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Trypanosoma cruzi causes Chagas disease and has a unique extranuclear genome enclosed in a structure called the kinetoplast, which contains circular genomes known as maxi- and minicircles. While the structure and function of maxicircles are well-understood, many aspects of minicircles remain to be discovered. Here, we performed a high-throughput analysis of the minicirculome (mcDNA) in 50 clones isolated from Colombia's diverse T. cruzi I populations. Results indicate that mcDNA comprises four diverse subpopulations with different structures, lengths, and numbers of interspersed semi-conserved (previously termed ultra-conserved regions mHCV) and hypervariable (mHVPs) regions. Analysis of mcDNA ancestry and inter-clone differentiation indicates the interbreeding of minicircle sequence classes is placed along diverse strains and hosts. These results support evidence of the multiclonal dynamics and random bi-parental segregation. Finally, we disclosed the guide RNA repertoire encoded by mcDNA at a clonal scale, and several attributes of its abundance and function are discussed.

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ABSTRACT Leishmania infantum is a protozoan that causes visceral leishmaniasis (VL) in the Americas and some regions of Europe. The disease is mainly characterized by hepatosplenomegaly and fever, and can be fatal. Factors related to the host and parasite can contribute to the transmission of Leishmania and the clinical outcome. The intraspecific genetic variability of L. infantum strains may be one of these factors. In this study, we evaluated the genetic variability of L. infantum obtained from bone marrow smear slides from patients in the Sao Paulo State, Brazil. For this, the minicircle of the kDNA hypervariable region was used as target by Sanger sequencing. By analyzing the similarity of the nucleotides and the maximum likelihood tree (Fasttree), we observed a high similarity (98%) among samples. Moreover, we identified four different profiles of L. infantum. In conclusion, L. infantum strains from Sao Paulo State, Brazil, showed low diversity measured by minicircle of the kDNA hypervariable region.

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Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10-2 (kDNA) or 5 × 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.

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Leishmaniases are neglected tropical diseases caused by Leishmania spp. parasites transmitted by the bite of phlebotomine sand flies. In Argentina, the most endemic area of American tegumentary leishmaniasis (ATL) has been Orán department, Province of Salta, where Leishmania (Viannia) braziliensis prevails and Nyssomyia neivai is considered as its vector, although there is no accurate and sufficient information in this regard. The aim of this work was to search for natural infection by Leishmania spp. in sand flies from peri-urban and rural sites with ATL background in Orán department. For this, sand flies were caught at five sites; female sand flies captured with Shannon trap were dissected to microscopically examine their gut contents, while females captured with CDC traps were molecularly analyzed by duplex PCR with two primer pairs to simultaneously amplify kinetoplast DNA (kDNA) and mammalian actin. A total of 1921 females were captured, with Ny. neivai being the most abundant species (89%), followed by Migonemyia migonei (6%) and cortelezzii complex (3%). No natural infection was found in any of them neither by dissection nor by PCR, although the detection limit of kDNA PCR was up to 25 promastigotes. The absence of infected females in peri-urban sites suggest that the transmission did not take place in those environments during the study period. Future searches for natural infection should focus on rural settings to deepen knowledge and elucidate the role of the circulating sand fly species as all have been linked to ATL transmission at other sites.

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Leishmania protozoa are the etiological agents of visceral, cutaneous and mucocutaneous leishmaniasis. In specific geographical regions, such as Latin America, several Leishmania species are endemic and simultaneously present; therefore, a diagnostic method for species discrimination is warranted. In this attempt, many qPCR-based assays have been developed. Recently, we have shown that L. (L.) infantum and L. (L.) amazonensis can be distinguished through the comparison of the Cq values from two qPCR assays (qPCR-ML and qPCR-ama), designed to amplify kDNA minicircle subclasses more represented in L. (L.) infantum and L. (L.) amazonensis, respectively. This paper describes the application of this approach to L. (L.) mexicana and introduces a new qPCR-ITS1 assay followed by high-resolution melt (HRM) analysis to differentiate this species from L. (L.) amazonensis. We show that L. (L.) mexicana can be distinguished from L. (L.) infantum using the same approach we had previously validated for L. (L.) amazonensis. Moreover, it was also possible to reliably discriminate L. (L.) mexicana from L. (L.) amazonensis by using qPCR-ITS1 followed by an HRM analysis. Therefore, a diagnostic algorithm based on sequential qPCR assays coupled with HRM analysis was established to identify/differentiate L. (L.) infantum, L. (L.) amazonensis, L. (L.) mexicana and Viannia subgenus. These findings update and extend previous data published by our research group, providing an additional diagnostic tool in endemic areas with co-existing species.

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RESUMEN En el Perú, la leishmaniasis es una enfermedad metaxénica que representa un serio problema de salud pública, debido a su amplia distribución y al número de personas en riesgo de contraer la enfermedad, siendo la población vulnerable principalmente las personas de bajos recursos económicos. El estudio se realizó a partir de pacientes que fueron derivados al Instituto Nacional de Salud entre el 2006 y el 2011 para que se les realizara el diagnóstico especializado. La identificación de la especie de Leishmania infectante se desarrolló mediante el análisis de las curvas de disociación (HRMA) obtenidas a partir del ADN genómico de promastigotes y amastigotes, lo que permitió identificar las especies de Leishmania (Viannia) braziliensis, Leishmania (V.) guyanensis, Leishmania (V.) peruviana como las más prevalentes, además de Leishmania (V.) lainsoni y Leishmania (L.) amazonensis.

ABSTRACT In Peru, leishmaniasis is a metaxenic disease that represents a serious public health problem, due to its wide distribution and the number of people in danger of contracting the disease, being the vulnerable population mainly those with low economic resources. The study was conducted from patients who were derived to Peru's National Institute of Health between 2006 and 2011 so that the specialized diagnosis could be carried out. The identification of the species of infectious Leishmania was developed through the analysis of the High-Resolution Melting Analysis obtained from the genomic DNA of promastigotes and amastigotes, which allows to identify the species of Leishmania (Viannia) braziliensis, Leishmania (V.) guyanensis, Leishmania (V.) peruviana as more prevalent, in addition to Leishmania (V.) lainsoni and Leishmania (L.) amazonensis.

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Leishmaniasis is a complex disease caused by Leishmania species belonging to subgenera Leishmania and Viannia. In South America, L. (L.) infantum is considered the most important causative agent of visceral leishmaniasis, while L. (L.) amazonensis and Viannia subgenus species are responsible for the different cutaneous or mucocutaneous forms. In our previous work, we developed a diagnostic approach for Leishmania species discrimination based on two qPCRs (qPCR-ML and qPCR-ama) targeting the minicircle kDNA followed by melting analysis. This approach allowed to (i) differentiate the subgenera Leishmania and Viannia, and (ii) distinguish between L. (L.) infantum and L. (L.) amazonensis. The aim of this work was to demonstrate the applicability of the approach previously described, using human and canine clinical samples and strains from a Brazilian region, where L. (L.) infantum, L. (L.) amazonensis and Viannia subgenus species coexist. After validation on New World strains, the diagnostic approach was applied blindly to 36 canine clinical samples (peripheral blood and bone marrow) and 11 human clinical samples (peripheral blood and bone marrow). The sensitivity was 95.6% (95% confidence interval 77.3-100%) and 100% (95% confidence interval 76.9-100%) in the canine bone marrow samples and human (peripheral blood and bone marrow) samples, respectively, compared to conventional PCR assays. Concerning the Leishmania species identification, the conventional and qPCR-based methods showed kappa value of 0.876 (95% confidence interval 0.638-1.000), indicating good agreement. Therefore, this approach proved to be useful in both veterinary and human clinical context in regions co-endemic for L. (L.) infantum, L. (L.) amazonensis, and Viannia subgenus, helping to provide rapid diagnosis and to allow studies of species distribution.

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Chagas disease (CD) is a human infection caused by Trypanosoma cruzi CD was traditionally endemic to the Americas; however, due to migration it has spread to countries where it is not endemic. The current chemotherapy to treat CD induces several side effects, and its effectiveness in the chronic phase of the disease is controversial. In this contribution, substituted phenylbenzothiazole derivatives were synthesized and biologically evaluated as trypanocidal agents against Trypanosoma cruzi The trypanocidal activities of the most promising compounds were determined through systematic in vitro screening, and their modes of action were determined as well. The physicochemical-structural characteristics responsible for the trypanocidal effects were identified, and their possible therapeutic application in Chagas disease is discussed. Our results show that the fluorinated compound 2-methoxy-4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl] phenol (BT10) has the ability to inhibit the proliferation of epimastigotes [IC50(Epi) = 23.1 ± 1.75 µM] and intracellular forms of trypomastigotes [IC50(Tryp) = 8.5 ± 2.9 µM] and diminishes the infection index by more than 80%. In addition, BT10 has the ability to selectively fragment 68% of the kinetoplastid DNA compared with 5% of nucleus DNA. The mode of action for BT10 on T. cruzi suggests that the development of fluorinated phenylbenzothiazole with electron-withdrawing substituent is a promising strategy for the design of trypanocidal drugs.

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Chagas disease (CD) is a human infection caused by Trypanosoma cruzi. CD was traditionally endemic to the Americas; however, due to migration it has spread to countries where it is not endemic. The current chemotherapy to treat CD induces several side effects, and its effectiveness in the chronic phase of the disease is controversial. In this contribution, substituted phenylbenzothiazole derivatives were synthesized and biologically evaluated as trypanocidal agents against Trypanosoma cruzi. The trypanocidal activities of the most promising compounds were determined through systematic in vitro screening, and their modes of action were determined as well. The physicochemical-structural characteristics responsible for the trypanocidal effects were identified, and their possible therapeutic application in Chagas disease is discussed. Our results show that the fluorinated compound 2-methoxy-4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl] phenol (BT10) has the ability to inhibit the proliferation of epimastigotes [IC50(Epi) = 23.1 ± 1.75 µM] and intracellular forms of trypomastigotes [IC50(Tryp) = 8.5 ± 2.9 µM] and diminishes the infection index by more than 80%. In addition, BT10 has the ability to selectively fragment 68% of the kinetoplastid DNA compared with 5% of nucleus DNA. The mode of action for BT10 on T. cruzi suggests that the development of fluorinated phenylbenzothiazole with electron-withdrawing substituent is a promising strategy for the design of trypanocidal drugs.

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Rbp38 is a protein exclusively found in trypanosomatid parasites, including Leishmania amazonensis, the etiologic agent of tegumentar leishmaniasis in the Americas. The protein was first described as a Leishmania tarentolae mitochondrial RNA binding protein. Later, it was shown that the trypanosomes Rbp38 orthologues were exclusively found in the mitochondria and involved in the stabilization and replication of kinetoplast DNA (kDNA). In contrast, L. amazonensis Rbp38 (LaRbp38), co-purifies with telomerase activity and interacts not only with kDNA but also with telomeric DNA, although shares with its counterparts high sequence identity and a putative N-terminal mitochondrial targeting signal (MTS). To understand how LaRbp38 interacts both with nuclear and kDNA, we have first investigated its subcellular localization. Using hydroxy-urea synchronized L. amazonensis promastigotes we could show that LaRbp38 shuttles from mitochondria to the nucleus at late S and G2 phases. Further, we identified a non-classical nuclear localization signal (NLS) at LaRbp38 C-terminal that binds with importin alpha, a protein involved in the nuclear transport of several proteins. Also, we obtained LaRbp38 truncated forms among which, some of them also showed an affinity for both telomeric DNA and kDNA. Analysis of these truncated forms showed that LaRbp38 DNA-binding region is located between amino acid residues 95-235. Together, our findings strongly suggest that LaRbp38 is multifunctional with dual subcellular localization.

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Rbp38 is a protein exclusively found in trypanosomatid parasites, including Leishmania amazonensis, the etiologic agent of tegumentar leishmaniasis in the Americas. The protein was first described as a Leishmania tarentolae mitochondrial RNA binding protein. Later, it was shown that the trypanosomes Rbp38 orthologues were exclusively found in the mitochondria and involved in the stabilization and replication of kinetoplast DNA (kDNA). In contrast, L. amazonensis Rbp38 (LaRbp38), co-purifies with telomerase activity and interacts not only with kDNA but also with telomeric DNA, although shares with its counterparts high sequence identity and a putative N-terminal mitochondrial targeting signal (MTS). To understand how LaRbp38 interacts both with nuclear and kDNA, we have first investigated its subcellular localization. Using hydroxy-urea synchronized L. amazonensis promastigotes we could show that LaRbp38 shuttles from mitochondria to the nucleus at late S and G2 phases. Further, we identified a non-classical nuclear localization signal (NLS) at LaRbp38?C-terminal that binds with importin alpha, a protein involved in the nuclear transport of several proteins. Also, we obtained LaRbp38 truncated forms among which, some of them also showed an affinity for both telomeric DNA and kDNA. Analysis of these truncated forms showed that LaRbp38 DNA-binding region is located between amino acid residues 95–235. Together, our findings strongly suggest that LaRbp38 is multifunctional with dual subcellular localization.

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BACKGROUND: Trypanosoma cruzi uses several strategies to survive in different hosts. A key step in the life-cycle of this parasite is metacyclogenesis, which involves various morphological, biochemical, and genetic changes that induce the differentiation of non-pathogenic epimastigotes into pathogenic metacyclic trypomastigotes. During metacyclogenesis, T. cruzi displays distinct morphologies and ultrastructural features, which have not been fully characterized. RESULTS: We performed a temporal description of metacyclogenesis using different microscopy techniques that resulted in the identification of three intermediate forms of T. cruzi: intermediates I, II and III. Such classification was based on morphological and ultrastructural aspects as the location of the kinetoplast in relation to the nucleus, kinetoplast shape and kDNA topology. Furthermore, we suggested that metacyclic trypomastigotes derived from intermediate forms that had already detached from the substrate. We also found that changes in the kinetoplast morphology and kDNA arrangement occurred only after the repositioning of this structure toward the posterior region of the cell body. These changes occurred during the later stages of differentiation. In contrast, changes in the nucleus shape began as soon as metacyclogenesis was initiated, while changes in nuclear ultrastructure, such as the loss of the nucleolus, were only observed during later stages of differentiation. Finally, we found that kDNA networks of distinct T. cruzi forms present different patterns of DNA topology. CONCLUSIONS: Our study of T. cruzi metacyclogenesis revealed important aspects of the morphology and ultrastructure of this intriguing cell differentiation process. This research expands our understanding of this parasite's fascinating life-cycle. It also highlights the study of T. cruzi as an important and exciting model system for investigating diverse aspects of cellular, molecular, and evolutionary biology.

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The taxon Trypanosoma cruzi, causative agent of Chagas disease, is composed of several discrete typing units (DTUs) named TcI-TcVI, and Tcbat. The history of the taxon T. cruzi is known, even though several controversial aspects remain as the relationships between TcIII and TcIV. We analyzed cloned T. cruzi stocks pertaining to the seven DTUs by filter hybridization tests of PCR amplicons from minicircle variable regions and kinetoplast DNA probes. Minicircle DNA blots from the cloned stocks and filter hybridization with one TcI, one TcII, one TcV, one TcVI, three TcIII, one TcIV from North America and one TcIV kinetoplast DNA probes from South America revealed minicircle variable region cross-reaction in some T. cruzi DTUs probed. TcIII was heterogeneous in minicircle class composition, even though two TcIII probes revealed that a small fraction of minicircles cross-hybridized with the minicircles from the TcIII, TcV and TcVI DTUs. The minicircles of TcIV from North America cross-reacted only with TcIV from North America but not with TcIV stocks from Brazil and Bolivia. The results on minicircle cross-hybridizations are discussed in the context of RNA editing, mitochondrial function in T. cruzi DTUs.

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BACKGROUND: Canine visceral leishmaniasis (CVL) is a worldwide parasitic zoonosis caused by Leishmania (Leishmania) infantum around the world. Canids are the definitive hosts and sand flies the intermediate hosts. OBJECTIVE: To test the hypothesis that a new species-specific primers (Lch14:Lch15, targeting a multiple alignment for L. infantum kDNA minicircle) is an efficient diagnostic tool for L. infantum. METHODS: The presence of L. infantum DNA was assessed in blood samples of 69 stray dogs using the conventional PCR (cPCR) and quantitative PCR (qPCR). Additional 50 lymph nodes and 50 bone marrow samples (positive and negative samples for parasitological tests) from dogs from endemic and nonendemic areas for CVL were also used. RESULTS: L. infantum strains, and all positive lymph node and bone marrow samples for parasitological test gave positive results for cPCR and qPCR, presenting analytical sensitivity of ∼100 parasite mL-1. For the blood samples, 40/69 (58%; CI 95%; 46%-69%) resulted positive for L. infantum in both tests. All positive samples were confirmed by sequencing. CONCLUSION: This study showed the importance of the specific detection of L. infantum based on species-specific primers by molecular techniques, highlighting the application as a confirmation method in epidemiological studies and to adopt the best control measures.

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Bahia was the last Brazilian state declared free of Chagas disease transmission by Triatoma infestans in 2006. The program designed to control vector transmission of Chagas is currently active, and all potential triatomines collected by the Bahia State Department of Health officials are most frequently diagnosed as negative for Trypanosoma cruzi when analyzed by the conventional parasitological direct method. The aim of the current study was to investigate whether triatomines from Bahia are free of T. cruzi infection using a more sensitive diagnostic methodology, namely the kinetoplastid-DNA polymerase chain reaction (kDNA-PCR). With the help of health officials, 51 triatomines were analyzed from peridomicile areas within the central north region of the state of Bahia. The majority (60.8%) were Triatoma brasiliensis, 29.4% were Triatoma pseudomaculata, and 9.8% were unidentified nymphs. Only one insect tested potentially positive for T. cruzi by the conventional parasitological direct method, and 31.4% were positive for T. cruzi DNA by kDNA-PCR. Almost half the infected insects (41.9%) were T. brasiliensis, a species with high potential for T. cruzi transmission. These results demonstrate that the number of infected triatomines with high transmission potential of T. cruzi may be greater than expected in four localities in the state of Bahia

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BACKGROUND: The study evaluated qualitative PCR, primers 121-122 as a tool to follow up evolution parasite load of Trypanosoma cruzi. METHODS: The study was conducted at the State University of Maringa, in 2015. Step 1, dilutions 1/10 were performed from T. cruzi-Y strain to obtain preparations of 50,000-0.05 parasites/mL from which DNA were extracted, quantified, and amplified. Step 2, the extracted DNA in the dilutions 5-0.05 parasites/mL was re-diluted 1/10, 1/100, 1/1000, quantified, and amplified. Polyacrylamide gels were photographed and thicknesses of the 330 bp kDNA fragments were measured. RESULTS: Step 1, in the dilutions 50,000-50 parasites/mL kDNA fragments had same thickness and, dilutions 5-0.05 parasites/mL showed progressive decrease in thicknesses and staining intensity of the 330 bp fragments. Step 2, demonstrated that dilutions of five (re-dilutions 1/10 and 1/100) and 0.5 (1/10) parasites/mL produced similar thicknesses of the 330 bp fragments obtained in Step 1. However, very dilute DNA samples make difficult to reproduce the fragments thicknesses. CONCLUSION: PCR, despite its limitations, was able to detect progressive decrease in thicknesses/staining intensity of kDNA fragments in the dilutions 5-0.05 parasites/mL. Hence, has the potential to be used to follow-up evolution of parasite load, not by quantifying the number of parasites, but by dynamic evolution of the fragments thicknesses during etiological treatment.

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SUMMARY This study evaluated the applicability of kDNA-PCR as a prospective routine diagnosis method for American tegumentary leishmaniasis (ATL) in patients from the Instituto de Infectologia Emílio Ribas (IIER), a reference center for infectious diseases in São Paulo - SP, Brazil. The kDNA-PCR method detected Leishmania DNA in 87.5% (112/128) of the clinically suspected ATL patients, while the traditional methods demonstrated the following percentages of positivity: 62.8% (49/78) for the Montenegro skin test, 61.8% (47/76) for direct investigation, and 19.3% (22/114) for in vitro culture. The molecular method was able to confirm the disease in samples considered negative or inconclusive by traditional laboratory methods, contributing to the final clinical diagnosis and therapy of ATL in this hospital. Thus, we strongly recommend the inclusion of kDNA-PCR amplification as an alternative diagnostic method for ATL, suggesting a new algorithm routine to be followed to help the diagnosis and treatment of ATL in IIER. .

RESUMO Este estudo avaliou a aplicabilidade do kDNA-PCR como método de rotina para diagnóstico de leishmaniose tegumentar americana (ATL) no Instituto de Infectologia Emílio Ribas (IIER), São Paulo, SP, Brasil. O método kDNA-PCR detectou DNA de Leishmania em 87,5% (112/128) dos pacientes com suspeita de ter leishmaniose e, os métodos tradicionais apresentaram as seguintes porcentagens de positividade: 62,8% (49/78) para o teste de Montenegro, 61,8% (47/76) para a pesquisa direta e 19,3% (22/114) para cultura in vitro. O método molecular confirmou a doença em amostras negativas ou inconclusivas pelos métodos laboratoriais tradicionais e, mostrou-se capaz de auxiliar na identificação de infecções causadas pela espécie Leishmania (V.) braziliensis. Além disso, a revisão dos prontuários médicos confirmou a importância do método PCR-RFLP no diagnóstico final de ATL, prognóstico e escolha do tratamento. Assim, recomendamos a inclusão do PCR como método diagnóstico de ATL na rotina hospitalar, e sugerimos um fluxograma para solicitação de exames laboratoriais. .

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Objetivo. Con el fin de aportar nueva información relevante para estudios de genotipificación y filogenética del género Leishmania, en este estudio se determinó y comparó la secuencia del maxicírculo de Leishmania braziliensis, cepa MHOM-BR-75-M2904, con las secuencias del maxicírculo reportadas para otras especies de tripanosomátidos. Materiales y métodos. La búsqueda de las secuencias del maxicírculo se realizó en las bases de datos de secuencias no ensambladas del GeneDB versión 2.1, así como en el GenBank, utilizando los genes ND8 y RPS12 de L. braziliensis como sonda inicial. Estas secuencias se ensamblaron y se compararon con sus homólogas en otros tripanosomátidos mediante el uso de herramientas bioinformáticas como LALIGN y ClustalW2. El tamaño total del maxicírculo se determinó mediante ensayos de Southern blot. Resultados. Se ensamblaron dos fragmentos del maxicírculo de L. braziliensis de 6535 y 4257 nucleótidos, cuyos genes presentaron elevada sintenia y similitud en sus secuencias con los previamente reportados en otras especies de Leishmania. Similitud que se extiende incluso a los patrones de edición de estas moléculas. Conclusiones. A pesar de ser L. braziliensis la especie más divergente del género Leishmania en cuanto a su genoma nuclear, el marxicírculo presenta una elevada conservación. Resultado que sugiere que el patrón de edición presente en las diferentes especies de Leishmania hasta ahora estudiadas se conserva también en el subgénero Viannia, lo que indica una elevada conservación en la edición de los transcritos mitocondriales a nivel de género.

Objective. With the aim to provide new insights for genotyping and phylogenetic studies of the Leishmania genus, in this study the sequence of the maxicircle in Leishmania braziliensis, strain MHOM-BR-75-M2904, was determined and compared with those reported in other trypanosomatids species. Materials and methods. Searches for maxicircle sequences were performed in the unassembled sequences of GeneDB database version 2.1, as well as in the GenBank, using the ND8 and RPS12 genes of L. braziliensis as the initial probes. These sequences were assembled and compared with the homologous sequences of trypanosomatids using the bioinformatics tools LALIGN and ClustalW2. The size of maxicircle was determined by Southern blot assays. Results. Two maxicircle fragments of 6535 and 4257 nucleotides were assembled. The sequences of these genes showed high synteny and similarity with the sequences in other Leishmania species. This similarity even was extended to the editing patterns of these molecules. Conclusions. Although L. braziliensis is the most divergent species of the Leishmania genus in their nuclear genome, the maxicicircle has a high conservation. This result suggests that the pattern of editing present in the different Leishmania species studied has been conserved also in the subgenus Viannia. These results indicate a high conservation in the editing of mitochondrial transcripts at the genus level.

Objetivo. Com o fim de contribuir nova informação relevante para estudos de genotipagem e filogenética do género Leishmania, neste estudo determinou-se a sequência do maxicirculo de Leishmania braziliensis, cepa MHOM-BR-75-M2904, comparandosecom as seqüências do maxicirculo reportadas para outras espécies de tripanossomatídeos. Materiais e Métodos. A busca das seqüências do maxicirculo foi realizada nas bases de dados para seqüências não alinhadas no GeneDB versão 2.1, assim como no GeneBank, utilizando o genes ND8 e RPS12 de L. braziliensis como sonda inicial. Essas seqüências foram alinhadas e comparadas com as suas homologas em outros tripanossomatídeos, mediante o uso de ferramentas bioinformáticas como L-ALIGN e ClustalW2. O tamanho total do maxicirculo foi determinado mediante ensaios de Southern blot. Resultados. Foram alinhados dois fragmentos do maxicirculo de L. braziliensis de 6535 e 4257 nucleotídeos, cujos genes apresentaram elevada sintenia e similaridade nas suas seqüências com os genes previamente reportados nas outras espécies de Leishmania. A similaridade vista estende-se, inclusive, aos padrões de edição para estas moléculas. Conclusões. Apesar de L. braziliensis ser a espécie mais divergente do gênero Leishmania, no que se refere ao seu genoma nuclear, o maxicirculo apresenta uma alta conservação. Esse resultado sugere que o padrão de edição apresentado nas espécies de Leishmania até agora estudadas, é conservado também no subgênero Viannia, o que indica uma alta conservação na edição dos transcritos mitocôndriais ao nível de gênero.