ABSTRACT
RESUMEN Con el objetivo del estudio fue determinar la coexistencia y fuentes de alimentación de mosquitos adultos (Diptera: Culicidae) en un centro de salud rural de Piura en Perú, se realizó un estudio descriptivo transversal. Se usaron técnicas entomológicas para capturar e identificar mosquitos, y técnicas de biotecnología molecular para identificar las fuentes de alimentación. Un total de 793 ejemplares de los géneros Culex y Aedes se encontraron coexistiendo, 789 (99,5%) corresponden a Culex quinquefasciatus, 607 (76,9%) fueron machos y 182 (23,1%) hembras. Así mismo, 4 (100%) corresponden a Aedes aegypti hembras. Las fuentes de alimentación de Aedes aegypti fueron Homo sapiens sapiens, y de Culex quinquefasciatus fueron Homo sapiens sapiens y Canis familiaris. Este estudio proporciona evidencia de que los centros de salud rurales estarían actuando como focos de arbovirosis, existiendo el riesgo de que personas que acuden por distintas dolencias, puedan contraer enfermedades transmitidas por C. quinquefasciatus y A. aegypti.
ABSTRACT This study aimed to determine the coexistence and food sources of adult mosquitoes (Diptera: Culicidae) in a rural health center in Piura, Peru by using a descriptive cross-sectional design. Entomological techniques were used to capture and identify mosquitoes, and molecular biotechnology techniques were used to identify food sources. A total of 793 specimens of the Culex and Aedes genera were found coexisting, 789 (99.5%) were Culex quinquefasciatus, 607 (76.9%) were males and 182 (23.1%) were females. Likewise, 4 (100%) corresponded to Aedes aegypti females. The food sources of Aedes aegypti were Homo sapiens sapiens, and Homo sapiens sapiens and Canis familiaris were the food sources of Culex quinquefasciatus. This study provides evidence that rural health centers could be acting as foci of arbovirosis, with the risk that people who come for different ailments could contract diseases transmitted by C. quinquefasciatus and A. aegypti.
Subject(s)
Zoonoses , Polymerase Chain Reaction , Aedes , Culicidae , Polymorphism, Restriction Fragment Length , Sex Characteristics , Vector Control of Diseases , Cytochromes bABSTRACT
RESUMEN Objetivos. Determinar la alimentación del Aedes aegypti en brotes de dengue de dos zonas rurales del Perú durante el ciclón Yaku y El Niño Global del 2023. Material y métodos. Se analizaron ocho muestras de sangre (8 pooles) obtenidas del abdomen de 80 especímenes Aedes aegypti capturados en los distritos rurales de Querecotillo y Marcavelica durante brotes de dengue acontecidos en el ciclón Yaku y en El Niño Global. Se extrajo ADN de las muestras analizadas, se llevó a cabo una PCR dirigida al gen CytB como marcador genético y los productos PCR fueron digeridos enzimáticamente con las restrictasas Hae III y Mwo I. Los productos PCR-RFLP fueron visualizados por electroforesis en gel de agarosa al 4%. Resultados. Se obtuvo ADN de todas las muestras y como producto PCR un amplicón de 358 pb. Así mismo, el único RFLP en Hae III observado fue el de Homo sapiens sapiens (233 y 125 pb). No se observó RFLP en Hae III de Gallus gallus y RFLP en Mwo I de Canis familiaris y Mus musculus. Conclusión. En brotes de dengue de zonas rurales, durante el ciclón Yaku y en El Niño Global, el Aedes aegypti presentó un comportamiento alimenticio antropofílico conservado.
ABSTRACT Objective. To determine the feeding behavior of Aedes aegypti in dengue outbreaks in two rural areas of Peru during the Yaku cyclone and El Niño phenomenon of 2023. Material and methods. Eight blood samples (8 pools) were obtained from the abdomen of 80 Aedes aegypti specimens captured in the rural districts of Querecotillo and Marcavelica during the Yaku cyclone and El Niño dengue outbreaks. DNA was extracted from the analyzed samples, then a PCR was directed at the CytB gene as a genetic marker and the PCR products were enzymatically digested with the restrictases Hae III and Mwo I. The PCR-RFLP products were visualized by agarose gel electrophoresis at 4%. Results. DNA was obtained from all samples and a 358 bp amplicon was obtained as a PCR product. Likewise, the only RFLP found in Hae III was from Homo sapiens sapiens (233 and 125 bp). RFLP was not found in Hae III of Gallus gallus and RFLP in Mwo I of Canis familiaris and Mus musculus. Conclusion. Aedes aegypti showed conserved anthropophilic feeding behavior in dengue outbreaks in rural areas during the Yaku cyclone and El Niño.
Subject(s)
Polymorphism, Restriction Fragment Length , Polymerase Chain Reaction , Aedes , Dengue , DNA , InfectionsABSTRACT
Single nucleotide polymorphisms (SNPs, rs12979860 e rs8099917) in the Interferon Lambda 4 gene (IFNL4, formerly IFNL3and/or IL28B) has been associated with failure in the innate immune response, sustained virological response in hepatitis C, and HTLV-1-associated myelopathy (HAM) development. To search for these polymorphisms several methodologies can be employed, such as sequencing, real-time or quantitative polymerase chain reaction (qPCR), restriction fragment length polymorphism analysis in PCR products (PCR-RFLP), and tetra-primer PCR. The present study compared the performance of the tetra-primer PCR in relation to the PCR-RFLP, both optimized in the Research HTLV Laboratory of the Center of Immunology of Instituto Adolfo Lutz in São Paulo. One hundred DNA samples obtained from patients of STD/Aids Reference Centre in São Paulo, previously analyzed for IL28B SNPs by PCR-RFLP were selected for analysis, after confirming that they represent all IL28B SNPs patterns described in the literature. The results obtained showed concordance between the PCR-RFLP and the tetra-primer PCR SNPs results, and because of the low cost, easy to perform, and minor employment of biological specimen and reagents, the tetra-primer PCR is of choice to be used in routine. (AU)
Polimorfismos de nucleotídeos únicos (single nucleotide polymorphisms, SNPs rs12979860 e rs8099917) no gene que codifica o Interferon Lambda 4 (IFNL4, antigamente IFNL3 e/ou IL28B) têm sido associados às falhas na resposta imune inata e resposta virológica sustentada na hepatite C, e a mielopatia associada ao HTLV-1 (HTLV-1-associated myelopathy, HAM). A pesquisa destes polimorfismos pode empregar diversas metodologias: sequenciamento, reação em cadeia da polimerase em tempo real ou quantitativa (quantitative polymerase chain reaction, qPCR), análise de fragmentos de restrição enzimática em produtos de PCR (restriction fragment length polymorphism in PCR products, PCR-RFLP) e a tetra-primer PCR. Este estudo comparou o desempenho da tetra-primer PCR em relação a PCR-RFLP, ambas otimizadas no Laboratório de Pesquisa em HTLV do Centro de Imunologia do Instituto Adolfo Lutz de São Paulo. Foram selecionadas 100 amostras de DNA obtidas de pacientes do Centro de Referência e Treinamento em DST/Aids de São Paulo cujos SNPs na IL28B foram anteriormente determinados por PCR-RFLP e representaram todos os perfis descritos em literatura. Os resultados obtidos mostraram concordância entre elas, e pelo fato da tetra-primer PCR ter menor custo, ser de fácil execução, empregar menos tempo, insumos e material biológico, é a técnica de escolha para uso em rotina. (AU)
Subject(s)
Polymorphism, Restriction Fragment Length , Polymerase Chain Reaction , Interleukins , Polymorphism, Single Nucleotide , Interferon LambdaABSTRACT
INTRODUÇÃO: A neoplasia sólida pseudopapilar do pâncreas (NSPP) é rara e de baixa malignidade. Constitui cerca de 1-2% de todas as neoplasias pancreáticas exócrinas e ocorre principalmente em mulheres jovens. A heterogeneidade intratumoral tem sido descrita em vários tipos de tumor, incluindo a NSPP. MÉTODOS: Neste trabalho, investigamos a heterogeneidade molecular de três diferentes áreas (periférica, intermediária e central) de uma NSPP em um paciente pediátrico submetido à cirurgia de Whipple no HUCFF/UFRJ. O polimorfismo dos genes de desintoxicação GSTP1, CYPA1m1 e CYPA1m2 foi investigado pela técnica de PCRpolimorfismo de fragmento de restrição (PCR-RFLP) e o perfil de metilação do promotor dos genes p14ARF, GSTP1, hTERT e MGMT foi acessado por PCR (do inglês, Polymerase Chain Reaction) específico de metilação (MSP)RESULTADOS E DISCUSSÃO: Nas 3 regiões estudadas, os resultados revelaram CYPA1m1 e CYPA1m2 com genótipo de tipo selvagem e GSPT1 com genótipo heterozigoto. Em relação ao estado de metilação dos genes investigados, o MGMT não apresentou metilação detectável em nenhuma das três regiões analisadas; em contraste, tanto p14ARF quanto hTERT mostraram metilação detectável em todas as três regiões. GSTP1 mostrou metilação nas áreas 1 e 2, porém foi negativa na 3. CONCLUSÃO: Com base em nosso estudo, concluímos que há heterogeneidade da biologia tumoral da NSPP, representada pela inativação epigenética de genes críticos e pela atividade de desintoxicação reduzida, o que pode contribuir para identificar novos marcadores tumorais e para a melhor compreensão da biologia dessa neoplasia.(AU)
INTRODUCTION: The solid pseudopapillary neoplasia of the pancreas (NSPP) is rare and of low malignancy. It constitutes about 1-2% of all exocrine pancreatic neoplasms and occurs mainly in young women. Intratumoral heterogeneity has been described in several tumor types, including an NSPP. METHODS: In this work, we investigated the molecular heterogeneity of three different areas (peripheral, intermediate and central) of an NSPP in a pediatric patient who underwent Whipple surgery at HUCFF/UFRJ. The polymorphism of the detoxification genes GSTP1, CYPA1m1 and CYPA1m2 was investigated by the technique of PCRrestriction fragment polymorphism (PCR-RFLP) and the promoter methylation profile of the genes p14ARF, GSTP1, hTERT and MGMT was accessed by PCR (Polymerase Chain Reaction) specific methylation (MSP). RESULTS AND DISCUSSION: In the 3 studied regions, the results revealed CYPA1m1 and CYPA1m2 with wild-type genotype and GSPT1 with heterozygous genotype. Regarding the methylation state of the investigated genes, MGMT did not show detectable methylation in any of the three analyzed regions; in contrast, both p14ARF and hTERT found detectable methylation in all three regions. GSTP1 showed methylation in areas 1 and 2, but was negative in 3. CONCLUSION: Based on our study, we conclude that there is heterogeneity in the tumor biology of NSPP, represented by the epigenetic inactivation of critical genes and reduced detoxification activity, which may contribute to the identification of new tumor markers and to a better understanding of the biology of this neoplasm.(AU)
Subject(s)
Pancreas , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/drug therapy , Polymorphism, Genetic , Polymorphism, Restriction Fragment Length/genetics , Genetic Heterogeneity , Epigenomics , Methylation , Molecular BiologyABSTRACT
Plasmodium vivax is the most common human malaria parasite in Asian countries including Pakistan. Present study was designed to explore the genetic diversity of plasmodium vivax genotypes based on Pvmsp-3α and Pvmsp-3βgenes using allelic specific nested PCR and RFLP assays markers from field isolates in district Mardan, Pakistan. Blood samples of 200 P. vivax malarial patients were collected after taking their written informed consent. Genetic diversity in nested PCR products was determined by Restriction Fragment Length Polymorphism (RFLP) utilizing Alu1 and PstI restriction enzymes for alpha and beta gene products digestion, respectively. For analysis the genetic diversity of the sub allelic variants of Pvmsp3α and Pvmsp3β genes, Chi-Square test was performed by utilizing Minitab programming software 18. The P value 0.05 was considered as statistically significant. For Pvmsp 3α genes after gel electrophoresis of digested products, four distinct genotypes were obtained from total of 50 samples; type A: 35 (70%) (1.5-2.0 kb), 12 of type B (24%) (1.5-1.7 kb), 2 of type C (4%) (0.5-1.5) and one for type D (2%) (0.5-0.65 kb) which could be characterized into 9 allelic pattern (A1-A4, B1-B3, C1, D), in which A3 remained the most predominant. For Pvmsp-3βgenes, three distinct genotypes were obtained from 50 samples; 40(80%) of type A (1.5-2.5 kb), 9 (18%) of type B (1.0-1.5kb) and 1(2%) of type C (0.65 kb) which could be characterized into 6 allelic patterns (A1-A3, B1-B2, and C1). Most dominant one in Type A was A1 alleles which were noted (46%), while in Type B, the most dominant were B1 (10%).This study is the first ever report of molecular epidemiology and genetic variation in Pvmsp-3α and Pvmsp-3β genes of P. vivax isolates by using PCR/RFLP from District Mardan and [...].
O Plasmodium vivax é o parasita da malária humana mais comum nos países asiáticos, incluindo o Paquistão. O presente estudo foi desenhado para explorar a diversidade genética de genótipos de Plasmodium vivax baseados nos genes Pvmsp-3α e Pvmsp-3β, usando marcadores de ensaios alélicos nested PCR e RFLP de isolados de campo no distrito de Mardan, Paquistão. Amostras de sangue de 200 pacientes com malária por P. vivax foram coletadas após assinatura do termo de consentimento livre e esclarecido. A diversidade genética em produtos de PCR nested foi determinada por polimorfismo de fragmento de restrição (RFLP) utilizando as enzimas de restrição Alu1 e PstI para a digestão dos produtos dos genes alfa e beta, respectivamente. Para análise da diversidade genética das variantes subalélicas dos genes Pvmsp3α e Pvmsp3β, o teste Qui-quadrado foi realizado utilizando o software de programação Minitab 18. O valor P = 0,05 foi considerado estatisticamente significativo. Para os genes Pvmsp 3α, após eletroforese em gel de produtos digeridos, quatro genótipos distintos foram obtidos de um total de 50 amostras; tipo A: 35 (70%) (1,5-2,0 kb), 12 do tipo B (24%) (1,5-1,7 kb), 2 do tipo C (4%) (0,5-1,5) e um para o tipo D (2%) (0,5-0,65 kb), que podem ser caracterizados em nove padrões alélicos (A1-A4, B1-B3, C1, D), em que A3 permaneceu como o mais predominante. Para Pvmsp-3βgenes, três genótipos distintos foram obtidos a partir de 50 amostras; 40 (80%) do tipo A (1,5-2,5 kb), 9 (18%) do tipo B (1,0-1,5 kb) e 1 (2%) do tipo C (0,65 kb), que podem ser caracterizados em seis padrões alélicos (A1-A3, B1-B2 e C1). Os mais dominantes no tipo A foram o alelo A1, observados em 46%, enquanto, no tipo B, os mais dominantes foram B1 (10%). Este estudo é o primeiro relato de epidemiologia molecular e variação genética em Pvmsp-3α. Os genes Pvmsp-3β de isolados de P. vivax utilizando PCR/RFLP do Distrito Mardan mostraram um nível notável de diversidade genética nos genes estudados [...].
Subject(s)
Humans , Merozoites , Plasmodium vivax/genetics , Plasmodium vivax/parasitology , Polymorphism, Restriction Fragment Length/genetics , Membrane Proteins/analysis , Membrane Proteins/geneticsABSTRACT
Plasmodium vivax is the most common human malaria parasite in Asian countries including Pakistan. Present study was designed to explore the genetic diversity of plasmodium vivax genotypes based on Pvmsp-3α and Pvmsp-3ßgenes using allelic specific nested PCR and RFLP assays markers from field isolates in district Mardan, Pakistan. Blood samples of 200 P. vivax malarial patients were collected after taking their written informed consent. Genetic diversity in nested PCR products was determined by Restriction Fragment Length Polymorphism (RFLP) utilizing Alu1 and PstI restriction enzymes for alpha and beta gene products digestion, respectively. For analysis the genetic diversity of the sub allelic variants of Pvmsp3α and Pvmsp3ß genes, Chi-Square test was performed by utilizing Minitab programming software 18. The P value 0.05 was considered as statistically significant. For Pvmsp3α genes after gel electrophoresis of digested products, four distinct genotypes were obtained from total of 50 samples; type A: 35 (70%) (1.5-2.0 kb), 12 of type B (24%) (1.5-1.7 kb), 2 of type C (4%) (0.5-1.5) and one for type D (2%) (0.5-0.65 kb) which could be characterized into 9 allelic pattern (A1-A4, B1-B3, C1, D), in which A3 remained the most predominant. For Pvmsp-3ßgenes, three distinct genotypes were obtained from 50 samples; 40(80%) of type A (1.5-2.5 kb), 9 (18%) of type B (1.0-1.5kb) and 1(2%) of type C (0.65 kb) which could be characterized into 6 allelic patterns (A1-A3, B1-B2, and C1). Most dominant one in Type A was A1 alleles which were noted (46%), while in Type B, the most dominant were B1 (10%).This study is the first ever report of molecular epidemiology and genetic variation in Pvmsp-3α and Pvmsp-3ß genes of P. vivax isolates by using PCR/RFLP from District Mardan and showed a remarkable level of genetic diversity in the studied genes of circulating parasites in the study area. The results of this study will contribute in future studies about the genetic structure of parasite and vaccine development against the malaria.
O Plasmodium vivax é o parasita da malária humana mais comum nos países asiáticos, incluindo o Paquistão. O presente estudo foi desenhado para explorar a diversidade genética de genótipos de Plasmodium vivax baseados nos genes Pvmsp-3α e Pvmsp-3ß, usando marcadores de ensaios alélicos nested PCR e RFLP de isolados de campo no distrito de Mardan, Paquistão. Amostras de sangue de 200 pacientes com malária por P. vivax foram coletadas após assinatura do termo de consentimento livre e esclarecido. A diversidade genética em produtos de PCR nested foi determinada por polimorfismo de fragmento de restrição (RFLP) utilizando as enzimas de restrição Alu1 e PstI para a digestão dos produtos dos genes alfa e beta, respectivamente. Para análise da diversidade genética das variantes subalélicas dos genes Pvmsp3α e Pvmsp3ß, o teste Qui-quadrado foi realizado utilizando o software de programação Minitab 18. O valor P = 0,05 foi considerado estatisticamente significativo. Para os genes Pvmsp3α, após eletroforese em gel de produtos digeridos, quatro genótipos distintos foram obtidos de um total de 50 amostras; tipo A: 35 (70%) (1,5-2,0 kb), 12 do tipo B (24%) (1,5-1,7 kb), 2 do tipo C (4%) (0,5-1,5) e um para o tipo D (2%) (0,5-0,65 kb), que podem ser caracterizados em nove padrões alélicos (A1-A4, B1-B3, C1, D), em que A3 permaneceu como o mais predominante. Para Pvmsp-3ßgenes, três genótipos distintos foram obtidos a partir de 50 amostras; 40 (80%) do tipo A (1,5-2,5 kb), 9 (18%) do tipo B (1,0-1,5 kb) e 1 (2%) do tipo C (0,65 kb), que podem ser caracterizados em seis padrões alélicos (A1-A3, B1-B2 e C1). Os mais dominantes no tipo A foram o alelo A1, observados em 46%, enquanto, no tipo B, os mais dominantes foram B1 (10%). Este estudo é o primeiro relato de epidemiologia molecular e variação genética em Pvmsp-3α. Os genes Pvmsp-3ß de isolados de P. vivax utilizando PCR/RFLP do Distrito Mardan mostraram um nível notável de diversidade genética nos genes estudados de parasitas circulantes na área de estudo. Os resultados desse estudo contribuirão em estudos futuros sobre a estrutura genética do parasita e o desenvolvimento de vacinas contra a malária.
Subject(s)
Humans , Plasmodium vivax/genetics , Protozoan Proteins/genetics , Pakistan , Genetic Variation , Polymorphism, Restriction Fragment Length , Polymerase Chain Reaction , GenotypeABSTRACT
Background: The intrinsic sensitivity limitations of basic parasitological methods, along with the particular biological characteristics of parasites, make these methods ineffective to differentiate morphologically indistinguishable species. Molecular detection and characterization techniques could be used to overcome these problems. The purpose of this work was to standardize molecular polymerase chain reaction (PCR) techniques, described in the literature, for the detection and molecular characterization of intestinal protozoa and other pathogens in humans. Methods: DNA was extracted from human or animal feces, previously washed or cultured in Boeck Drbohlav's Modified Medium. DNA extraction was performed with Machery-Nagel extraction kits. The standardization of the PCR, nested-PCR or RFLP techniques was carried out according to the literature. For each molecular technique performed, the sensitivity of the test was determined based on the minimun quantity required of DNA (sensitivity A) and the minimum quantity of life forms that the test detected (sensitivity B). Results: Sensitivity A was 10 fg for G. duodenalis, 12.5 pg for Entamoeba histolytica or Entamoeba dispar, 50 fg for Cryptosporidium spp., 225 pg for Cyclospora spp. and 800 fg or 8 fg for Blastocystis spp. after performing a 1780 bp PCR or 310 bp nested PCR, respectively. The sensitivity B was 100 cysts for G. duodenalis, 500 cysts for E. histolytica or E. dispar, 1000 oocysts for Cyclospora spp. and 3600 or four vegetatives forms for PCR or nested PCR of Blastocystis spp., respectively. Conclusions: The molecular detection of protozoa and chromist was achieved and the molecular characterization allowed the genotyping of some of the parasites such as Giardia duodenalis, Cryptosporidium spp., and Blastocystis spp. This study summarizes the molecular techniques for epidemiological studies in humans and animals, and helps in the investigation of their transmission sources in countries where intestinal parasites are a public health problem.(AU)
Subject(s)
Humans , Animals , Polymerase Chain Reaction/standards , Intestinal Diseases, Parasitic/diagnosis , Intestines/parasitology , Polymorphism, Restriction Fragment Length , Epidemiologic Studies , Giardia lamblia , Blastocystis , CryptosporidiumABSTRACT
BACKGROUND The genetic heterogeneity of Leishmania parasites is a major factor responsible for the wide variety of Leishmania-associated manifestations. Consequently, understanding the genetic make-up of Leishmania species using suitable molecular markers is an important component of realising local and regional scale disease risk. The cytochrome b (cytb) is frequently used to type New World Leishmania species. However, its potential to discriminate Leishmania species and variants requires further evaluation. OBJECTIVES To explore the capacity of cytb gene to identify New World Leishmania species and variants and to develop an approach able to type local Leishmania species and variants. METHODS We retrieved 360 partial and complete Leishmania cytb gene sequences publicly available in GenBank database to study all single nucleotide polymorphisms (SNPs) across the cytb gene that differentiate New World Leishmania species. This information was used to develop an approach based upon the polymorphisms found in a DNA segment of 948bp. We also compared the typing results found with this technique with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiling obtained using HSP70 gene as target. One hundred Panamanian isolates were used to both typed Leishmania species and assess local genetic variability. FINDINGS We found complete agreement between our cytb approach and the PCR-RFLP profiling method based on HSP70 for Leishmania species identification. Ninety-two isolates were identified as L. panamensis, although other Viannia species were found circulating at a lower frequency. Three L. panamensis haplotypes were identified in Panamanian provinces. We also provide an initial report of L. guyanensis haplotypes circulating in Panama. MAIN CONCLUSIONS Cytb gene sequence encompasses key main SNPs that aid to identify Leishmania species. The cytb approach developed with this information was able to identify and assess genetic variability of local Leishmania species found in this study.
Subject(s)
Humans , Leishmaniasis, Cutaneous , Leishmania/genetics , Panama , Polymorphism, Restriction Fragment Length , Polymerase Chain Reaction , DNA, Protozoan/genetics , Cytochromes b/geneticsABSTRACT
Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease in cats. However, scarce data on its prevalence are available in Brazil. Persian cats and Persian-related breeds were assessed by molecular genotyping for a C to A transversion in exon 29 of PKD1 gene to determine ADPKD prevalence in a Brazilian population. Genomic DNA extracted from peripheral whole blood or oral swabs samples was used to amplify exon 29 of PKD1 gene employing a PCR-RFLP methodology. From a total of 616 animals, 27/537 Persian and 1/17 Himalayan cats showed the single-nucleotide variant (C to A) at position 3284 in exon 29 of feline PKD1. This pathogenic variation has been identified only in heterozygous state. The prevalence of ADPKD in Persian cats and Persian-related breeds was 5.03% and 1.6%, respectively. There was no significant association between feline breed, gender or age with ADPKD prevalence. Of note, the observed ADPKD prevalence in Persian cats and Persian-related breeds in Brazil was lower than the ones reported in other parts of the world. This finding may be related to genetic counseling and consequent selection of ADPKD-free cats for reproduction.
A doença renal policística autossômica dominante (DRPAD) é a doença genética mais comum em gatos. No entanto, poucos dados sobre sua prevalência estão disponíveis no Brasil. Gatos Persas e de raças relacionadas foram avaliados por genotipagem molecular para a transversão C→A no exon 29 do gene PKD1 felino para determinar a prevalência de DRPAD. DNA genômico extraído de sangue total periférico ou amostras de swabs orais foram utilizados para amplificar o exon 29 do gene PKD1 pela técnica de PCR-RFLP. De um total de 616 gatos, 27/537 Persas e 1/17 Himalaia mostraram a variante de nucleotídeo único (C→A) na posição 3284 no exon 29 do gene PKD1. Esta variante patogênica foi identificada apenas em heterozigose. A prevalência de DRPAD em gatos Persas e raças relacionadas foram de 5,03% e 1,6%, respectivamente. Não houve associações significativas entre raça, gênero ou idade dos felinos e incidência de DRPAD. A prevalência de DRPAD em gatos Persas e raças relacionadas no Brasil foi menor do que em outras partes do mundo, o que pode estar relacionado ao aconselhamento genético e consequente seleção de gatos sem ADPKD para reprodução.
Subject(s)
Animals , Cats , Polycystic Kidney, Autosomal Dominant/genetics , Polycystic Kidney, Autosomal Dominant/veterinary , Polycystic Kidney, Autosomal Dominant/epidemiology , Polymorphism, Restriction Fragment Length , Brazil/epidemiology , Polymerase Chain Reaction/veterinary , Prevalence , Genotyping Techniques/veterinary , MutationABSTRACT
Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.
Subject(s)
Humans , Polymorphism, Restriction Fragment Length/genetics , Minisatellite Repeats/genetics , Mycobacterium tuberculosis/genetics , Brazil/epidemiology , Bacterial Typing Techniques , Molecular Epidemiology , Whole Genome Sequencing , Genotype , Mycobacterium tuberculosis/isolation & purificationABSTRACT
Abstract INTRODUCTION: The clinical manifestations of cryptococcosis are usually associated with the infecting agents Cryptococcus neoformans (CN) and C. gattii (CG) species complexes and the host. In this study, non-HIV-infected patients, at a university hospital in southeastern Brazil, had epidemiological and clinical data associated with cryptococcal disease and isolated Cryptococcus species: CN - 24 patients and CG - 12 patients. METHODS: The comparison was comprised of demographic data, predisposing factors, clinical and laboratory manifestations, and outcomes of cryptococcosis patients treated between 2000 and 2016. Immunocompetent and immunosuppressed patients were also compared, irrespective of the infecting species. Cryptococcus spp. were genotyped by PCR-RFLP analysis of the URA5 gene. RESULTS: Infections by the CN species complex (100% VNI genotype) were associated with drug immunosuppression and fungemia, and patients infected with the CG species complex (83% VG II and 17% VGI genotypes) had more evident environmental exposure and higher humoral response. CN and CG affected patients with or without comorbidities. CONCLUSIONS: Diabetes mellitus, other chronic non-infectious diseases, and alcoholism were likely predisposing factors for infection by both CN and CG species. Immunocompetent patients, independent of the infecting Cryptococcus species complexes, showed a higher occurrence of meningitis and a trend toward less fungal dissemination and longer survival than immunosuppressed hosts.
Subject(s)
Humans , Cryptococcosis/diagnosis , Cryptococcosis/epidemiology , Cryptococcus neoformans/genetics , Cryptococcus gattii/genetics , Polymorphism, Restriction Fragment Length , Brazil/epidemiology , GenotypeABSTRACT
ABSTRACT Objective: To evaluate the possible association of a polymorphism in the gene encoding methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), 1958G>A, with the susceptibility to orofacial cleft in an Indonesian population. Material and Methods: A total of 200 stored secondary biological samples from 30 cases of orofacial cleft and 170 unaffected controls were analyzed to determine the polymorphism status at base 1958. The analysis was conducted using the PCR-restriction fragment length polymorphism technique after digestion with the Msp1 restriction enzyme. The samples were then subjected to agarose gel electrophoresis to investigate the presence or absence of the following fragments: genotype GG, 196, 86 and 40 base pairs (bp); genotype AA, 282 and 28 bp and genotype AG, 282, 196, 86, 40 and 28 bp. The test groups were compared using the Chi-square test. Results: The wild-type allele containing 1958G, as well as the genotype GG, were significantly more common in the control group than in the orofacial cleft group. Conclusion: The MTHFD1 1958G>A polymorphism was significantly associated with orofacial cleft susceptibility in the tested Indonesian population.
Subject(s)
Polymorphism, Genetic , Genetic Variation , Cleft Lip/pathology , Cleft Palate/pathology , Methylenetetrahydrofolate Dehydrogenase (NADP) , Polymorphism, Restriction Fragment Length , Chi-Square Distribution , IndonesiaABSTRACT
Abstract Introduction: Car painters are routinely exposed to organic solvents classified as carcinogenic and mutagenic substances. Objective: To characterize the population susceptibility and evaluate the genotoxic effects of exposure to organic solvents. Methods: A cross-sectional study comparing a group of car painters exposed to organic solvents with a non-exposed group. CYP2E1 polymorphisms and the presence of micronuclei in lymphocytes were determined. Results: One hundred twenty-two workers participated in the study: 62 who worked in car paint shops and were exposed to solvents, and 60 who were not exposed. There were statistically significant differences between the two groups regarding micronucleated cells and nucleoplasmic bridges frequencies (p=0.042 and p=0.046, respectively; exact likelihood ratio). Significant differences were found at the interaction between the CYP2E1 genotype c1c1 and occupational exposure to solvents, with higher frequencies of micronuclei (p= 0.013) and micronucleated cells (p= 0.015). However, when the frequencies of micronuclei, micronucleated cells and nucleoplasmic bridges in the exposure group were compared between the c1c1 and c2c2/c1c2 allele groups of the CYP2E1 polymorphism, statistically significant differences were found. Conclusions: This study confirms that when workers with CYP2E1 polymorphisms, specifically the c1c1 genotype, are exposed to organic solvents, they are more likely to have somatic cell mutations, a condition associated with increased susceptibility to diseases such as cancer
Resumen Introducción: Los pintores de vehículos automotores están rutinariamente expuestos a agentes como los solventes orgánicos, capaces de producir efectos mutágenos y carcinógenos. Objetivo: Caracterizar la susceptibilidad poblacional y evaluar los efectos genotóxicos debidos a la exposición a solventes orgánicos. Métodos: Estudio de corte transversal que comparó a un grupo de pintores de carros expuestos a solven tes orgánicos con un grupo de personas no expuestas. Fueron determinados tanto los polimorfismos de CYP2E1 como la presencia de micronúcleos en linfocitos. Resultados: Participaron 122 personas, 62 trabajadores de talleres de pintura de autos expuestos a solventes y 60 personas no expuestas. Con relación al cuestionario Q 16, 32% de los expuestos refirieron síntomas sugestivos de neurotoxicidad. Las frecuencias de células micronucleadas y de puentes nucleoplásmicos fueron significativamente mayores en los expuestos que en los no expuestos: p= 0.042 y p= 0.046, respectivamente, Razón de verosimilitud exacta). Fueron halladas diferencias significativas en la interacción de CYP2E1 (c1c1) y la exposición ocupacional a solventes, con mayores frecuencias de micronúcleos (p= 0.013) y de células micronucleadas (p= 0.015). Conclusiones: Este estudio reafirma que los trabajadores expuestos a solventes orgánicos con polimorfismos de CYP2E1, específicamente con genotipo c1c1, tienen mayor probabilidad de presentar mutaciones en las células somáticas, condición asociada con una mayor susceptibilidad a enfermedades como el cáncer
Subject(s)
Adult , Humans , Male , Middle Aged , Paint/toxicity , Solvents/toxicity , Carcinogens/toxicity , Occupational Exposure/adverse effects , Cytochrome P-450 CYP2E1/genetics , Polymorphism, Genetic , Automobiles , Polymorphism, Restriction Fragment Length , Lymphocytes/drug effects , Lymphocytes/ultrastructure , Micronucleus Tests/methods , Case-Control Studies , Cross-Sectional Studies , Colombia , Neurotoxicity Syndromes/diagnosis , Alleles , Personal Protective Equipment , Mutagenicity TestsABSTRACT
OBJECTIVE@#To explore the genetic basis for a patient featuring multiple carboxylase deficiency (MCD).@*METHODS@#PCR and Sanger sequencing were used to detect variant in the coding region of BT and HLCS genes in the patient. Suspected variants were verified in her parents and 80 unrelated healthy controls by a PCR-restriction fragment length polymorphism (PCR-RFLP) method.@*RESULTS@#The patient was found to carry compound heterozygous variants of the HLCS gene, namely c.286delG (p.Val96Leufs*162) and c.1648G>A (p.Val550Met). The c.286delG (p.Val96Leufs*162) was verified to be novel variant based on the result of PCR-RFLP analysis. No variant was found in the coding regions of BT gene in the patient.@*CONCLUSION@#The compound c.286delG (p.Val96Leufs*162) and c.1648G>A (p.Val550Met) variants probably underlie the MCD disorder in this patient. Above results have enriched the variant spectrum of MCA.
Subject(s)
Female , Humans , Carbon-Nitrogen Ligases , Genetics , Exons , Multiple Carboxylase Deficiency , Genetics , Mutation , Open Reading Frames , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Sequence Analysis, DNAABSTRACT
OBJECTIVE@#To study the association of the polymorphisms of the serum amyloid A1 (SAA1) gene at rs4638289 and rs7131332 loci with Kawasaki disease (KD) and its complication coronary artery lesion (CAL) in children.@*METHODS@#A total of 105 Han children with KD who were hospitalized and treated from 2013 to 2017 were enrolled as the KD group. A total of 100 Han children who underwent physical examination were enrolled as the control group. According to the presence or absence of CAL, the KD group was further divided into a CAL group with 23 children and a non-CAL (NCAL) group with 82 children. Polymerase chain reaction-restriction fragment length polymorphism was used to investigate the polymorphisms of the SAA1 gene at rs4638289 and rs7131332 loci.@*RESUKTS@#For the locus rs4638289 of the SAA1 gene, there were no significant differences between the KD and control groups in the genotype frequencies of AA, AT, and TT and the allele frequencies of A and T (P>0.05). But there were significant differences between the CAL and NCAL groups in the genotype frequencies of AA, AT, and TT (P=0.016), while there were no significant differences in the allele frequencies of A and T (P>0.05). AT genotype was a protective factor against CAL (OR=0.276, 95%CI: 0.099-0.772, P=0.011). For the locus rs7131332 of the SAA1 gene, there were no significant differences between the KD and control groups in the genotype frequencies of AA, AG, and GG and the allele frequencies of A and G (P>0.05). There were also no significant differences between the CAL and NCAL groups in the genotype frequencies of AA, AG, and GG and the allele frequencies of A and G (P>0.05).@*CONCLUSIONS@#Polymorphisms of the SAA1 gene at loci rs4638289 and rs7131332 are not associated with the onset of KD, while the polymorphism at the locus rs4638289 is associated with CAL in KD patients. KD patients with genotype AT may have a reduced risk of CAL.
Subject(s)
Child , Humans , Case-Control Studies , Gene Frequency , Genetic Predisposition to Disease , Genotype , Mucocutaneous Lymph Node Syndrome , Genetics , Polymorphism, Restriction Fragment Length , Polymorphism, Single Nucleotide , Serum Amyloid A Protein , GeneticsABSTRACT
Molecular biology is a new subject that clarifies the phenomena and nature of life at the molecular level. Its development provides new biotechnology and methods for the study of traditional pharmacognosy. The formation of molecular biology has brought the development of pharmacognosy into a new era of gene research. Lonicerae Japonicae Flos is a classical Chinese medicine. Many scholars of home and abroad have carried out relevant studies on its molecular biology on the basis of the in-depth study with traditional methods, and have achieved certain results. In order to provide references on the method, technical for promoting the modernization of Lonicerae Japonicae Flos, and the development, protection, and utilization of other traditional Chinese medicine resources. This article summarized the application status of molecular biology methods and techniques on the identification, biosynthesis of active constituents, and molecular mechanism of secondary metabolite under stress conditions of Lonicerae Japonicae Flos in recent years. In hybridization technology of tag(RFLP), molecular markers based on PCR(RAPD, AFLP, SSR and ISSR), based on DNA sequence analysis of SNP and DNA barcode for the variety identification, diagnosis, identification of Lonicerae Japonicae Flos, and so forth in detail. At the same time, it is proposed that multi-omics technology can be used to build systems biology technology and platforms, and establish related models of secondary metabolite biosynthesis, so as to deepen acknowledge the molecular mechanism of the active component biosynthesis of Lonicerae Japonicae Flos and the accumulation of metabolites, life activities of other medicinal plants under adverse environment, then to regulate them.
Subject(s)
Amplified Fragment Length Polymorphism Analysis , Chromatography, High Pressure Liquid , DNA Barcoding, Taxonomic , Drugs, Chinese Herbal/pharmacology , Lonicera/chemistry , Medicine, Chinese Traditional , Microsatellite Repeats , Plants, Medicinal/chemistry , Polymorphism, Restriction Fragment Length , Polymorphism, Single Nucleotide , Random Amplified Polymorphic DNA Technique , Secondary MetabolismABSTRACT
BACKGROUND: BMPR-1B is part of the transforming growth factor ß super family and plays a pivotal role in ewe litter size. Functional loss of exon-8 mutations in the BMPR-1B gene (namely the FecB gene) can increase both the ewe ovulation rate and litter size. RESULTS: This study constructed a eukaryotic expression system, prepared a monoclonal antibody, and characterized BMPR-1B/FecB protein-protein interactions (PPIs). Using Co-immunoprecipitation coupled to mass spectrometry (Co-IP/MS), 23 proteins were identified that specifically interact with FecB in ovary extracts of ewes. Bioinformatics analysis of selected PPIs demonstrated that FecB associated with several other BMPs, primarily via signal transduction in the ovary. FecB and its associated interaction proteins enriched the reproduction process via BMP2 and BMP4 pathways. Signal transduction was identified via Smads proteins and TGF-beta signaling pathway by analyzing the biological processes and pathways. Moreover, other target proteins (GDF5, GDF9, RhoD, and HSP 10) that interact with FecB and that are related to ovulation and litter size in ewes were identified. CONCLUSIONS: In summary, this research identified a novel pathway and insight to explore the PPi network of BMPR-1B.
Subject(s)
Animals , Female , Ovary/metabolism , Bone Morphogenetic Protein Receptors, Type I/genetics , Eukaryota/genetics , Protein Interaction Maps/genetics , Mass Spectrometry , Polymorphism, Restriction Fragment Length , Sheep , Signal Transduction , Polymerase Chain Reaction , Computational Biology , Bone Morphogenetic Protein Receptors, Type I/metabolism , Eukaryota/metabolism , Genotype , MutationABSTRACT
Abstract INTRODUCTION: We aimed to estimate the prevalence and transmission of drug-resistant tuberculosis in a high-burden Brazilian setting under directly observed therapy short-course strategy. METHODS: Isolates of culture-confirmed pulmonary tuberculosis patients from Guarulhos, Brazil, diagnosed in October 2007-2011 were subjected to drug susceptibility and IS6110-restriction fragment length polymorphism testing. RESULTS: The overall resistance prevalence was 11.5% and the multi-drug resistance rate was 4.2%. Twenty-six (43.3%) of 60 drug-resistant isolates were clustered. Epidemiological relationships were identified in 11 (42.3%) patients; 30.8% of the cases were transmitted in households. CONCLUSIONS: Drug-resistant tuberculosis was relatively low and transmitted in households and the community.
Subject(s)
Humans , Male , Female , Adolescent , Adult , Young Adult , Polymorphism, Restriction Fragment Length , Brazil/epidemiology , Prevalence , Cross-Sectional Studies , Tuberculosis, Multidrug-Resistant , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/epidemiology , Directly Observed Therapy/methods , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/geneticsABSTRACT
Abstract Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. Objective: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and −149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. Methodology: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. Results: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. Conclusion: The polymorphism −149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Periodontitis/genetics , Polymorphism, Genetic , DNA Methylation/genetics , MicroRNAs/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , Polymorphism, Restriction Fragment Length , Catalase/genetics , Case-Control Studies , Polymerase Chain Reaction , Methylenetetrahydrofolate Reductase (NADPH2)/genetics , Genetic Association Studies , Superoxide Dismutase-1/genetics , GenotypeABSTRACT
ABSTRACT Objective: To identify phenylalanine hydroxylase (PAH) mutations in patients with phenylketonuria (PKU) from the Newborn Screening Service in Mato Grosso, Midwest Brazil. Methods: This is a cross-sectional descriptive study. The sample consisted of 19 PKU patients diagnosed by newborn screening. Molecular analysis: DNA extraction using the "salting-out" method. Detection of IVS10nt-11G>A, V388M, R261Q, R261X, R252W, and R408W mutations by the restriction fragment length polymorphism (RFLP) technique. Results: Two mutant alleles were identified in four patients (21.1%), one allele in five patients (26.2%), and none in the remaining ten patients (52.6%). A total of 13/38 alleles were detected, corresponding to 34.2% of the PAH alleles present. The most prevalent variant was V388M (13.2% of the alleles), followed by R261Q (10.1%) and IVS10nt-11G>A (7.9%). Three variants (R261X, R252W, and R408W) were not found. The most frequent mutation types were: missense mutation in eight alleles (18.4%) and splicing in four alleles (10.5%). The model proposed by Guldberg to determine a genotype/phenotype correlation was applied to four classical PKU patients with two identified mutations. In three of them, the predicted moderate/moderate or moderate PKU phenotype did not coincide with the actual diagnosis. The prediction coincided with the diagnosis of one classic PKU patient. The estimated incidence of PKU for Mato Grosso, Brazil, was 1:33,342 live births from 2003 to 2015. Conclusion: The only mutations found in the analyzed samples were the IVS10nt-11G>A, V388M, and R261Q. The genotype/phenotype correlation only occurred in four (5.3%) patients.
RESUMO Objetivo: Identificar mutações da fenilalanina hidroxilase (PAH) em pacientes com PKU (fenilcetonúria) do Serviço de Triagem Neonatal em Mato Grosso. Métodos: Estudo de corte transversal. Amostra composta de 19 pacientes com PKU através do exame de triagem neonatal biológica. Análise molecular: a) extração de DNA pela metodologia "salting out". B) detecção de mutações IVS10nt-11G>A, V388M, R261Q, R261X, R252W e R408W pela técnica de polimorfismo de comprimento de fragmento de restrição (RFLP). Resultados: Dois alelos foram identificados em quatro pacientes (21,1%), um alelo em cinco pacientes (26,2%) e nenhum nos dez pacientes restantes (52,6%). Um total de 13/38 alelos foram identificados, correspondendo a 34,2% dos alelos PAH presentes. A variante mais prevalente foi a V388M (13,2% dos alelos), seguida de R261Q (10,1%) e IVS10nt-11G>A (7,9%). Três variantes (R261X, R252W e R408W) não foram encontradas. Os tipos de mutações mais frequentes foram: troca de sentido em oito alelos (18,4%) e emenda em quatro alelos (10,5%). O modelo proposto por Guldberg para determinar uma correlação genótipo/fenótipo foi aplicado para quatro pacientes clássicos de PKU, com duas mutações identificadas. Em três, o fenótipo previsto de PKU moderada/moderada ou moderada não coincidiu com o diagnóstico real. A predição coincidiu com o diagnóstico de um paciente PKU clássico. A incidência de PKU estimada para Mato Grosso, Brasil foi de 1:33.342 nascidos vivos para o período de 2003 a 2015. Conclusões: Foram encontradas apenas as mutações IVS10nt-11G>A, V388M, R261Q nas amostras analisadas. A correlação genótipo/fenótipo ocorreu em quatro (5,3%) pacientes.