Estudio comparativo de medición entre lecturas visuales y espectrofotométricas en pruebas de susceptibilidad in vitrode aislamientos de candida

Se realizó un estudio de susceptibilidad in vitrofrente a itraconazol, ketoconazol y clotrimazol de 144 cepas de Candida, conservadas y previamente identificadas, aisladas de la cavidad oral de pacientes infectados por elvirus de inmunodeficiencia humana (VIH) con cuadros clínicos de candidiasis orofaríngea (COF). El estudio se llevó a cabo mediante dos metodologías; la primera, utilizando los requerimientos del Clinical and Laboratory Standard Institute (CLSI), en el cual está establecida la lectura visual para determinar los patrones de susceptibilidad; y la segunda, mediante la propuesta de la European Committee on Antibiotic Susceptibility Testing, el cual tiene fijado la lectura espectrofotométrica para eliminar las posibles subjetividades de la metodología del CLSI. Los resultados obtenidos mediante ambas lecturas no mostraron diferencias mayores a dos diluciones en los valores de concentración mínima inhibitoria, y demostraron que ambos métodos se correlacionan y es importante para aquellos laboratorios de pocos recursos económicos.Palabras clave: Susceptibilidad in vitr,; Candida, lectura visual, CLSI, lectura espectrofotométrica, EUCAST azoles, VIH/sida.</p

Molecular Characterization and Risk Factors of Giardia duodenalis among School Children from La Habana, Cuba

Giardia duodenalis is considered the most common protozoan infecting humans worldwide. Molecular characterization of G. duodenalis isolates has revealed the existence of eight groups (assemblages A to H) which differ in their host distribution. A cross-sectional study was conducted in 639 children from La Habana between January and December 2013. Two assemblage-specific PCRs were carried out for the molecular characterization. The overall prevalence of Giardia infection was 11.9%. DNA from 63 of 76 (82.9%) samples was successfully amplified by PCR-tpi, while 58 from 76 (76.3%) were detected by PCRE1-HF. Similar results by both PCRs were obtained in 54 from 76 samples (71%). According to these analyses, assemblage B and mixed assemblages A + B account for most of the Giardia infections in the cohort of children tested. Our current study identified assemblage B as predominant genotype in children infected with Giardia. Univariate analysis indicated that omission of washing hands before eating and keeping dogs at home were significant risk factors for a Giardia infection. In the future, novel molecular tools for a better discrimination of assemblages at the subassemblages level are needed to verify possible correlations between Giardia genotypes and symptomatology of giardiasis

Aspectos epidemiológicos y relación de hallazgos moleculares con características clínicas en la giardiosis pediátrica. La Habana y Fomento, 2010-2013

Giardia lamblia representa uno de los par&amp;aacute;sitos intestinales m&amp;aacute;s frecuentes en nuestro pa&amp;iacute;s, asociado principalmente con infecciones en ni&amp;ntilde;os. Se realiz&amp;oacute; un estudio de corte transversal en el periodo 2010-2013, con el objetivo de describir la infecci&amp;oacute;n de G. lamblia, en ni&amp;ntilde;os de La Habana y del municipio Fomento, y relacionar los resultados de la caracterizaci&amp;oacute;n molecular de este protozoo intestinal mediante dos marcadores gen&amp;eacute;ticos con variables cl&amp;iacute;nicas recogidas en encuestas epidemiol&amp;oacute;gicas. La infecci&amp;oacute;n por G. lamblia fue muyfrecuente en los ni&amp;ntilde;os estudiados, con un riesgo incrementado en aquellos que tomaban agua no hervida y con tenencia de perros en el hogar. Los marcadores gen&amp;eacute;ticos triosafosfato isomerasa (tpi) y las secuencias interg&amp;eacute;nicas del ADNr (IGS-ADNr) mostraron resultados id&amp;eacute;nticos de concordancia en la clasificaci&amp;oacute;n de los ensambles gen&amp;eacute;ticos de G. lamblia y un mejor porcentaje de tipificaci&amp;oacute;n de las muestras de heces estudiadas. La identificaci&amp;oacute;n del ensamble B y de los sub ensambles AII y BIII de G. lamblia fue predominante en los ni&amp;ntilde;os infectados por este protozoo intestinal, en comparaci&amp;oacute;n con el ensamble A y mixto A+B y los sub ensambles AI y BIV. En los ni&amp;ntilde;os solo infectados con G. lamblia la infecci&amp;oacute;n por el ensamble gen&amp;eacute;tico B de G. lamblia estuvo asociado significativamente con diarrea o dolor abdominal o flatulencia, especialmente en ni&amp;ntilde;os menores de 5 a&amp;ntilde;os de edad

Aspectos epidemiológicos y relación de hallazgos moleculares con características clínicas en la giardiosis pediátrica. La Habana y Fomento, 2010-2013

Giardia lamblia representa uno de los par&amp;aacute;sitos intestinales m&amp;aacute;s frecuentes en nuestro pa&amp;iacute;s, asociado principalmente con infecciones en ni&amp;ntilde;os. Se realiz&amp;oacute; un estudio de corte transversal en el periodo 2010-2013, con el objetivo de describir la infecci&amp;oacute;n de G. lamblia, en ni&amp;ntilde;os de La Habana y del municipio Fomento, y relacionar los resultados de la caracterizaci&amp;oacute;n molecular de este protozoo intestinal mediante dos marcadores gen&amp;eacute;ticos con variables cl&amp;iacute;nicas recogidas en encuestas epidemiol&amp;oacute;gicas. La infecci&amp;oacute;n por G. lamblia fue muyfrecuente en los ni&amp;ntilde;os estudiados, con un riesgo incrementado en aquellos que tomaban agua no hervida y con tenencia de perros en el hogar. Los marcadores gen&amp;eacute;ticos triosafosfato isomerasa (tpi) y las secuencias interg&amp;eacute;nicas del ADNr (IGS-ADNr) mostraron resultados id&amp;eacute;nticos de concordancia en la clasificaci&amp;oacute;n de los ensambles gen&amp;eacute;ticos de G. lamblia y un mejor porcentaje de tipificaci&amp;oacute;n de las muestras de heces estudiadas. La identificaci&amp;oacute;n del ensamble B y de los sub ensambles AII y BIII de G. lamblia fue predominante en los ni&amp;ntilde;os infectados por este protozoo intestinal, en comparaci&amp;oacute;n con el ensamble A y mixto A+B y los sub ensambles AI y BIV. En los ni&amp;ntilde;os solo infectados con G. lamblia la infecci&amp;oacute;n por el ensamble gen&amp;eacute;tico B de G. lamblia estuvo asociado significativamente con diarrea o dolor abdominal o flatulencia, especialmente en ni&amp;ntilde;os menores de 5 a&amp;ntilde;os de edad

Prevalence of intestinal parasites and molecular characterization of Giardia duodenalis from dogs in La Habana, Cuba

Giardia duodenalis is a worldwide protozoan parasite that infects humans and other mammals including dogs. Due to the risk of zoonotic transmission between dogs and humans, we aimed in this study to determine the prevalence of the intestinal parasites and the distribution of assemblages of G. duodenalis among dogs analysed. A descriptive cross-sectional study was carried out in La Habana from June 2014 to March 2015 in the Zoonosis Unit of La Lisa municipality. A total of 98 dogs were analysed by three different techniques (microscopy with faecal concentration, Enzyme-Linked ImmunoSorbent Assay, and Polymerase Chain Reaction) in order to detect Giardia in stool samples. Out of 98 dogs studied, 43 (43.9%) were infected with intestinal parasites. The zoonotic parasites Ancylostoma caninum (21.4%), Trichuris vulpis (16.3%) and the protozoan Giardia duodenalis (11.2%) were the most prevalent parasites. In regards to the G. duodenalis, seven dogs were positive by microscopy after faecal concentration, nine by NOVITEC® Giardia Microplate Assay, and ten and eleven samples were amplified by the β-giardin and SSU-RNA PCRs, respectively. After PCR sequence analysis of both genes only zoonotic assemblages (A and B) were detected. The SSU-RNA sequence results revealed a distribution of 8 assemblage A and 4 assemblage B, whereas only assemblage A were identified by the β-giardin analysis. Among subassemblage classification by β-giardin phylogenetic tree, four isolates showed an AI pattern and one isolate displayed an AII distribution. Mixed infections were detected in three isolates. These findings highlight the risk of zoonotic transmission of Giardia duodenalis between dogs and humans

Diagnosis of intestinal protozoan infections in patients in Cuba by microscopy and molecular methods: advantages and disadvantages

publishedVersio

Concordance of Giardia duodenalis assemblages determined by different PCR methodologies in three observational studies in Cuba

Giardia duodenalis is one of the most important intestinal parasites globally, especially in children, and in Cuba is the leading cause of chronic paediatric diarrhoea in this population. G. duodenalis is composed of eight genetic groups (or assemblages), two of which (A and B) are apparently zoonotic, occurring in both humans and other animals. However, consensus on the most appropriate genotyping scheme for optimal characterization of G. duodenalis isolates is lacking. In this article we present the results of three descriptive observational studies conducted in Havana, Cuba between 2010 and 2013, with the aim of comparing the results from molecular (PCR) approaches targeting different genes in order to assign with confidence 224 isolates of G. duodenalis to the correct assemblages. In each sub-study, following DNA isolation by the phenol/chloroform/isoamyl alcohol extraction method, PCR targeting the triose phosphate isomerase (tpi) gene was used for molecular characterization, as well as one additional PCR-method targeting another gene or pair of genes. DNA amplification was obtained in 87%, 83%, and 80% in the three sub-studies. Although excellent agreement (kappa index = 1) was recorded between results from some pairs of genes, for other combinations only moderate or substantial agreement was achieved. These results highlight the importance of interpretation of genotyping data, especially when single genetic markers are used. From the results of our studies, PCR targeting a combination of the tpi gene and the intergenic spacer region of rDNA may be a useful approach for the molecular characterization of G. duodenalis isolates

Estudio comparativo de medición entre lecturas visuales y espectrofotométricas en pruebas de susceptibilidad in vitro de aislamientos de candida Comparative study of measure visual and spectrophotometer lectures in a in vitro antifungal susceptibility testing in candida isolates

Se realizó un estudio de susceptibilidad in vitro frente a itraconazol, ketoconazol y clotrimazol de 144 cepas de Candida, conservadas y previamente identificadas, aisladas de la cavidad oral de pacientes infectados por el virus de inmunodeficiencia humana (VIH) con cuadros clínicos de candidiasis orofaríngea (COF). El estudio se llevó a cabo mediante dos metodologías; la primera, utilizando los requerimientos del Clinical and Laboratory Standard Institute (CLSI), en el cual está establecida la lectura visual para determinar los patrones de susceptibilidad; y la segunda, mediante la propuesta de la European Committee on Antibiotic Susceptibility Testing, el cual tiene fijado la lectura espectrofotométrica para eliminar las posibles subjetividades de la metodología del CLSI. Los resultados obtenidos mediante ambas lecturas no mostraron diferencias mayores a dos diluciones en los valores de concentración mínima inhibitoria, y demostraron que ambos métodos se correlacionan y es importante para aquellos laboratorios de pocos recursos económicos.<br>A study of in vitro susceptibility was realized opposite to itraconazol, ketoconazol and clotrimazol of 144 strains of Candida, conserved and previously identified, isolated of the oral cavity of patients infected by the virus of human immunodeficiency (VIH) with clinical pictures of oropharingeal candidiasis (COF). The study was realized by means of two methodologies; the first one, using the requests of the Clinical and Laboratory Standard Institute (CLSI), in which the visual reading is established to determine the patterns of susceptibility; and the second one, by means of the proposal of the European Committee on Antibiotic Susceptibility Testing, which has fixed the spectrophotometric to eliminate the possible subjectivities of the methodology of the CLSI. The results obtained by means of both readings did not show differences bigger than two dilutions in the values of minimal inhibitory concentration, demonstrating that both methods are correlated and it is important for those laboratories of few economic resources

A dual PCR-based sequencing approach for the identification and discrimination of Echinococcus and Taenia taxa

Reliable and rapid molecular tools for the genetic identification and differentiation of Echinococcus species and/or genotypes are crucial for studying spatial and temporal transmission dynamics. Here, we describe a novel dual PCR targeting regions in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rRNA) genes, which enables (i) the specific identification of species and genotypes of Echinococcus (rrnS + L-PCR) and/or (ii) the identification of a range of taeniid cestodes, including different species of Echinococcus, Taenia and some others (17 species of diphyllidean helminths). This dual PCR approach was highly sensitive, with an analytical detection limit of 1 pg for genomic DNA of Echinococcus. Using concatenated sequence data derived from the two gene markers (1225 bp), we identified five unique and geographically informative single nucleotide polymorphisms (SNPs) that allowed genotypes (G1 and G3) of Echinococcus granulosus sensu stricto to be distinguished, and 25 SNPs that allowed differentiation within Echinococcus canadensis (G6/7/8/10). In conclusion, we propose that this dual PCR-based sequencing approach can be used for molecular epidemiological studies of Echinococcus and other taeniid cestodes