Identificación y selección de Saccharomyces cerevisiae nativas para mejorar el proceso productivo del Pisco a partir de uva Quebranta

El Pisco es el destilado del Perú, elaborado a partir de mostos recientemente fermentados con uvas criollas denominadas "pisqueras". Las levaduras son los microorganismos clave en la fermentación y el uso de cepas nativas seleccionadas presenta ventajas competitivas para la tipicidad del producto, así como también para la estandarización del producto y el control microbiológico del proceso. El objetivo fue identificar y seleccionar las cepas de levaduras nativas para la producción del Pisco de uva Quebranta aisladas de procesos productivos de Pisco en el valle de Ica. Para ello, se emplearon técnicas microbiológicas y moleculares mediante el análisis de ITS1-5.8S-ITS2 PCR-RFLP para la identidad taxonómica. La evaluación de las cepas para producir Pisco consistió en el análisis fisicoquímico y organoléptico del destilado obtenido con las cepas seleccionadas. Se evaluaron 3 aislados para la producción de Pisco identificados como Saccharomyces cerevisiae: UNA SC - 25, UNA SC - 49 y UNA SC - 54, de los cuales la cepa UNA SC-49 destacó por mostrar aptitudes enológicas diferentes a las otras cepas. Este trabajo constituye el primer registro de levaduras nativas del Perú para mostos procedentes de uva Quebranta.

El Pisco stands as Peru's distilled spirit, crafted from recently fermented musts derived from native grape varieties known as "pisqueras". Yeasts serve as decisive microorganisms in the fermentation process, and the utilization of selected native strains confers competitive advantages for product typicity, standardization, and microbiological control within the production process. The aim was to identify and select native yeast strains for Quebranta grape Pisco production, isolated from Pisco production processes in the Ica Valley. Microbiological and molecular techniques were employed, utilizing ITS1-5.8S-ITS2 PCR-RFLP analysis for taxonomic identification. The assessment of yeast strains for Pisco production involved the physicochemical and organoleptic analysis of the distilled product obtained using the selected strains. Three isolates were evaluated for Pisco production, identified as Saccharomyces cerevisiae: UNA SC - 25, UNA SC - 49, and UNA SC - 54. Among these, the UNA SC-49 strain stood out due to displaying oenological characteristics distinct from the other strains. This work is the first documentation of native yeasts in Peru for musts derived from Quebranta grapes.

The Impact of Human Genetic Factors (G6pd and Type of Hemoglobin) on the Course of Uncomplicated Malaria Infection in Children Aged from 2 to 10 Years Living in the Banfora Health District in Burkina Faso

Aims: The aim of this study was to assess the impact of hemoglobin polymorphisms and G6PD deficiency on the course of uncomplicated malaria infection in children aged from 2 to 10 years in Burkina Faso.Study Design: The study was conducted as a longitudinal study in Banfora health district. A total of 150 children aged from 2 to 10 years was enrolled and followed up between May 2015 and February 2016. Blood samples were collected at four different time points: before infection (Visit 1), during asymptomatic parasitemia (Visit 2), during symptomatic parasitemia (Visit 3) and three weeks after treatment (Visit 4). Clinical examination, hematology parameters and malaria diagnosis using microscopy were performed. Hemoglobin and G6PD typing were done using PCR-RFLP. Hemoglobin AA genotypes were defined as normal hemoglobin while Hemoglobin AC, AS and SS were defined as abnormal hemoglobin (hb non-AA).Results:The prevalence of hemoglobin (hb) genotypes was 81.21% for AA while hb non-AA genotypes were estimated at 18.79% (12.08% for hbAC, 6.04% for hbAS and 0.67% for HbSC). The prevalence of G6PD genotypes was 89.26% and 10.74% for normal G6PDn and G6PD deficiency respectively. The prevalence of asymptomatic carriers of P. falciparumwas not affected neither by the genotypes of Hemoglobin, nor by the G6PD deficiency. Conversely, the risks of developing uncomplicated malaria in G6PD deficiency (G202A) group, was significantly lower (p=0.04).The results showeda significant difference (p˂0.0001) in the means of P. falciparumparasite densities between asymptomatic and symptomatic phase in Hemoglobin AA genotypes carriers while the means of parasite density was comparable in non-Hemoglobin AA carriers. Conclusion:Our study showed that G6PD deficiency protects against clinical malaria while P. falciparumparasite density increasing was correlated with carrying hemoglobin genotypes AA.

GENOTYPE CHARACTERIZATION OF Leishmania (Viannia) braziliensis ISOLATED FROM HUMAN AND CANINE BIOPSIES WITH AMERICAN CUTANEOUS LEISHMANIASIS / Caracterização genotípica de isolados de Leishmania (Viannia) braziliensis provenientes de biopsias de humanos e cães com leishmaniose tegumentar americana

Introduction: American tegumentary leishmaniasis (ATL) can be caused by Leishmania (Viannia) braziliensis complex. The evolution of ATL initially results in lesions and can develop into disseminated or diffuse forms. The genetic diversity of L. (V.) braziliensis in some endemic areas of Brazil has been poorly studied, such as in the state of São Paulo. This study analyzed the genetic diversity of L. (V.) braziliensis isolates collected from patients and dogs with LTA from the state of São Paulo. Methods: Leishmaniasis diagnosis was determined by PCR. The 132 biopsies were collected in different regions of Sao Paulo State, Brazil (36 municipalities). The genetic characterization of L. (V.) braziliensis isolates was tested by RFLP-PCR using DNA extracted from biopsies. The primer set amplified a specific region of Leishmania internal transcribed spacers of the ribosomal DNA locus. Results: Of the 132 samples, 52 (40%) were completely genotyped by RFLP-PCR (44 from human patients and eight from dogs). The results showed nine distinct patterns. The majority of the genotyped samples were from Sorocaba (30), and the others were distributed among 14 other municipalities. The first pattern was more frequent (29 samples), followed by pattern 2 (nine samples) and pattern 3 (three samples). Patterns 4, 6, 7, 8 and 9 were composed of two samples each and pattern 5 of one sample. Conclusion: These results suggest that polymorphic strains of L. (V.) braziliensis circulate in the state of São Paulo. These data agree with studies from other regions of Brazil, showing great variability among the natural populations of endemic foci. .

Introdução: A leishmaniose tegumentar americana (LTA) é causada pelo sub-gênero Leishmania (Viannia) braziliensis. A evolução da LTA resulta com a evolução das lesões iniciais. A diversidade genética de L. (V.) braziliensis em algumas áreas endêmicas brasileiras, como no estado de São Paulo, é pouco conhecida. Assim, este estudo teve como objetivo analisar a variabilidade genética de isolados de L. (V.) braziliensis coletados de biopsias de pacientes e cães com LTA no estado de São Paulo. Métodos: O diagnóstico da leishmaniose foi realizado por PCR. As 132 biópsias analisadas foram coletadas em diferentes regiões do Estado de São Paulo, Brasil (36 municípios). A caracterização genética de L. (V.) braziliensis foi realizada por RFLP-PCR utilizando DNA extraído das biopsias. O conjunto de iniciadores utilizado amplificou a região ITS de Leishmania. Resultados: Das 132 amostras analisadas, 52 (40%) foram completamente genotipadas por RFLP-PCR (44 de pacientes e oito de cães). Os resultados mostraram nove padrões distintos. A maioria das amostras genotipadas foi de Sorocaba (30), e as demais foram distribuídas entre 14 outros municípios. O primeiro padrão foi mais frequente (29 amostras), seguido pelo padrão 2 (nove amostras), padrão 3 (três amostras). Padrões 4, 6, 7, 8 e 9 foram compostos de duas amostras de cada um e o padrão 5, com uma amostra. Conclusão: Estes resultados sugerem que cepas polimórficas de L. (V.) braziliensis circulam no estado de São Paulo. Estes dados são concordantes com estudos em outras regiões do Brasil, mostrando grande variabilidade destas populações naturais de focos endêmicos. .

Detection and molecular analysis of Toxoplasma gondii and Neospora caninum from dogs with neurological disorders / Detecção e análise molecular de Toxoplasma gondii e Neospora caninum em cães com distúrbios neurológicos

INTRODUCTION: Toxoplasma gondii and Neospora caninum are related Apicomplexa parasites responsible for systemic diseases in many species of animals, including dogs. METHODS: This study aimed to determine the occurrence of T. gondii and N. caninum infections in 50 dogs with neurological signs that were admitted to the Veterinary Hospital of Universidade Estadual Paulista, City of Botucatu, Brazil. All animals were screened for antibodies using an immunofluorescent antibody test for both parasites. Tissues of positive animals were bioassayed in mice (T. gondii) and gerbils (N. caninum), and DNA was analyzed using the polymerase chain reaction (PCR). Positive samples for T. gondii by PCR were typed using restriction fragment length polymorphism-PCR for 11 markers: SAG1, SAG2 (5′-3′-SAG2 and alt.SAG2), SAG3, Btub, GRA6, L358, c22-8, c29-6, PK1 and Apico, and CS3 marker for virulence analysis. RESULTS: Specific antibodies were detected in 11/50 (22%; 95% confidence interval (CI95%), 12.8-35.3%) animals for T. gondii and 7/50 (14%; CI95%, 7.02-26.3%) for N. caninum. In the bioassay and PCR, 7/11 (63.6%; CI95%, 34.9-84.8%) samples were positive for T. gondii and 3/7 (42.9%; CI95%I, 15.7-75.5%) samples were positive for N. caninum. Three different genotypes were identified, but only 1 was unique. CONCLUSIONS: These data confirm the presence of T. gondii and N. caninum in dogs from Brazil, indicating the importance of this host as a sentinel of T. gondii for human beings, and the genotypic variation of this parasite in Brazil.

INTRODUÇÃO: Toxoplasma gondii e Neospora caninum são parasitas Apicomplexa responsáveis por doenças sistêmicas em muitas espécies de animais, incluindo cães, o que representa grande importância em animais de estimação. MÉTODOS: Este estudo teve como objetivo determinar a prevalência da infecção de T. gondii e N. caninum em 50 cães com sinais neurológicos internados no Hospital Veterinário da Universidade Estadual Paulista (UNESP) na Cidade de Botucatu, Brasil. Todos os animais foram examinados para detecção de anticorpos por IFAT para ambos os parasitas. Tecidos de animais positivos foram analisados por bioensaio em camundongos (T. gondii) e gerbilos (N. caninum) e o DNA foi pesquisado por PCR. Amostras positivas para T. gondii por PCR foram analisadas por meio de análise de restrição de fragmentos polimórficos (restriction fragment length polymorphism-polymerase chain reaction - RFLP-PCR), utilizando-se 11 marcadores: SAG1, SAG2 (5'-3'SAG2 e, alt.SAG2), SAG3, Btub, GRA6, L358, c22 -8, C29-6, PK1 e Apico e o marcador CS3 para análise de virulência. RESULTADOS: Os anticorpos específicos foram detectados em 11/50 animais (22%; IC95% 12,8-35,3%) para T. gondii, e 7/50 (14%; IC95% 7,0-26,3%) para N. caninum. No bioensaio e PCR, 7/11 (63,6%; IC95% 34,9-84,8%) das amostras foram positivas para T. gondii, e 3/7 (42,9%; IC95% 15,7-75,5%) para N. caninum. Três diferentes genótipos foram identificados. Apenas um foi único. CONCLUSÕES: Estes dados confirmam a presença de T. gondii e N. caninum em cães do Brasil, e demonstra a importância do T. gondii como sentinela para a infecção e a variação genotípica deste parasita no Brasil.

The genomic identification of Colombian Acinetobacter baumannii clinical isolates by RFLP-PCR analysis of the 16S-23S rRNA gene spacer region / Identificación genómica de aislamientos colombianos de Acinetobacter baumannii mediante RFLP-PCR de la región intergénica espaciadora de los genes 16S y 23S rRNA

The 16S-23S rRNA gene intergenic spacer (ITS) was analysed by RFLP in this study to identify A. baumannii from 139 isolates from four hospitals (identified as A, B, C and D). One hundred and twenty of these isolates (86.3%) belonged to the A. baumannii species; those identified as being A. baumannii were found to be polyclonal (19 clone groups) when determining the genetic relationships, 16 of them being found in hospital C. Hospitals A, B and D shared two clone groups isolated during different years. This study describes a rapid and easy method for genospecies identification of Acinetobacter baumannii.

Con el objeto de identificar la genomoespecie Acinetobacter baumannii, se estudiaron 189 aislamientos pertenecientes al Complejo Acinetobacter baumannii-Acinetobacter calcoaceticus provenientes de cuatro hospitales colombianos (denominados A,B,C,D) mediante el análisis por RFLP-PCR de la región intergénica espaciador (ITS) de los genes 16S y 23S rRNA. Se encontraron 120 aislamientos (86.3%) pertenecientes a la especie A. baumannii. La estructura de la población fue policlonal, con 19 grupos clonales, 16 de los cuales se hallaron en el hospital C. En los hospitales A,B y D se encontraron 2 grupos clonales aislados durante diferentes años. En este estudio se propone un método rápido y fácil para la identificación de Acinetobacter baumannii.

Infección por Trypanosoma cruzi y polimorfismo del Citocromo B del ADN mitocondrial en Triatoma maculata de Anzoategui y Portuguesa, Venezuela / Trypanosoma cruzi infection and cytochrome b polimorphism of mitochondrial dna in Triatoma maculata of Anzoátegui and Portuguesa states, Venezuela

Con el fin de entender la dinámica poblacional de Triatoma maculata, se analizó el polimorfismo genético y los índices de infección con Trypanosoma cruzi, utilizando triatominos provenientes de ecotopos y regiones geográficas diferentes. El índice de infección parasitaria para T. maculata, fue de 29.8% a través de la observación directa al microscopio y 40.3% utilizando el método de reacción en cadena de la polimerasa. Los niveles de infección encontrados incrementan la importancia de T. maculata como vector involucrado en el ciclo de transmisión de T. cruzi. El análisis del polimorfismo de longitud de fragmentos de restricción de una región del gen Cyt B, permitió establecer en forma preliminar, diferencias en los patrones de bandas de este gen, según el origen geográfico de cada población. Esto puede asociarse a cambios relacionados con procesos adaptativos involucrados en la colonización de nuevos hábitats. No se observó variación genética para vectores capturados en diferentes ecotopos de una misma localidad. Sin embargo es evidente la participación del vector en el ciclo de transmisión, mostrando que la presencia de T. maculata en las casas no puede ser ignorada

In order to understand more about the populational dynamics of Triatoma maculata, the genetic polimorphism and the infection indexes of Trypanosoma cruzi were analysed, using triatomine obtained from different ecotopes and geographical regions. The parasitic infection index of T. maculata was 29.8% using the microscope direct observation, and 40.3% by the polymerase chain reaction method. Both methods were important for epidemiological screening of the vectors with low potential of infection. The amplification of one region the Cyt B gene of these organisms, followed by a restriction fragments length polymorphism analysis, allowed us to establish different patterns of bands according to the geographic origin of each population, which indicates the lack of migration between individuals of Portuguesa and Anzoátegui states. These genetic differences may be associated with changes in adaptative events involved in the colonization of new habitats. The lack of polymorphism among vectors collected in different habitats of the same region showed an important genetic flow which has epidemiological implications in the risk of transmission of the disease, showing that the presence of T. maculata in houses cannot be ignored

Identification of Candida species using PCR-RFLP in cancer patients in Iran.

Opportunistic infections caused by Non-Candida albicans. have been increasing. Traditional methods that are used to identify clinical isolates of Candida species are time-consuming and not appropriate for rapid, accurate and reliable identification. Purpose: To identify Candida spp isolated from cancer patients using PCR-restriction enzyme. Materials and ethods: Using universal primers, ITS1 and ITS4, in this study, we could amplify ITS1-5.8S-ITS2 rDNA regions at both 80 clinical isolates and 3 standard strains. The PCR products were digested with two restriction enzymes MspI and BlnI separately. Result: We successfully identified all isolated species using two restriction enzymes (MspI, BlnI). Candida albicans was the most common species (77.5%), followed by C. glabrata (15%), C. tropicalis (5%), C. krusei (2.5%). Although the primers and enzyme had the ability to identify C. parapsilosis, C. guilliermondii, C. dubliniensis, present isolates did not include these among identified ones. Conclusion: RFLP-PCR using ITSI and ITS4 primers and restriction enzyme is a rapid, easy, reliable and also applicable method in clinical laboratory for identification of medically important Candida spp.

Comparación de las pruebas de dilución en Agar y PCR para determinación de susceptibilidad antimicrobiana de Helicobacter pylori. Revisión sistemática de la literatura / Comparation of Agar dilution test with Polymerase Chain Reaction (PCR) to determinate antimicrobial susceptiblility of Helicobacter Pylori. A systematic literature review

Objetivo: determinar las características operativas de la prueba RFLP-PCR frente a la prueba dilución en agar para evaluar la susceptibilidad antimicrobiana a claritromicina en aislamientos clínicos de H. pylori. Metodología: la búsqueda de estudios de pruebas diagnósticas sobre resistencia antimicrobiana de H. pylori a claritromicina con técnicas de dilución en agar y RFLP-PCR se realizó en Medline, Science direct, Ovid y Cochrane. Se elaboraron tablas de contingencia para calcular las características operativas, en el programa RevMan 5. La heterogeneidad fue evaluada por la gráfica Forest Plot y el estadístico de Q. La presencia de sesgos de publicación se evaluó con Funnel Plot. Resultados: doce artículos cumplieron con los criterios de inclusión. El overall de especificidad fue 100% (IC 95% 91-100), demostrando baja probabilidad de falsos positivos. Para sensibilidad el valor fue de 91% (IC 95% 88-94) indicando una mayor probabilidad de resultados falsamente negativos. En la gráfica de “Funnel Plot” se observó asimetría para ambas características demostrando sesgo de publicación. Conclusiones: la técnica RFLP-PCR no presentó características operativas iguales o superiores al 95%, comparada con el estándar de referencia dilución en agar. Por lo anterior esta técnica de se debe considerar la prueba de elección cuando se estudie la susceptibilidad antimicrobiana de H. pylori.

Aim: Establish the available scientific evidence of the operational characteristics of PCR-RFLP test with Agar Dilution for the determination of antimicrobial susceptibility in clarithromycin clinical isolates of Helicobacter pylori. Methods: We have performed the search bibliography about diagnostic tests on antimicrobial resistance of H. pylori to clarithromycin by using Agar Dilution and PCR-RFLP techniques over Medline, Science direct, Ovid and Cochrane of studies. The information was validated by two observers who checked the inclusion criteria and quality. We obtained the operational characteristics of the studies on contingency tables; analysis was performed on the RevMan 5 program. Results: A total of 50 references were tested from those 12 has been chosen in accord with the inclusion criteria and analyzed as summary measures. The specificity “overall” was a 100 % (CI 95% 91-100) that demonstrate a low probability of positive false. The projected overall sensitivity was 91% (CI 95% 88-94%) which indicated a high probability of negative results. The test showed heterogeneity studies homogeneous in sensitivity and specificity (p = 0.78) (p = 0.99). The graphics of “Funnel Plot” revealed asymmetry for both of those characteristics that showed a publications bias. In the group analysis have not found antibiotics different from clarithromycin and was evident that the continent was more publications Europe, followed by Asia and Latin America. Conclusion: The sensitivity and specificity of PCR-RFLP technique for clarithromycin not have values equal to or higher than 95% compared proof Agar Dilution.

Comparação genotípica de isolados de Corynebacterium pseudotuberculosis de caprinos e ovinos do sertão de Pernambuco / Genotypic comparison between Corynebacterium pseudotuberculosis samples obtained from sheep and goats with caseous lymphadenitis, raised in the semi-arid region of Pernambuco

Objetivou-se com este estudo comparar genotipicamente 35 isolados de Corynebacterium pseudotuberculosis recuperados de conteúdo de abscessos de caprinos e ovinos com linfadenite caseosa, procedentes de cinco municípios localizados no Sertão de Pernambuco, Brasil. Utilizou-se a técnica de fingerprint RFLP-PCR com as enzimas de restrição Hpy-Ch4 e Msp1 aplicada ao gene rpoB e as enzimas Pst I e Msp I para o gene pld. Não houve diferença nos padrões de fragmentos de bandas entre os isolados, independente da espécie hospedeira ou da área geográfica estudada, definindo-se um padrão genotípico homogêneo de C. pseudotuberculosis responsável por abscessos superficiais na região.(AU)

The objective was to genotypically compare 35 samples of Corynebacterium pseudotuberculosis obtained from abscesses of sheep and goats diagnosed with caseous lymphadenitis originated from 5 different municipalities in the semi-arid region of Pernambuco, Brazil. The RFLP-PCR technique with Hpy-Ch4 and Msp I and Pst I Msp I restriction enzimes was used to fingerprint the genes rpoB and pld, respectively. The results demonstrate that there was no difference on the fragments banding pattern among samples, independently of the host species or geographic area studied, defining a homogeneous profile of C. pseudotuberculosis responsible for superficial abscesses for the region.(AU)

Diagnosis of Pancreatic Cancer by Detecting K-ras Gene Mutations / 中国医师杂志

Objective To detect plasma K-ras gene mutation by using CED-RFLP/PCR to diagnose pancreatic cancer. Methods CED-RFLP/PCR technique was used to detect K-ras gene mutation in the plasma specimens of pancreatic cancer patients, patients with benign pancreatic diseases and healthy subjects. Results In pancreatic cancer patients the positive rate of plasma K-ras gene mutation was 73%, without false positivity, and higher than that in the pancreatic juice and duodenal juice, but lower than that in the fine-needle aspirates. Plasma K-ras gene mutation was not found in patients with benign pancreatic disease and healthy subjects. Conclusion The detection of plasma K-ras gene mutation by CED-RFLP/PCR is simple and effective, and could avoid the faults of other detection methods. It is helpful for the diagnosis and identification of pancreatic cancer.

The Carrier Detection and Genetic Counseling of Duchenne and Becker Muscular Dystrophy Using Linkage Analysis / 대한정형외과학회잡지

PURPOSE: Restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) is a widely accepted method for carrier detection of Duchenne and Becker muscular dystrophy (DMD and BMD) . This study was done to evaluate the clinical value of linkage analysis of RFLP-PCR using five polymorphic markers selected and the heterozygote frequency of those markers in DMD/BMD patients and their family members. MATERIALS AND METHODS: RFLP-PCR test was performed in twenty clinically diagnosed male DMD/BMD patients from 13 families who have been confirmed to have dystrophin gene defect from 1994 to 1997 and their 47 female family members and the results were evaluated by linkage analysis to detect carriers. RESULTS: The heterozygote frequency of pERT 87-15/XmnI, pERT87-15/BamHI, pERT87-8/TaqI, 5'-dysIII (CA) and 3'-dys (CA) markers were 55%, 49%, 45%, 32% and 26% respectively. Fourty-four (91%) out of 47 female family members had heterozygosity to at least one of those five markers. Since the obligate carriers from two families showed homozygocity to all five markers, carrier detection was possible in eleven families (85%) by the linkage analysis. CONCLUSION: RFLP-PCR using markers with high heterozygote frequency could be the first line modality of carrier detection that is crucial in genetic counseling of DMD/BMD patients and their families.