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1.
Nucleic Acids Res ; 47(1): 152-167, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30321403

RESUMO

DNMT3L (DNMT3-like), a member of the DNMT3 family, has no DNA methyltransferase activity but regulates de novo DNA methylation. While biochemical studies show that DNMT3L is capable of interacting with both DNMT3A and DNMT3B and stimulating their enzymatic activities, genetic evidence suggests that DNMT3L is essential for DNMT3A-mediated de novo methylation in germ cells but is dispensable for de novo methylation during embryogenesis, which is mainly mediated by DNMT3B. How DNMT3L regulates DNA methylation and what determines its functional specificity are not well understood. Here we show that DNMT3L-deficient mouse embryonic stem cells (mESCs) exhibit downregulation of DNMT3A, especially DNMT3A2, the predominant DNMT3A isoform in mESCs. DNA methylation analysis of DNMT3L-deficient mESCs reveals hypomethylation at many DNMT3A target regions. These results confirm that DNMT3L is a positive regulator of DNA methylation, contrary to a previous report that, in mESCs, DNMT3L regulates DNA methylation positively or negatively, depending on genomic regions. Mechanistically, DNMT3L forms a complex with DNMT3A2 and prevents DNMT3A2 from being degraded. Restoring the DNMT3A protein level in DNMT3L-deficient mESCs partially recovers DNA methylation. Thus, our work uncovers a role for DNMT3L in maintaining DNMT3A stability, which contributes to the effect of DNMT3L on DNMT3A-dependent DNA methylation.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Animais , DNA Metiltransferase 3A , Estabilidade Enzimática/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica/genética
2.
J Biol Chem ; 293(33): 12770-12780, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29959229

RESUMO

Set7/9 (also known as Set7, Set9, Setd7, and Kmt7) is a lysine methyltransferase that catalyzes the methylation of multiple substrates, including histone H3 and non-histone proteins. Although not essential for normal development and physiology, Set7/9-mediated methylation events play important roles in regulating cellular pathways involved in various human diseases, making Set7/9 a promising therapeutic target. Multiple Set7/9 inhibitors have been developed, which exhibit varying degrees of potency and selectivity in vitro However, validation of these compounds in vivo has been hampered by the lack of a reliable cellular biomarker for Set7/9 activity. Here, we report the identification of Rpl29, a ribosomal protein abundantly expressed in all cell types, as a major substrate of Set7/9. We show that Rpl29 lysine 5 (Rpl29K5) is methylated exclusively by Set7/9 and can be demethylated by Lsd1 (also known as Kdm1a). Rpl29 is not a core component of the ribosome translational machinery and plays a regulatory role in translation efficiency. Our results indicate that Rpl29 methylation has no effect on global protein synthesis but affects Rpl29 subcellular localization. Using an Rpl29 methylation-specific antibody, we demonstrate that Rpl29K5 methylation is present ubiquitously and validate that (R)-PFI-2, a Set7/9 inhibitor, efficiently reduces Rpl29K5 methylation in cell lines. Thus, Rpl29 methylation can serve as a specific cellular biomarker for measuring Set7/9 activity.


Assuntos
Fatores de Coagulação Sanguínea/genética , Metilação de DNA , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/química , Proteínas Ribossômicas/fisiologia , Animais , Fatores de Coagulação Sanguínea/metabolismo , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Histona-Lisina N-Metiltransferase/genética , Humanos , Masculino , Camundongos Knockout , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA , Transcrição Gênica
3.
J Biol Chem ; 292(6): 2255-2265, 2017 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28031468

RESUMO

PRMT5 is the primary enzyme responsible for the deposition of the symmetric dimethylarginine in mammalian cells. In an effort to understand how PRMT5 is regulated, we identified a threonine phosphorylation site within a C-terminal tail motif, which is targeted by the Akt/serum- and glucocorticoid-inducible kinases. While investigating the function of this posttranslational modification, we serendipitously discovered that its free C-terminal tail binds PDZ domains (when unphosphorylated) and 14-3-3 proteins (when phosphorylated). In essence, a phosphorylation event within the last few residues of the C-terminal tail generates a posttranslational modification-dependent PDZ/14-3-3 interaction "switch." The C-terminal motif of PRMT5 is required for plasma membrane association, and loss of this switching capacity is not compatible with life. This signaling phenomenon was recently reported for the HPV E6 oncoprotein but has not yet been observed for mammalian proteins. To investigate the prevalence of PDZ/14-3-3 switching in signal transduction, we built a protein domain microarray that harbors PDZ domains and 14-3-3 proteins. We have used this microarray to interrogate the C-terminal tails of a small group of candidate proteins and identified ERBB4, PGHS2, and IRK1 (as well as E6 and PRMT5) as conforming to this signaling mode, suggesting that PDZ/14-3-3 switching may be a broad biological paradigm.


Assuntos
Proteínas 14-3-3/metabolismo , Domínios PDZ , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas 14-3-3/química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Camundongos , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/química
4.
Commun Biol ; 7(1): 442, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38600349

RESUMO

Aryl hydrocarbon receptor (AHR) signalling integrates biological processes that sense and respond to environmental, dietary, and metabolic challenges to ensure tissue homeostasis. AHR is a transcription factor that is inactive in the cytosol but upon encounter with ligand translocates to the nucleus and drives the expression of AHR targets, including genes of the cytochrome P4501 family of enzymes such as Cyp1a1. To dynamically visualise AHR activity in vivo, we generated reporter mice in which firefly luciferase (Fluc) was non-disruptively targeted into the endogenous Cyp1a1 locus. Exposure of these animals to FICZ, 3-MC or to dietary I3C induced strong bioluminescence signal and Cyp1a1 expression in many organs including liver, lung and intestine. Longitudinal studies revealed that AHR activity was surprisingly long-lived in the lung, with sustained Cyp1a1 expression evident in discrete populations of cells including columnar epithelia around bronchioles. Our data link diet to lung physiology and also reveal the power of bespoke Cyp1a1-Fluc reporters to longitudinally monitor AHR activity in vivo.


Assuntos
Citocromo P-450 CYP1A1 , Receptores de Hidrocarboneto Arílico , Camundongos , Animais , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Luciferases/genética , Fígado/metabolismo , Pulmão/metabolismo
5.
Exp Parasitol ; 134(3): 281-9, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23562705

RESUMO

Protozoa of the Leishmania genus are transmitted to humans by the bite of infected sandflies, and are the causative agents of leishmaniasis which ranges from cutaneous to visceral clinical forms. The definitive diagnosis of leishmaniasis has relied traditionally on parasite demonstration, either by microscopy or culture; in the last years, diagnosis based on PCR methods has overcome some drawbacks of traditional methods, increasing sensitivity and allowing using less invasive sampling for diagnosis. However, there are not defined protocols and almost each laboratory applies its own in-house method. Although there are several studies comparing the performance of different methods within the same laboratory, those addressing interlaboratory comparison are scarce, in spite of the growing number of collaborative projects between partners from different leishmaniasis endemic and non-endemic countries. In this work we propose a protocol for interlaboratory comparison of conventional and real-time PCR methods involving four participant laboratories from four different endemic regions in four continents; the protocol includes a quality control step and reduces the variability among the samples tested by each participant. A panel of 77 samples from human origin and 9 from different parasite strains was blindly tested by the participants, aiming to assess the sensitivity of the different methods as well as their usefulness for species identification. Real-time PCR methods targeting the kDNA minicircles returned the highest sensitivity, while both PCR targeting ITS-1 and further HaeIII digestion and a combined algorithm including hsp70 PCR and restriction fragment length polymorphism analysis were the most appropriate approaches for species identification.


Assuntos
DNA de Protozoário/análise , Leishmania/isolamento & purificação , Leishmaniose/diagnóstico , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Estudos de Casos e Controles , DNA/sangue , DNA/química , DNA de Cinetoplasto/análise , DNA de Protozoário/química , Humanos , Leishmania/classificação , Leishmania/genética , Reação em Cadeia da Polimerase/normas , Polimorfismo de Fragmento de Restrição , Controle de Qualidade , Reação em Cadeia da Polimerase em Tempo Real/normas , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas , Especificidade da Espécie
6.
Nat Struct Mol Biol ; 30(4): 489-501, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36941433

RESUMO

Recent studies have shown that repressive chromatin machinery, including DNA methyltransferases and polycomb repressor complexes, binds to chromosomes throughout mitosis and their depletion results in increased chromosome size. In the present study, we show that enzymes that catalyze H3K9 methylation, such as Suv39h1, Suv39h2, G9a and Glp, are also retained on mitotic chromosomes. Surprisingly, however, mutants lacking histone 3 lysine 9 trimethylation (H3K9me3) have unusually small and compact mitotic chromosomes associated with increased histone H3 phospho Ser10 (H3S10ph) and H3K27me3 levels. Chromosome size and centromere compaction in these mutants were rescued by providing exogenous first protein lysine methyltransferase Suv39h1 or inhibiting Ezh2 activity. Quantitative proteomic comparisons of native mitotic chromosomes isolated from wild-type versus Suv39h1/Suv39h2 double-null mouse embryonic stem cells revealed that H3K9me3 was essential for the efficient retention of bookmarking factors such as Esrrb. These results highlight an unexpected role for repressive heterochromatin domains in preserving transcription factor binding through mitosis and underscore the importance of H3K9me3 for sustaining chromosome architecture and epigenetic memory during cell division.


Assuntos
Proteômica , Fatores de Transcrição , Animais , Camundongos , Fatores de Transcrição/metabolismo , Histonas/metabolismo , Heterocromatina , Metilação de DNA , Mitose , Proteínas do Grupo Polycomb/genética , Metiltransferases/metabolismo
7.
J Clin Microbiol ; 50(2): 495-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22116151

RESUMO

We present an algorithm based on three PCR assays for Leishmania (Viannia) species identification and assessed its performance using 70 specimens from Peruvian patients. The succession of the assayed targets can be ordered according to species prevalence. Sequential progression through the algorithm reduced the number of samples here studied by approximately 30% after each step.


Assuntos
Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Parasitologia/métodos , Reação em Cadeia da Polimerase/métodos , Algoritmos , Humanos , Peru
8.
J Clin Microbiol ; 49(3): 1097-100, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21177908

RESUMO

We compared traditional cutaneous leishmaniasis diagnostic methods to filter paper lesion impression (FPLI) PCR for secondarily infected ulcers and nonulcerative lesions. The sensitivity and specificity of FPLI PCR for secondarily infected lesions (n = 8) were 100%. In primarily nonulcerative lesions (n = 15), the sensitivity of FPLI PCR was inferior to that of pooled-invasive-specimen PCR (72.7% versus 100%) (P = 0.10). FPLI PCR is sensitive, specific, and unlike invasive procedures, can be used in secondarily infected ulcers. Invasive specimen collection is superior in nonulcerative lesions.


Assuntos
Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Parasitologia/métodos , Reação em Cadeia da Polimerase/métodos , Úlcera Cutânea/patologia , Úlcera Cutânea/parasitologia , Manejo de Espécimes/métodos , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Humanos , Leishmania/genética , Sensibilidade e Especificidade
9.
Clin Infect Dis ; 50(1): e1-6, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19947858

RESUMO

BACKGROUND: Traditional detection of Leishmania from ulcers involves collection of invasive specimens that cause discomfort, require technical expertise, and carry risks of invasive procedures. We compared traditional diagnostic methods with a molecular noninvasive filter paper-based method for the diagnosis of cutaneous leishmaniasis. METHODS: Consecutive patients presenting to the Leishmania Clinic at Hospital Nacional Cayetano Heredia were enrolled. Polymerase chain reaction (PCR) was performed on lesion scrapings, aspirates, and filter paper impressions. The reference standard was any 2 of 5 tests positive: smear, aspirate culture, invasive-specimen PCR (scrapings and aspirates), filter paper PCR, and leishmanin skin test. Outcome measures were sensitivity and specificity. Leishmania speciation was performed by PCR-restriction fragment length polymorphism (RFLP) of positive specimens. RESULTS: Forty-five patients with 66 lesions were enrolled. Of 52 lesions diagnosed as cutaneous leishmaniasis, 50 were positive by PCR of invasive specimens versus 48 by PCR of filter papers (P=.930). Sensitivity and specificity of PCR on invasively obtained specimens were 94.2% (95% confidence interval [CI], 87.9%-100%) and 92.9% (95% CI, 79.4%-100%). Sensitivity and specificity of filter paper PCR were 92.3% (95% CI, 85.1%-99.5%) and 100%. Culture, smear, and leishmanin skin test all had inferior sensitivities, compared with PCR of invasive or noninvasive specimens (P<.001). Of 50 specimens positive by PCR, 19 had sufficient DNA for PCR-RFLP analysis. CONCLUSIONS: Filter paper PCR constitutes a sensitive and specific alternative to traditional diagnostic assays. This novel, rapid, well-tolerated method has the potential for widespread use in the field and in pediatric populations where traditional specimen collection is most difficult to perform, and can potentially be used for rapid species identification.


Assuntos
DNA/análise , Leishmania/classificação , Leishmaniose Cutânea/parasitologia , Reação em Cadeia da Polimerase/métodos , Adolescente , Idoso , Criança , Pré-Escolar , DNA/genética , Feminino , Humanos , Leishmania/genética , Leishmaniose Cutânea/diagnóstico , Masculino , Pessoa de Meia-Idade , Peru , Sensibilidade e Especificidade , Pele/parasitologia , Especificidade da Espécie , Manejo de Espécimes/métodos
10.
Nat Commun ; 11(1): 4118, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807789

RESUMO

Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers such as PRC2 and DNA methyl-transferases, and proteins governing chromosome architecture. Deletion of PRC2, Dnmt1/3a/3b or Mecp2 in ESCs leads to an increase in the size of individual mitotic chromosomes, consistent with de-condensation. Similar results were obtained by the experimental cleavage of cohesin. Thus, we identify chromosome-bound factors in pluripotent stem cells during mitosis and reveal that PRC2, DNA methylation and Mecp2 are required to maintain chromosome compaction.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Metilação de DNA/fisiologia , DNA Metiltransferase 3A , Imunofluorescência , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Proteômica , DNA Metiltransferase 3B
11.
Cell Rep ; 20(8): 1936-1949, 2017 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-28834755

RESUMO

Proper telomere length is essential for embryonic stem cell (ESC) self-renewal and pluripotency. Mouse ESCs (mESCs) sporadically convert to a transient totipotent state similar to that of two-cell (2C) embryos to recover shortened telomeres. Zscan4, which exhibits a burst of expression in 2C-like mESCs, is required for telomere extension in these cells. However, the mechanism by which Zscan4 extends telomeres remains elusive. Here, we show that Zscan4 facilitates telomere elongation by inducing global DNA demethylation through downregulation of Uhrf1 and Dnmt1, major components of the maintenance DNA methylation machinery. Mechanistically, Zscan4 recruits Uhrf1 and Dnmt1 and promotes their degradation, which depends on the E3 ubiquitin ligase activity of Uhrf1. Blocking DNA demethylation prevents telomere elongation associated with Zscan4 expression, suggesting that DNA demethylation mediates the effect of Zscan4. Our results define a molecular pathway that contributes to the maintenance of telomere length homeostasis in mESCs.


Assuntos
Metilação de DNA , Células-Tronco Embrionárias Murinas/fisiologia , Telômero/genética , Fatores de Transcrição/genética , Animais , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Telômero/metabolismo , Fatores de Transcrição/metabolismo , Transfecção
12.
Cell Rep ; 21(12): 3390-3397, 2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29262320

RESUMO

DNA methylation plays crucial roles in chromatin structure and gene expression. Aberrant DNA methylation patterns, including global hypomethylation and regional hypermethylation, are associated with cancer and implicated in oncogenic events. How DNA methylation is regulated in developmental and cellular processes and dysregulated in cancer is poorly understood. Here, we show that PRMT6, a protein arginine methyltransferase responsible for asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a), negatively regulates DNA methylation and that PRMT6 upregulation contributes to global DNA hypomethylation in cancer. Mechanistically, PRMT6 overexpression impairs chromatin association of UHRF1, an accessory factor of DNMT1, resulting in passive DNA demethylation. The effect is likely due to elevated H3R2me2a, which inhibits the interaction between UHRF1 and histone H3. Our work identifies a mechanistic link between protein arginine methylation and DNA methylation, which is disrupted in cancer.


Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Código das Histonas , Histonas/metabolismo , Humanos , Células MCF-7 , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Ubiquitina-Proteína Ligases
15.
PLoS One ; 7(11): e49738, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23185421

RESUMO

BACKGROUND: Traditional methods of detecting Leishmania from cutaneous lesions involve invasive diagnostic procedures, such as scrapings, which cause discomfort, require technical expertise, and carry risks of invasive procedures. We compared the performance of 2 novel, molecular-based non-invasive methods for the diagnosis of cutaneous leishmaniasis (CL). METHODS: Consecutive patients presenting to the Leishmania Clinic at the Hospital Nacional Cayetano Heredia were enrolled. PCR was performed on filter paper lesion impressions (FPLIs), cytology brushes, and lancets for detection of Leishmania DNA. Smears from lesion scrapings and leishmanin skin test were also performed. Outcome measures were sensitivity and specificity. Composite reference standard was any 2 of 5 tests positive. Species identification was performed by PCR assays of positive specimens. RESULTS: Ninety patients with 129 lesions were enrolled, 117 of which fulfilled reference criteria for a diagnosis of CL. Of these 117 lesions, 113 were positive by PCR of lancets used for lesion scrapings versus 116 by PCR of FPLIs (p=0.930) or 116 by PCR of cytology brushes (p=0.930). Sensitivity and specificity of PCR on lancets were 96.6% [95% CI 93.3-99.9%] and 100%, respectively. Sensitivity and specificity of FPLI PCR were 99.1% [95% CI 97.4-100%] and 100%, respectively. Sensitivity and specificity of cytology brush PCR were 99.1% [95% CI 97.4-100%] and 100%, respectively. Giemsa-stained lesion smear and leishmanin skin test had inferior sensitivities at 47.9% [95% CI 38.9-57.0%] and 82.3% [95% CI 73.9-90.7%], respectively, compared to PCR of invasive or non-invasive specimens (p<0.001). CONCLUSIONS: Cytology brush PCR constitutes a sensitive and specific alternative to traditional diagnostic assays performed on invasive specimens such as lesion scrapings. It performs comparatively to non-invasive FPLI PCR. This novel, rapid, and well-tolerated method has the potential for widespread use in the field and in pediatric populations where traditional specimen collection is difficult.


Assuntos
Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/genética , Reação em Cadeia da Polimerase/métodos , DNA/metabolismo , DNA de Cinetoplasto/metabolismo , Humanos , Papel , Peru , Polimorfismo de Fragmento de Restrição , Sensibilidade e Especificidade , Pele/química , Pele/microbiologia , Testes Cutâneos/métodos , Especificidade da Espécie , Manejo de Espécimes/métodos
16.
Infect Genet Evol ; 12(8): 1994-2002, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22516226

RESUMO

In order to get more insight into its evolution and geographical distribution, we investigated the Leishmania (Viannia) braziliensis species complex using amplified fragment length polymorphisms and sequencing of a heat-shock protein 70 gene fragment. Previously, several assays had alluded to the high genetic diversity of the group, and single-locus assays typically identified two species, i.e. L. braziliensis and Leishmania peruviana, with occasional genetic signatures of both in the same strain. By analysis of 53 parasite isolates from Peru, and eight additional ones from other countries, we identified an atypical L. braziliensis cluster, and confirmed the origin of L. peruviana from the L. braziliensis cluster during the colonization of the western Andean coastal valleys. We discuss the clinical and taxonomical implications of our findings in relation to currently used species typing assays.


Assuntos
Leishmania braziliensis/genética , Leishmaniose Cutânea/parasitologia , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Bolívia/epidemiologia , Análise por Conglomerados , Genótipo , Proteínas de Choque Térmico HSP70/genética , Humanos , Leishmania braziliensis/classificação , Leishmaniose Cutânea/epidemiologia , Peru/epidemiologia , Filogenia , Reprodutibilidade dos Testes
17.
Diagn Microbiol Infect Dis ; 74(2): 142-50, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22819605

RESUMO

The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the Old and New World, using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis. Three new Leishmania-specific hsp70 PCRs were recently described, and we applied 2 of these on 89 clinical samples from a total of 73 Peruvian patients with either cutaneous or mucocutaneous leishmaniasis. The new PCRs on average showed a 2- to 3-fold improved sensitivity in the tested sample types (lesion biopsies, aspirates, and scrapings), for both genus detection and species typing, and were most successful in biopsies. Leishmania braziliensis, L. peruviana, and L. guyanensis were encountered. About one third of the L. braziliensis parasites contained 2 hsp70 alleles. This study is a paradigm for the implementation of a globally applicable upgraded tool for the identification of Leishmania directly on human specimens from cutaneous and mucocutaneous lesions in the New World.


Assuntos
Leishmania/classificação , Leishmania/genética , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/parasitologia , Reação em Cadeia da Polimerase/métodos , Genótipo , Proteínas de Choque Térmico HSP70/genética , Humanos , Leishmania/isolamento & purificação , Peru , Proteínas de Protozoários/genética
18.
PLoS One ; 6(10): e26395, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22046280

RESUMO

BACKGROUND: Traditional methods of diagnosing mucosal leishmaniasis (ML), such as biopsy with histopathology, are insensitive and require collection of an invasive diagnostic specimen. METHODS: We compared standard invasive procedures including biopsy histopathology, biopsy PCR, and leishmanin skin test (LST) to a novel, non-invasive, cytology-brush based PCR for the diagnosis of ML in Lima, Peru. Consensus reference standard was 2/4 tests positive, and outcome measures were sensitivity and specificity. Leishmania species identification was performed by PCR-based assays of positive specimens. RESULTS: Twenty-eight patients were enrolled, 23 of whom fulfilled criteria for a diagnosis of ML. Sensitivity and specificity of biopsy with histopathology were 21.7% [95% CI 4.9-38.5%] and 100%; 69.6% [95% CI 50.8-88.4%] and 100% for LST; 95.7% [95% CI 87.4-100%] and 100% for biopsy PCR; and 95.7% [95% CI 87.4-100%] and 90% [95% CI 71.4-100%] for cytology brush PCR using both Cervisoft® and Histobrush® cervical cytology brushes. Represented species identified by PCR-RFLP included: L. (V). braziliensis (n = 4), and L. (V). peruviana (n = 3). CONCLUSIONS: Use of commercial grade cytology brush PCR for diagnosis of ML is sensitive, rapid, well tolerated, and carries none of the risks of invasive diagnostic procedures such as biopsy. Further optimization is required for adequate species identification. Further evaluation of this method in field and other settings is warranted.


Assuntos
Leishmaniose Mucocutânea/diagnóstico , Reação em Cadeia da Polimerase/métodos , Biópsia , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Peru , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/normas , Sensibilidade e Especificidade , Testes Cutâneos
19.
Am J Trop Med Hyg ; 84(4): 556-61, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21460009

RESUMO

We hypothesized that Leishmania kDNA may be present in urine of patients with cutaneous leishmaniasis (CL). Urine samples and standard diagnostic specimens were collected from patients with skin lesions. kDNA polymerase chain reaction (PCR) was performed on samples from patients and 10 healthy volunteers from non-endemic areas. Eighty-six of 108 patients were diagnosed with CL and 18 (21%) had detectable Leishmania Viannia kDNA in the urine. Sensitivity and specificity were 20.9% (95% confidence interval [CI] 12.3-29.5%) and 100%. Six of 8 patients with mucocutaneous involvement had detectable kDNA in urine versus 12 of 78 patients with isolated cutaneous disease (P < 0.001). L. (V.) braziliensis (N = 3), L. (V.) guyanensis (N = 6), and L. (V.) peruviana (N = 3) were identified from urine. No healthy volunteer or patient with an alternate diagnosis had detectable kDNA in urine. Sensitivity of urine PCR is sub-optimal for diagnosis. On the basis of these preliminary data in a small number of patients, detectable kDNA in urine may identify less localized forms of infection and inform treatment decisions.


Assuntos
DNA de Cinetoplasto/isolamento & purificação , DNA de Cinetoplasto/urina , Leishmania/genética , Leishmaniose Cutânea/urina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , DNA de Cinetoplasto/genética , Feminino , Humanos , Leishmania/isolamento & purificação , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Mucocutânea/epidemiologia , Leishmaniose Mucocutânea/parasitologia , Leishmaniose Mucocutânea/urina , Masculino , Pessoa de Meia-Idade , Peru/epidemiologia , Adulto Jovem
20.
Am J Trop Med Hyg ; 83(2): 345-50, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20682880

RESUMO

We evaluated performance characteristics of five diagnostic methods for cutaneous leishmaniasis. Patients who came to the Leishmania Clinic of Hospital Nacional Cayetano Heredia in Lima, Peru, were enrolled in the study. Lesion smears, culture, microculture, polymerase chain reaction (PCR), and leishmanin skin test (LST) were performed. A total of 145 patients with 202 lesions were enrolled: 114 patients with 161 lesions fulfilled criteria for cutaneous leishmaniasis. Sensitivity and specificity were 57.8% (95% confidence interval [CI] = 50.2-65.4%) and 100.0% for culture, 78.3% (95% CI = 71.9-84.7%) and 100.0% for microculture, 71.4% (95% CI = 64.4-78.4%) and 100.0% for smears, 78.2% (95% CI = 70.6-85.8%) and 77.4% (95% CI = 62.7-92.1%) for LST, and 96.9% (95% CI = 94.2-99.6%) and 65.9% (95% CI = 51.4-80.4%) for PCR. PCR was more sensitive than the other assays (P < 0.001). Sensitivities of culture, smears, and LST varied by lesion duration and appearance. PCR offers performance advantages over other assays, irrespective of patient age, sex, lesion duration, or appearance. That clinical factors influence performance of non-molecular assays offers clinicians a patient-focused approach to diagnostic test selection.


Assuntos
Leishmaniose Cutânea/diagnóstico , Testes Cutâneos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Protozoários , Criança , Pré-Escolar , Técnicas de Cultura , DNA de Cinetoplasto/genética , Feminino , Humanos , Lactente , Leishmania braziliensis/isolamento & purificação , Leishmania guyanensis/isolamento & purificação , Leishmaniose Cutânea/epidemiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Pele/parasitologia , Adulto Jovem
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