. Different evidence shows that in vivo or in vitro culture of T. cruzi in the laboratory can induce changes in the genetic profile of cells by positively selecting less frequent genotypes that are eventually better adapted to those environments and not necessarily the most prevalent genotype in human infection.5353. Lima FM, Souza RT, Santori FR, Santos MF, Cortez DR, Barros RM, et al. Interclonal variations in the molecular karyotype of Trypanosoma cruzi: chromosome rearrangements in a single cell-derived clone of the G strain. PLoS One. 2013; 8(5): e63738.,8787. Deane MP, Jansen AM, Mangia RHR, Gonçalves AM, Morel CM. Are our laboratory "strains" representative samples of Trypanosoma cruzi populations that circulate in nature? Mem Inst Oswaldo Cruz. 1984; 79(Suppl.): 19-24.,8888. Valadares HMS, Pimenta JR, Segatto M, Veloso VM, Gomes ML, Chiari E, et al. Unequivocal identification of subpopulations in putative multiclonal Trypanosoma cruzi strains by FACs single cell sorting and genotyping. PLoS Negl Trop Dis. 2012; 6(7): e1722. Some authors even hypothesise if T. cruzi, like other trypanosomatids,8989. Mannaert A, Downing T, Imamura H, Dujardin J-C. Adaptive mechanisms in pathogens: universal aneuploidy in Leishmania. Trends Parasitol. 2012; 28(9): 370-6.,9090. Tan Z, Hays M, Cromie GA, Jeffery EW, Scott AC, Ahyong V, et al. Aneuploidy underlies a multicellular phenotypic switch. Proc Natl Acad Sci. 2013; 110(30): 12367-72. could be susceptible to aneuploidy induced by environmental stress.4343. Reis-Cunha JL, Rodrigues-Luiz GF, Valdivia HO, Baptista RP, Mendes TAO, de Morais GL, et al. Chromosomal copy number variation reveals differential levels of genomic plasticity in distinct Trypanosoma cruzi strains. BMC Genomics. 2015; 16(1): 499.,5353. Lima FM, Souza RT, Santori FR, Santos MF, Cortez DR, Barros RM, et al. Interclonal variations in the molecular karyotype of Trypanosoma cruzi: chromosome rearrangements in a single cell-derived clone of the G strain. PLoS One. 2013; 8(5): e63738. The genetic profiles observed in sequencing of polyclonal infection samples frequently represent the most abundant infecting genotype but, depending on the abundancy of the different clones, it can result in a mosaic genome that is not representative of any of the real infecting genotypes. To overcome this, improved sample preparation protocols and optimised methods for sequencing data analysis and genome assembly are required.

ACKNOWLEDGEMENTS

To Rafael Soares, Mayla Abrahim and Louise Castro for insightful and useful discussions.

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Publication Dates

  • Publication in this collection
    20 Jan 2023
  • Date of issue
    2022

History

  • Received
    13 July 2022
  • Accepted
    29 Nov 2022
Instituto Oswaldo Cruz, Ministério da Saúde Av. Brasil, 4365 - Pavilhão Mourisco, Manguinhos, 21040-900 Rio de Janeiro RJ Brazil, Tel.: (55 21) 2562-1222, Fax: (55 21) 2562 1220 - Rio de Janeiro - RJ - Brazil
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