A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits.

Brown rot, caused by different Monilinia species, is a most economically important disease of pome and stone fruits worldwide. In Europe and in Italy, the quarantine pathogen M. fructicola was recently introduced and rapidly spread and, by competing with the main indigenous species Monilinia fructigena and Monilinia laxa, caused relevant changes in Monilinia populations. As a result, in most areas, the pathogen almost replaced M. fructigena and now coexists with M. laxa. The availability of specific and easy-of-use quantification methods is essential to study the population dynamics, and in this work, a new method for the simultaneous quantification of M. fructicola and M. laxa based on droplet digital PCR (ddPCR) technique was established. Under the optimized reaction conditions, consisting of 250/500 nM of primers/probe sets concentration, 58°C as annealing temperature and 50 PCR cycles, the duplex-ddPCR assay was 200-fold more sensitive than duplex-real-time quantitative PCR (qPCR) assay, quantifying < 1 copy µL-1 of target DNA in the PCR mixture. The results obtained with the validation assay performed on apricot and peach fruits, artificially inoculated with conidial suspensions containing different ratios of M. fructicola and M. laxa, showed a high correlation (R 2 = 0.98) between the relative quantity of DNA of the two species quantified by ddPCR and qPCR and a more accurate quantification by ddPCR compared to qPCR at higher concentrations of M. fructicola. The herein described method represents a useful tool for the early detection of Monilinia spp. on stone fruits and for the improving knowledge on the epidemiology of brow rot and interactions between the two prevalent Monilinia species.

Transcriptome sequence resource for the cucurbit powdery mildew pathogen Podosphaera xanthii.

Podosphaera xanthii is the main causal agent of cucurbit powdery mildew in Southern Italy. Illumina sequencing of mRNA from two P. xanthii isolates of opposite mating types (MAT1-1 and MAT1-2) and their sexual cross was used to obtain a detailed de novo Trinity-based assembly of the transcriptome of the fungus. Over 60 million of high-quality paired-end reads were obtained and assembled into 71,095 contigs corresponding to putative transcripts that were functionally annotated. More than 55% of the assembled transcripts (40,221 contigs) had a significant hit in BLASTx search and included sequences related to sexual compatibility and reproduction, as well as several classes of transposable elements and putative mycoviruses. The availability of these new transcriptomic data and investigations on potential source of genetic variation in P. xanthii will promote new insights on the pathogen and its interactions with host plants and associated microbiome.

Chemical Constituents with Leishmanicidal Activity from a Pink-Yellow Cultivar of Lantana camara var. aculeata (L.) Collected in Central Mexico.

Lantana camara (L.) is employed by several ethnical groups to treat numerous diseases. Although there are no ethnomedical reports on its use against leishmaniasis, organic extracts prepared from L. camara were shown to display leishmanicidal activity. In the present study, we carried out a bioassay-guided fractionation of the dichloromethane extract from Mexican L. camara in order to identify the compounds responsible for the leishmanicidal activity. Eighteen chromatographic fractions (FI⁻FXVIII) were evaluated in vitro against Leishmania mexicana and L. amazonensis. FII, FX, FXI, FXV, and FXVI showed significant activity against both Leishmania strains, the most potent of which was FXV. Eicosane (1), squalene (2), ß-ionone (3), caryophyllene oxide (4), ß-caryophyllene (5), hexanoic acid (6), tiglic acid (7), a mixture of lantanilic (8) and camaric (9) acids, and lantadene B (10) were identified and obtained from the active fractions and evaluated for their leishmanicidal activity. The mixture of lantanilic (8) and camaric (9) acids (79%/21%) was the most potent one (half maximal inhibitory concentration (IC50) = 12.02 ± 0.36 µM). This study indicates that this cultivar of L. camara has high potential for the development of phytomedicines or as a source of natural products, which might represent lead compounds for the design of new drugs against leishmaniasis.

Diagnostic Accuracy of Pleural Fluid Adenosine Deaminase for Diagnosing Tuberculosis. Meta-analysis of Spanish Studies. / Eficacia diagnóstica de la adenosina desaminasa en líquido pleural para diagnosticar tuberculosis. Metaanálisis de estudios españoles.

OBJECTIVE: To evaluate the usefulness of pleural fluid adenosine deaminase (ADA) for diagnosing tuberculous pleural effusions in the Spanish population, according to laboratory technique and cut-off point, and to compare the results with other populations. METHODS: Meta-analysis of diagnostic studies on pleural fluid ADA in the Spanish population, extracted from the PubMed and Embase databases from inception until July 2017, with no language restrictions. The overall diagnostic accuracy of ADA and that of each of the measurement techniques (Giusti, manual and automated kinetic methods) and selected cut-offs were analyzed. The QUADAS-2 tool was used to evaluate the quality of studies. A bivariate random effects model was used. Results were compared with those obtained from previous meta-analyses in non-Spanish populations. RESULTS: Sixteen studies in a total of 4,147 patients, 1,172 of whom had tuberculous pleural effusions, were included. ADA had 93% sensitivity, 92% specificity, positive likelihood ratio of 12, negative likelihood ratio of 0.08, and an area-under-the-curve of 0.968 for identifying tuberculosis. There were no differences in diagnostic accuracy between the techniques used for ADA measurement or the selected cut-offs. In 73 studies from non-Spanish populations a trend toward lower ADA sensitivity (88%, 95% CI:86%-90%) and specificity (88%, 95% CI: 86%-90%) was noted, but differences did not reach statistical significance. CONCLUSIONS: Pleural fluid ADA in the Spanish population shows good diagnostic accuracy (regardless of the measurement technique or cut-off), similar to that reported in non-Spanish populations.

Eficacia diagnóstica de la adenosina desaminasa en líquido pleural para diagnosticar tuberculosis. Metaanálisis de estudios españoles / Diagnostic Accuracy of Pleural Fluid Adenosine Deaminase for Diagnosing Tuberculosis. Meta-analysis of Spanish Studies

Objetivo: Determinar la utilidad de la adenosina desaminasa (ADA) pleural para diagnosticar derrame pleural tuberculoso en población española, según la técnica de medición y punto de corte utilizados, y compararla con la descrita para otras poblaciones. Métodos: Metaanálisis de estudios diagnósticos sobre ADA pleural en población española, extraídos de PubMed y Embase desde sus comienzos hasta julio de 2017, sin restricciones de lenguaje. Se analizó la eficacia diagnóstica global de la ADA, según sus técnicas de medición (Giusti, métodos cinéticos manuales y métodos cinéticos automatizados) y el punto de corte seleccionado. La herramienta QUADAS-2 evaluó la calidad de los estudios. Se utilizó un método bivariante de efectos aleatorios. Se compararon los resultados con los descritos en metaanálisis previos sobre población no española. Resultados: Se incluyeron 16 estudios, con 4.147 pacientes, de los que 1.172 tenían derrame pleural tuberculoso. La ADA tuvo una sensibilidad del 93%, especificidad del 92%, likelihood ratio positiva de 12, likelihood ratio negativa de 0,08, y área bajo la curva de 0,968 para identificar tuberculosis. No hubo diferencias de eficacia diagnóstica entre las técnicas de medición de ADA o el punto de corte escogido. En 73 estudios de población no española se observó una tendencia hacia una menor sensibilidad (88%, IC95%: 86-90%) y especificidad (88%, IC95% 86-90%) de la ADA, pero las diferencias no alcanzaron significación estadística. Conclusiones: La ADA pleural en población española tiene una buena precisión diagnóstica (independientemente de la técnica de medición o punto de corte empleados), similar a la reportada en población no española

Objective: To evaluate the usefulness of pleural fluid adenosine deaminase (ADA) for diagnosing tuberculous pleural effusions in the Spanish population, according to laboratory technique and cut-off point, and to compare the results with other populations. Methods: Meta-analysis of diagnostic studies on pleural fluid ADA in the Spanish population, extracted from the PubMed and Embase databases from inception until July 2017, with no language restrictions. The overall diagnostic accuracy of ADA and that of each of the measurement techniques (Giusti, manual and automated kinetic methods) and selected cut-offs were analyzed. The QUADAS-2 tool was used to evaluate the quality of studies. A bivariate random effects model was used. Results were compared with those obtained from previous meta-analyses in non-Spanish populations. Results: Sixteen studies in a total of 4,147 patients, 1,172 of whom had tuberculous pleural effusions, were included. ADA had 93% sensitivity, 92% specificity, positive likelihood ratio of 12, negative likelihood ratio of 0.08, and an area-under-the-curve of 0.968 for identifying tuberculosis. There were no differences in diagnostic accuracy between the techniques used for ADA measurement or the selected cut-offs. In 73 studies from non-Spanish populations a trend toward lower ADA sensitivity (88%, 95% CI:86%-90%) and specificity (88%, 95% CI: 86%-90%) was noted, but differences did not reach statistical significance. Conclusions: Pleural fluid ADA in the Spanish population shows good diagnostic accuracy (regardless of the measurement technique or cut-off), similar to that reported in non-Spanish populations

TTF-1 and napsin A on cell blocks and supernatants of pleural fluids for labeling malignant effusions.

In this retrospective study of 80 pleural effusions, the combination of thyroid transcription factor 1 (TTF-1) and napsin A immunostaining on fluid cell blocks was positive in 80% of lung adenocarcinomas. Although measuring TTF-1 pleural fluid concentrations was of no value, quantification of napsin A levels allowed the identification of one third of the double-negative stained lung adenocarcinomas, with an overall accuracy similar to classical tumour markers for malignant-benign discrimination (sensitivity 40%, specificity 100%).

Empiema por Actinomyces meyeri / Empyema by Actinomyces meyeri

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[Leg ulcer: conservative treatment]. / Úlcera flebostática: estudo prospectivo de 202 doentes.

During 16 years 202 patients with leg ulcer have been studied prospectively. Whenever possible, cure has been obtained in ambulatory, which was the main objective. Surgery was preferentially made after the cure of the ulcer. From the 202 patients, 166 have made a prolonged follow-up. Cure in ambulatory has been obtained in 91% of the patients. We stress the importance of the direct control of all the treatment by the responsible doctor.