Evolutionary relationships among protein lysine deacetylases of parasites causing neglected diseases.

The availability of the genomic data of diverse parasites provides an opportunity to identify new drug candidates against neglected tropical diseases affecting people worldwide. Histone modifying enzymes (HMEs) are potential candidates since they play key roles in the regulation of chromatin modifications, thus globally regulating gene expression. Furthermore, aberrant epigenetic states are often associated with human diseases, leading to great interest in HMEs as therapeutic targets. Our work focused on two families of protein lysine deacetylases (HDACs and sirtuins). First, we identified potential homologues in the predicted proteomes of selected taxa by using hidden Markov model profiles. Then, we reconstructed the evolutionary relationships of protein sequences by Bayesian inference and maximum likelihood method. In addition, we constructed homology models for five parasite HDACs to provide information for experimental validation and structure-based optimization of inhibitors. Our results showed that parasite genomes code for diverse HDACs and sirtuins. The evolutionary pattern of protein deacetylases with additional experimental data points to these enzymes as common drug targets among parasites. This work has improved the functional annotation of approximately 63% HDACs and 51% sirtuins in the selected taxa providing insights for experimental design. Homology models pointed out structural conservation and differences among parasite and human homologues and highlight potential candidates for further inhibitor development. Some of these parasite proteins are undergoing RNA interference or knockout analyses to validate the function of their corresponding genes. In the future, we will investigate the main functions performed by these proteins, related phenotypes, and their potential as therapeutic targets.

Whole genome sequencing of Guzerá cattle reveals genetic variants in candidate genes for production, disease resistance, and heat tolerance.

In bovines, artificial selection has produced a large number of breeds which differ in production, environmental adaptation, and health characteristics. To investigate the genetic basis of these phenotypical differences, several bovine breeds have been sequenced. Millions of new SNVs were described at every new breed sequenced, suggesting that every breed should be sequenced. Guzerat or Guzerá is an indicine breed resistant to drought and parasites that has been the base for some important breeds such as Brahman. Here, we describe the sequence of the Guzerá genome and the in silico functional analyses of intragenic breed-specific variations. Mate-paired libraries were generated using the ABI SOLiD system. Sequences were mapped to the Bos taurus reference genome (UMD 3.1) and 87% of the reference genome was covered at a 26X. Among the variants identified, 2,676,067 SNVs and 463,158 INDELs were homozygous, not found in any database searched, and may represent true differences between Guzerá and B. taurus. Functional analyses investigated with the NGS-SNP package focused on 1069 new, non-synonymous SNVs, splice-site variants (including acceptor and donor sites, and the conserved regions at both intron borders, referred to here as splice regions) and coding INDELs (NS/SS/I). These NS/SS/I map to 935 genes belonging to cell communication, environmental adaptation, signal transduction, sensory, and immune systems pathways. These pathways have been involved in phenotypes related to health, adaptation to the environment and behavior, and particularly, disease resistance and heat tolerance. Indeed, 105 of these genes are known QTLs for milk, meat and carcass, production, reproduction, and health traits. Therefore, in addition to describing new genetic variants, our approach provided groundwork for unraveling key candidate genes and mutations.

ZIKV - CDB: A Collaborative Database to Guide Research Linking SncRNAs and ZIKA Virus Disease Symptoms.

BACKGROUND: In early 2015, a ZIKA Virus (ZIKV) infection outbreak was recognized in northeast Brazil, where concerns over its possible links with infant microcephaly have been discussed. Providing a causal link between ZIKV infection and birth defects is still a challenge. MicroRNAs (miRNAs) are small noncoding RNAs (sncRNAs) that regulate post-transcriptional gene expression by translational repression, and play important roles in viral pathogenesis and brain development. The potential for flavivirus-mediated miRNA signalling dysfunction in brain-tissue development provides a compelling hypothesis to test the perceived link between ZIKV and microcephaly. METHODOLOGY/PRINCIPAL FINDINGS: Here, we applied in silico analyses to provide novel insights to understand how Congenital ZIKA Syndrome symptoms may be related to an imbalance in miRNAs function. Moreover, following World Health Organization (WHO) recommendations, we have assembled a database to help target investigations of the possible relationship between ZIKV symptoms and miRNA-mediated human gene expression. CONCLUSIONS/SIGNIFICANCE: We have computationally predicted both miRNAs encoded by ZIKV able to target genes in the human genome and cellular (human) miRNAs capable of interacting with ZIKV genomes. Our results represent a step forward in the ZIKV studies, providing new insights to support research in this field and identify potential targets for therapy.

Draft Genome Sequence of Hydrotalea flava Strain CCUG 51397T.

We report the draft genome sequence of Hydrotalea flava CCUG 51397(T), the type strain of the genus Hydrotalea (family Chitinophagaceae), isolated from water samples in southern Sweden.

Estimation of genetic diversity in viral populations from next generation sequencing data with extremely deep coverage.

BACKGROUND: In this paper we propose a method and discuss its computational implementation as an integrated tool for the analysis of viral genetic diversity on data generated by high-throughput sequencing. The main motivation for this work is to better understand the genetic diversity of viruses with high rates of nucleotide substitution, as HIV-1 and Influenza. Most methods for viral diversity estimation proposed so far are intended to take benefit of the longer reads produced by some next-generation sequencing platforms in order to estimate a population of haplotypes which represent the diversity of the original population. The method proposed here is custom-made to take advantage of the very low error rate and extremely deep coverage per site, which are the main features of some neglected technologies that have not received much attention due to the short length of its reads, which precludes haplotype estimation. This approach allowed us to avoid some hard problems related to haplotype reconstruction (need of long reads, preliminary error filtering and assembly). RESULTS: We propose to measure genetic diversity of a viral population through a family of multinomial probability distributions indexed by the sites of the virus genome, each one representing the distribution of nucleic bases per site. Moreover, the implementation of the method focuses on two main optimization strategies: a read mapping/alignment procedure that aims at the recovery of the maximum possible number of short-reads; the inference of the multinomial parameters in a Bayesian framework with smoothed Dirichlet estimation. The Bayesian approach provides conditional probability distributions for the multinomial parameters allowing one to take into account the prior information of the control experiment and providing a natural way to separate signal from noise, since it automatically furnishes Bayesian confidence intervals and thus avoids the drawbacks of preliminary error filtering. CONCLUSIONS: The methods described in this paper have been implemented as an integrated tool called Tanden (Tool for Analysis of Diversity in Viral Populations) and successfully tested on samples obtained from HIV-1 strain NL4-3 (group M, subtype B) cultivations on primary human cell cultures in many distinct viral propagation conditions. Tanden is written in C# (Microsoft), runs on the Windows operating system, and can be downloaded from: http://tanden.url.ph/.

HIV-1 Tropism Determines Different Mutation Profiles in Proviral DNA.

In order to establish new infections HIV-1 particles need to attach to receptors expressed on the cellular surface. HIV-1 particles interact with a cell membrane receptor known as CD4 and subsequently with another cell membrane molecule known as a co-receptor. Two major different co-receptors have been identified: C-C chemokine Receptor type 5 (CCR5) and C-X-C chemokine Receptor type 4 (CXCR4) Previous reports have demonstrated cellular modifications upon HIV-1 binding to its co-receptors including gene expression modulations. Here we investigated the effect of viral binding to either CCR5 or CXCR4 co-receptors on viral diversity after a single round of reverse transcription. CCR5 and CXCR4 pseudotyped viruses were used to infect non-stimulated and stimulated PBMCs and purified CD4 positive cells. We adopted the SOLiD methodology to sequence virtually the entire proviral DNA from all experimental infections. Infections with CCR5 and CXCR4 pseudotyped virus resulted in different patterns of genetic diversification. CCR5 virus infections produced extensive proviral diversity while in CXCR4 infections a more localized substitution process was observed. In addition, we present pioneering results of a recently developed method for the analysis of SOLiD generated sequencing data applicable to the study of viral quasi-species. Our findings demonstrate the feasibility of viral quasi-species evaluation by NGS methodologies. We presented for the first time strong evidence for a host cell driving mechanism acting on the HIV-1 genetic variability under the control of co-receptor stimulation. Additional investigations are needed to further clarify this question, which is relevant to viral diversification process and consequent disease progression.

Revisiting molecular serotyping of Streptococcus pneumoniae.

BACKGROUND: Ninety-two Streptococcus pneumoniae serotypes have been described so far, but the pneumococcal conjugate vaccine introduced in the Brazilian basic vaccination schedule in 2010 covers only the ten most prevalent in the country. Pneumococcal serotype-shifting after massive immunization is a major concern and monitoring this phenomenon requires efficient and accessible serotyping methods. Pneumococcal serotyping based on antisera produced in animals is laborious and restricted to a few reference laboratories. Alternatively, molecular serotyping methods assess polymorphisms in the cps gene cluster, which encodes key enzymes for capsular polysaccharides synthesis in pneumococci. In one such approach, cps-RFLP, the PCR amplified cps loci are digested with an endonuclease, generating serotype-specific fingerprints on agarose gel electrophoresis. METHODS: In this work, in silico and in vitro approaches were combined to demonstrate that XhoII is the most discriminating endonuclease for cps-RFLP, and to build a database of serotype-specific fingerprints that accommodates the genetic diversity within the cps locus of 92 known pneumococci serotypes. RESULTS: The expected specificity of cps-RFLP using XhoII was 76% for serotyping and 100% for serogrouping. The database of cps-RFLP fingerprints was integrated to Molecular Serotyping Tool (MST), a previously published web-based software for molecular serotyping. In addition, 43 isolates representing 29 serotypes prevalent in the state of Minas Gerais, Brazil, from 2007 to 2013, were examined in vitro; 11 serotypes (nine serogroups) matched the respective in silico patterns calculated for reference strains. The remaining experimental patterns, despite their resemblance to their expected in silico patterns, did not reach the threshold of similarity score to be considered a match and were then added to the database. CONCLUSION: The cps-RFLP method with XhoII outperformed the antisera-based and other molecular serotyping methods in regard of the expected specificity. In order to accommodate the genetic variability of the pneumococci cps loci, the database of cps-RFLP patterns will be progressively expanded to include new variant in vitro patterns. The cps-RFLP method with endonuclease XhoII coupled with MST for computer-assisted interpretation of results may represent a relevant contribution to the real time detection of changes in regional pneumococci population diversity in response to mass immunization programs.

Regulation of Schistosoma mansoni development and reproduction by the mitogen-activated protein kinase signaling pathway.

BACKGROUND: Protein kinases are proven targets for drug development with an increasing number of eukaryotic Protein Kinase (ePK) inhibitors now approved as drugs. Mitogen-activated protein kinase (MAPK) family members connect cell-surface receptors to regulatory targets within cells and influence a number of tissue-specific biological activities such as cell proliferation, differentiation and survival. However, the contributions of members of the MAPK pathway to schistosome development and survival are unclear. METHODOLOGY/PRINCIPAL FINDINGS: We employed RNA interference (RNAi) to elucidate the functional roles of five S. mansoni genes (SmCaMK2, SmJNK, SmERK1, SmERK2 and SmRas) involved in MAPK signaling pathway. Mice were injected with post-infective larvae (schistosomula) subsequent to RNAi and the development of adult worms observed. The data demonstrate that SmJNK participates in parasite maturation and survival of the parasites, whereas SmERK are involved in egg production as infected mice had significantly lower egg burdens with female worms presenting underdeveloped ovaries. Furthermore, it was shown that the c-fos transcription factor was overexpressed in parasites submitted to RNAi of SmERK1, SmJNK and SmCaMK2 indicating its putative involvement in gene regulation in this parasite's MAPK signaling cascade. CONCLUSIONS: We conclude that MAPKs proteins play important roles in the parasite in vivo survival, being essential for normal development and successful survival and reproduction of the schistosome parasite. Moreover SmERK and SmJNK are potential targets for drug development.

Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identification of the parasite species.

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

Sensitivity and reproducibility of a PCR assay for Leishmania detection using skin biopsy imprints on filter paper.

The sensitivity and reproducibility of a PCR targeted to amplify the conserved 120 base-pair region of minicircles from Leishmania kDNA was defined using DNA extracted from skin biopsy imprints on filter paper. Seventy-seven patients with cutaneous leishmaniasis from an endemic region of Leishmania (Viannia) braziliensis in Brazil underwent skin biopsy of the ulcer border. Tissue samples were imprinted on filter paper and then, they were stored at -20 degrees C. Imprints on filter paper were stored at 4 degrees C. Samples were processed at three laboratories; Lab1 and Lab2 performed the PCR-kDNA assay using DNA extracted from the filter paper, and Lab3 processed PCR-kDNA using DNA from fresh-frozen tissue used as a gold standard. All samples were codified to maintain blinding during lab processing. Fifty-three (68.8%) patients had parasites isolated and identified by isoenzymes as L. (V.) braziliensis. The positivity of PCR-kDNA was similar between the three laboratories: 87.0, 85.7 and 88.3% (Lab1, Lab2 and Lab3, respectively). The sensitivity of PCR-kDNA in culture-proven cases was better, and showed similar results in all laboratories: 95.8, 95.8 and 97.9% (Lab1, Lab2 and Lab3, respectively). Data from the 77 enrolled patients showed an overall percent agreement of 80.5% (Kappa=0.173) for the filter-paper approach between Lab1 and Lab2. Percent agreement between Lab1 and Lab3 was 83.1% (Kappa=0.22), and it was 94.8% between Lab2 and Lab3 (Kappa=0.77). Fifteen patients were diagnosed in just one of the two laboratories that used DNA extracted from filter paper. We conclude that the sensitivity of the filter paper approach is satisfactory and could be used in clinical trials and field work. Reproducibility could be improved using two separate imprints from the same biopsy sample.

Species diversity causing human cutaneous leishmaniasis in Rio Branco, state of Acre, Brazil.

OBJECTIVE: Information on Leishmania species diversity in western Brazilian Amazon and the clinical picture of human cutaneous leishmaniasis it causes is scarce. We describe clinical findings, diagnostic procedures and identification of Leishmania species in patients from that region. METHODS: The sample consisted of 50 patients, prospectively evaluated for epidemiological and clinical characteristics by means of a structured questionnaire. Conventional and molecular tools were applied to confirm the parasitological diagnosis and identify the species responsible for the disease. RESULTS: Patients were predominantly male (76.5%) and living in rural areas. Median average age was 18 years and median average disease evolution was 8 weeks. For the diagnostic procedures of leishmanin skin test, direct visualization of amastigotes in dermal scrapings and parasite culture of aspirates of the ulcer border were positive for 98%, 52% and 34%, respectively. Molecular methods applied to DNA extracted from skin biopsies of the 50 patients yielded 100%, 82% and 44% positivity by PCR minicircle kDNA, PCR-RFLP ITS1rDNA and PCR-glucose-6-phosphate (G6P), respectively. Fourteen samples from 13 patients were successfully isolated and identified. Multilocus enzyme electrophoresis, PCR-RFLP ITS1rDNA and PCR-G6P permitted identification of the Leishmania species responsible for the aetiology of American tegumentary leishmaniasis in 60% of the examined patients: 16 Leishmania (Viannia) braziliensis, 12 Leishmania (Viannia) lainsoni, 1 Leishmania (Viannia) guyanensis and 1 putative hybrid of Leishmania (Viannia) naiffi and L. (V.) lainsoni. CONCLUSION: The clinical and epidemiological behaviour of cutaneous leishmaniasis in Acre, Brazil, is similar to other Amazon scenarios previously described; however Acre's complex parasite diversity may be contributed to the concomitant circulation of at least three distinct Leishmania species. The implementation of control interventions in the studied area must take into consideration the possibility of various expected phlebotomine vectors and reservoirs.

Use of PCR-RFLP to identify Leishmania species in naturally-infected dogs.

Tissue imprints on Giemsa stained slides from dogs were used to investigate the presence of Leishmania amastigotes by either optical microscopy (OM) or Polymerase chain reaction (PCR) detection of DNA. Samples from skin, spleen, lymph node, liver and bone marrow from a Leishmaniasis endemic area dogs where Leishmania (Leishmania) chagasi and Leishmania (Viannia) braziliensis are sympatric were studied. Dogs were initially diagnosed by Indirect Immunofluorescence (IIF), as which 39 were IIF positive (> or = 1:40) and 16 negative. The IIF positive dogs were clinically grouped as symptomatic (n = 15), oligosymptomatic (n = 12) and asymptomatic (n = 12). Although PCR positivity was higher in symptomatic dogs, specially their skin samples, there was no significant difference among clinical groups or organs examined. Ten (62.5%) out of 16 IIF and OM negative animals were positive for PCR in at least one organ. Forty-eight positive PCR amplicons were further submitted to RFLP for Leishmania identification. All dogs were infected with L. (L.) chagasi except one, infected with L. (V.) braziliensis. PCR was more efficient than IIF and OM to diagnose canine visceral Leishmaniasis (CVL), regardless of the organ examined and the clinical form present. The use of PCR together with serology helps determining the extension of sub clinical infection in CVL endemic areas and provides a better estimate of the number of dogs to be targeted for control measures. In conclusion, our data reinforce the need for a specific diagnosis of canine infection in areas where diverse Leishmania species are sympatric and demonstrate that PCR-RFLP can be used to identify Leishmania species in dog tissue imprint stained slides.

Comparison of polymerase chain reaction with other laboratory methods for the diagnosis of American cutaneous leishmaniasis: diagnosis of cutaneous leishmaniasis by polymerase chain reaction.

An evaluation of 5 laboratory methods for diagnosing American cutaneous leishmaniasis (ACL) was carried out on patients from an endemic area of Brazil. From 164 patients presenting cutaneous lesions, and suspected to have ACL, 133 (81.1%) were confirmed for the disease by Montenegro skin test (MST) and/or parasitologic examination (PE). In both groups of patients, the positivity of polymerase chain reaction (PCR) was similar to that of immunofluorescence assay and enzyme-linked immunosorbent assay, and higher than that of MST and PE (P < .05). In the group of patients suspected to have ACL, PCR presented the same positivity as PE and MST together. No correlation between positivity of the laboratory methods and clinical or epidemiologic aspects was observed. Our data confirmed the value of PCR as an alternative laboratory method for diagnosing ACL, especially for those patients with negative PE and MST.

PCR-RFLP to identify Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis causing American cutaneous leishmaniasis.

A PCR-RFLP based method was developed to diagnose and identify the Leishmania species causing American cutaneous leishmaniasis (ACL) in a panel of clinical samples obtained from an endemic region of Brazil. The comparison of the results obtained by PCR-RFLP and PCR-hybridization in the identification of Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis were highly concordant (kappa=91.5%). The PCR-RFLP method was reliable, fast and easy to conduct on biopsies and presents potential value of utmost importance for the diagnosis and identification of Leishmania in clinical specimens, infected reservoirs and vectors.

PCR-RFLP mkDNA no diagnóstico e identificação das espécies de Leishmania causadoras de Leishmaniose cutânea no Brasil / PCR-RFLP mkDNA in the diagnosis and identification of causing species of Leishmania of Leishmaniasis cutaneous in Brazil

Neste trabalho estudamos a aplicabilidade e a capacidade da PCR-RFLP mkDNA na identificação e diferenciação de espécies de Leishmania que circulam no Brasil. Esta técnica diferencia-se das outras PCRs seguidas de restrição no produto de amplificação, por utilizar como alvo a região conservada dos minicírculos de kDNA das leishmânias... Em seguida, testamos a capacidade da PCR-RFLP mkDNA em diferenciar, também, as espécies L. (V.) guyanensis, L. (V.) shawi, L. (V.) naiffi, L. (V.) lainsoni e L. (L.) chagasi, todas presentes no Brasil. Para isto, fizemos um mapa de restrição nas seqüências de DNA da região conservada dos minicírculos de Leishmania depositadas no GenBank e testamos a PCR-RFLP mkDNA em cepas referência destas espécies e em 23 isolados de Leishmania de diferentes zimodemas, que confirmaram a identidade dos parasitos. Propomos um esquema de diferenciação que contempla a maioria das espécies de Leishmania presentes no Brasil, entretanto, não conseguimos diferenciar L. (V.) braziliensis de L. (V.) guyanensis. A partir destes resultados partimos para a aplicação da metodologia em dois conjuntos de amostras clínicas. No primeiro conjunto, testamos a PCR-RFLP mkDNA na identificação de Leishmania em amostras da região endêmica para LTA do Vale do Rio Doce - MG. Os parasitos foram extraídos de uma amostra de 475 lâminas coradas pelo Giemsa, arquivadas entre 1965-2000. A PCR-RFLP mkDNA foi positiva em 395 (83,2 por cento) laminas e L. (V.) braziliensis a única espécie presente na amostra. No segundo conjunto, avaliamos o uso da PCR-RFLP mkDNA na detecção e identificação dos agentes etiológicos da LTA de diferentes áreas endêmicas do Brasil em amostras clínicas de 329 pacientes, de 6 estados do Brasil, preservadas de diversas formas. Independente da forma de preservação, a PCR-RFLP mkDNA detectou e identificou L. (V.) braziliensis (89,0 por cento), L. (L.) amazonensis (6,4 por cento) e L. (V.) lainsoni (4,6 por cento) nas amostras clínicas. Por ser uma técnica muito sensível, de rápida execução, de fácil leitura, confirmar as caracterizações dos parasitos feitas previamente por outros métodos, sugerimos a utilização da PCR-RFLP mkDNA como um método alternativo na detecção e identificação dos agentes etiológicos da LTA no Brasil.

PCR-RFLP mkDNA no diagnóstico e identificação das espécies de Leishmania causadoras de Leishmaniose cutânea no Brasil

Neste trabalho estudamos a aplicabilidade e a capacidade da PCR-RFLP mkDNA na identificação e diferenciação de espécies de Leishmania que circulam no Brasil. Esta técnica diferencia-se das outras PCRs seguidas de restrição no produto de amplificação, por utilizar como alvo a região conservada dos minicírculos de kDNA das leishmânias. Inicialmente, testamos a técnica num conjunto de amostras que haviam sido previamente caracterizadas por hibridização com sondas radioativas, de uma região endêmica para Leishmaniose Tegumentar Americana (LTA) onde apenas L. (V.) braziliensis e L. (L.) amazonensis haviam sido encontradas. A comparação dos resultados obtidos com a PCR-RFLP mkDNA e hibridização foram 91,5 porcento concordantes, mostrando que a técnica apresentava potencial para ser usada diretamente em material clínico

Em seguida, testamos a capacidade da PCR-RFLP mkDNA em diferenciar, também, as espécies L. (V.) guyanensis, L. (V.) shawi, L. (V.) naiffi, L. (V.) lainsoni e L. (L.) chagasi, todas presentes no Brasil. Para isto, fizemos um mapa de restrição nas seqüências de DNA da região conservada dos minicírculos de Leishmania depositadas no GenBank e testamos a PCR-RFLP mkDNA em cepas referência destas espécies e em 23 isolados de Leishmania de diferentes zimodemas, que confirmaram a identidade dos parasitos. Propomos um esquema de diferenciação que contempla a maioria das espécies de Leishmania presentes no Brasil, entretanto, não conseguimos diferenciar L. (V.) braziliensis de L. (V.) guyanensis. A partir destes resultados partimos para a aplicação da metodologia em dois conjuntos de amostras clínicas.No primeiro conjunto, testamos a PCR-RFLP mkDNA na identificação de Leishmania em amostras da região endêmica para LTA do Vale do Rio Doce - MG. Os parasitos foram extraídos de uma amostra de 475 lâminas coradas pelo Giemsa, arquivadas entre 1965-2000. A PCR-RFLP mkDNA foi positiva em 395 (83,2 porcento) laminas e L. (V.) braziliensis a única espécie presente na amostra. No segundo conjunto, avaliamos o uso da PCR-RFLP mkDNA na detecção e identificação dos agentes etiológicos da LTA de diferentes áreas endêmicas do Brasil em amostras clínicas de 329 pacientes, de 6 estados do Brasil, preservadas de diversas formas. Independente da forma de preservação, a PCR-RFLP mkDNA detectou e identificou L. (V.) braziliensis (89,0 porcento), L. (L.) amazonensis (6,4 porcento) e L. (V.) lainsoni (4,6 porcento) nas amostras clínicas. Por ser uma técnica muito sensível, de rápida execução, de fácil leitura, confirmar as caracterizações dos parasitos feitas previamente por outros métodos, sugerimos a utilização da PCR-RFLP mkDNA como um método alternativo na detecção e identificação dos agentes etiológicos da LTA no Brasil

PCR-RFLP mkDNA no diagnóstico e identificação das espécies de Leishmania causadoras de Leishmaniose cutânea no Brasil / PCR-RFLP mkDNA in the diagnosis and identification of Leishmania species causing cutaneous leishmaniasis in Brazil

Neste trabalho estudamos a aplicabilidade e a capacidade da PCR-RFLP mkDNA na identificação e diferenciação de espécies de Leishmania que circulam no Brasil. Esta técnica diferencia-se das outras PCRs seguidas de restrição no produto de amplificação, por utilizar como alvo a região conservada dos minicírculos de kDNA das leishmânias. Inicialmente, testamos a técnica num conjunto de amostras que haviam sido previamente caracterizadas por hibridização com sondas radioativas, de uma região endêmica para Leishmaniose Tegumentar Americana (LTA) onde apenas L. (V.) braziliensis e L. (L.) amazonensis haviam sido encontradas. A comparação dos resultados obtidos com a PCR-RFLP mkDNA e hibridização foram 91,5 por cento concordantes, mostrando que a técnica apresentava potencial para ser usada diretamente em material clínico. Em seguida, testamos a capacidade da PCR-RFLP mkDNA em diferenciar, também, as espécies L. (V.) guyanensis, L. (V.) shawi, L. (V.) naiffi, L. (V.) lainsoni e L. (L.) chagasi, todas presentes no Brasil. Para isto, fizemos um mapa de restrição nas seqüências de DNA da região conservada dos minicírculos de Leishmania depositadas no GenBank e testamos a PCR-RFLP mkDNA em cepas referência destas espécies e em 23 isolados de Leishmania de diferentes zimodemas, que confirmaram a identidade dos parasitos. Propomos um esquema de diferenciação que contempla a maioria das espécies de Leishmania presentes no Brasil, entretanto, não conseguimos diferenciar L. (V.) braziliensis de L. (V.) guyanensis. A partir destes resultados partimos para a aplicação da metodologia em dois conjuntos de amostras clínicas. No primeiro conjunto, testamos a PCR-RFLP mkDNA na identificação de Leishmania em amostras da região endêmica para LTA do Vale do Rio Doce - MG. Os parasitos foram extraídos de uma amostra de 475 lâminas coradas pelo Giemsa, arquivadas entre 1965-2000. A PCR-RFLP mkDNA foi positiva em 395 (83,2 porcento) laminas e L. (V.) braziliensis a única espécie presente na amostra. No segundo conjunto, avaliamos o uso da PCR-RFLP mkDNA na detecção e identificação dos agentes etiológicos da LTA de diferentes áreas endêmicas do Brasil em amostras clínicas de 329 pacientes, de 6 estados do Brasil, preservadas de diversas formas. Independente da forma de preservação, a PCR-RFLP mkDNA detectou e identificou L. (V.) braziliensis (89,0 por cento), L. (L.) amazonensis (6,4 por cento) e L. (V.) lainsoni (4,6 por cento) nas amostras clínicas. Por ser uma técnica muito sensível, de rápida execução, de fácil leitura, confirmar as caracterizações dos parasitos feitas previamente por outros métodos, sugerimos a utilização da PCR-RFLP mkDNA como um método alternativo na detecção e identificação dos agentes etiológicos da LTA no Brasil.