A simplified molecular tool for detecting the Chagas etiological agent using a vector feces sample in field conditions.

Triatomine bugs are vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease in the American continent. Here, we have tested a loop-mediated isothermal amplification (LAMP) test for a direct detection of T. cruzi in feces of Triatoma infestans, the main vector of this parasite in the Southern Cone of America. The analytical evaluation showed positive results with samples of triatomine feces artificially inoculated with DNA from strains of T. cruzi corresponding to each Discrete Typing Units (I-VI), with a sensitivity of up to one parasite per reaction. Conversely, the reaction yielded negative results when tested with DNA from Trypanosoma rangeli and other phylogenetically related and unrelated organisms. In triatomines captured under real field conditions (from urban households), and defined as positive or negative for T. cruzi using the reference microscopy technique, the LAMP test achieved a concordance of 100 %. Our results demonstrate that this LAMP reaction exhibits excellent analytical specificity and sensitivity without interference from the fecal matrix, since all the reactions were conducted without purification steps. This simple molecular diagnostic technique can be easily used by vector control agencies under field conditions.

Establishing a Receptor Binding Assay for Ciguatoxins: Challenges, Assay Performance and Application.

Ciguatera, a global issue, lacks adequate capacity for ciguatoxin analysis in most affected countries. The Caribbean region, known for its endemic ciguatera and being home to a majority of the global small island developing states, particularly needs established methods for ciguatoxin detection in seafood and the environment. The radioligand receptor binding assay (r-RBA) is among the in vitro bioassays currently used for ciguatoxin analysis; however, similarly to the other chemical-based or bioassays that have been developed, it faces challenges due to limited standards and interlaboratory comparisons. This work presents a single laboratory validation of an r-RBA developed in a Cuban laboratory while characterizing the performance of the liquid scintillation counter instrument as a key external parameter. The results obtained show the assay is precise, accurate and robust, confirming its potential as a routine screening method for the detection and quantification of ciguatoxins. The new method will aid in identifying high-risk ciguatoxic fish in Cuba and the Caribbean region, supporting monitoring and scientific management of ciguatera and the development of early warning systems to enhance food safety and food security, and promote fair trade fisheries.

SSVEP detection assessment by combining visual stimuli paradigms and no-training detection methods.

Introduction: Brain-Computer Interfaces (BCI) based on Steady-State Visually Evoked Potentials (SSVEP) have great potential for use in communication applications because of their relatively simple assembly and in some cases the possibility of bypassing the time-consuming training stage. However, among multiple factors, the efficient performance of this technology is highly dependent on the stimulation paradigm applied in combination with the SSVEP detection algorithm employed. This paper proposes the performance assessment of the classification of target events with respect to non-target events by applying four types of visual paradigms, rectangular modulated On-Off (OOR), sinusoidal modulated On-Off (OOS), rectangular modulated Checkerboard (CBR), and sinusoidal modulated Checkerboard (CBS), with three types of SSVEP detection methods, Canonical Correlation Analysis (CCA), Filter-Bank CCA (FBCCA), and Minimum Energy Combination (MEC). Methods: We set up an experimental protocol in which the four types of visual stimuli were presented randomly to twenty-seven participants and after acquiring their electroencephalographic responses to five stimulation frequencies (8.57, 10.909, 15, 20, and 24 Hz), the three detection methods were applied to the collected data. Results: The results are conclusive, obtaining the best performance with the combination of either OOR or OOS visual stimulus and the FBCCA as a detection method, however, this finding contrasts with the opinion of almost half of the participants in terms of visual comfort, where the 51.9% of the subjects felt more comfortable and focused with CBR or CBS stimulation. Discussion: Finally, the EEG recordings correspond to the SSVEP response of 27 subjects to four visual paradigms when selecting five items on a screen, which is useful in BCI navigation applications. The dataset is available to anyone interested in studying and evaluating signal processing and machine-learning algorithms for SSVEP-BCI systems.

Protocol optimization for the detection of Trypanosoma cruzi DNA in açai (Euterpe oleraceae) pulp / Otimização de protocolo para detecção de DNA de Trypanosoma cruzi em polpa de açaí (Euterpe oleracea)

Chagas disease, caused by the protozoan Trypanosoma cruzi, has often been linked to oral transmission through açai consumption. Molecular methods that allow fast and accurate identification of the pathogen are important for the detection of the presence of the parasite in this food. This study aimed to optimize polymerase chain reaction (PCR)-based detection of T. cruzi DNA in açai pulp. Several dilutions of T. cruzi DTU TcI trypomastigote forms were cultured in liver infusion tryptose (LIT) medium. Trypanosoma cruzi DNA was extracted from the cells and subjected to PCR. Subsequently, culture dilutions were added to açai pulp to evaluate the detection threshold of the optimized PCR assay. We demonstrate that our assay can detect T. cruzi DNA in açai pulp at a concentration of 1.08 × 10-10 ng µL-1. We conclude that our optimized protocol is effective and can be used as an important tool for the detection of T. cruzi contamination in açaí.(AU)

A doença de Chagas, enfermidade causada pelo protozoário Trypanosoma cruzi, tem sido relacionada com frequência à transmissão oral pelo consumo de açaí. Métodos moleculares que fornecem uma identificação rápida e precisa do patógeno para a detecção da presença do parasita são de extrema importância para a detecção da presença do parasita neste alimento. Este estudo teve como objetivo otimizar a detecção de DNA de T. cruzi em polpa de açaí por meio da reação em cadeia da polimerase (PCR). Foram preparadas várias diluições das formas tripomastigotas de T. cruzi DTU TcI cultivadas em meio de cultivo Liver Infusion Tryptose. O DNA de T. cruzi foi extraído das células e submetido à PCR. Posteriormente, as diluições da cultura foram adicionadas às polpas de açaí para avaliar o limite de detecção do novo ensaio de PCR otimizado. Mostramos que nosso ensaio pode detectar DNA de T. cruzi em polpas de açaí na concentração de 1.08 × 10-10 ng µL-1. Concluímos que a metodologia desenvolvida se mostra eficaz e pode ser uma ferramenta importante para a detecção de contaminação por T. cruzi em açaí.(AU)

Analysis of Triphenylmethane Dye Residues and their Leuco-Forms in Frozen Fish by LC-MS/MS, Fish Microbial Quality, and Effect of Immersion in Whole Milk on Dye Removal.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated and used to quantify crystal violet (CV), leucocrystal violet (LCV), malachite green (MG), leucomalachite green (LMG), and brilliant green (BG) residues in frozen fish (121 samples) from various countries, in order to detect the use of prohibited antibiotic dyes in fish for human consumption. The microbial quality of the fish was also assessed along with the effectiveness of a simple treatment with whole fat milk to reduce the levels of CV and LCV contamination. CV and LCV were the only two residues detected. They were found in farmed Pangasius (0.362 to 41.34 µg/kg and 0.178 to 10.58 µg/kg, respectively) and Tilapia (1.24 to 9.48 µg/kg and 1.29 to 2.81 µg/kg). Based on aerobic plate count (APC), 74%, 59%, and 55% of the samples of Tilapia fillets (from China) and Pangasius fillets (United Arab Emirates and Vietnam), and 100% and 50% of the skin samples of Hake (Argentina and U.S.A.) were of unacceptable microbial quality (APC > 107 cfu/g). Human pathogens, namely Escherichia coli, Staphylococcus aureus, and Vibrio spp., were detected in most fish. A significant reduction in CV and LCV concentrations by more than a third was achieved after immersing Pangasius and Tilapia fillets in whole fat milk for 120 minutes. These findings support the necessity of regular inspections and monitoring of CV and other antibiotic dye residues in fish, along with routine assessments of fish microbial quality, in order to protect public health. PRACTICAL APPLICATION: The described LC-MS/MS method can be used to rapidly and simultaneously quantify antibiotic dye residues in frozen fish. CV and LCV were detected in farmed Pangasius and Tilapia fillets and their concentrations was reduced by more than one third after immersing the fillets in whole milk for 120 min, a treatment which is not intended to replace safe fish farming practices upstream to artificially lower the level of banned dyes in fish. The findings support the necessity of regular inspections and monitoring of CV and other antibiotic dye residues in fish, along with assessments of fish microbial quality, to protect public health.

A radioligand receptor binding assay for ciguatoxin monitoring in environmental samples: Method development and determination of quality control criteria.

Ciguatoxins are algal toxins responsible for tens of thousands of human intoxications yearly, both in tropical and subtropical endemic regions as well as worldwide through fish exportation. Previously developed methods for biotoxin surveillance in the environment and seafood include analytical methods and in vivo and in vitro bioassays. The radioligand receptor binding assay (r-RBA) is among the in vitro methodologies currently used for the detection and quantification of marine biotoxins. For the ciguatoxin group, the r-RBA has been widely used as a means to characterize the mode of action and as detection method in various biological matrices. Yet, screening methods have not been standardized, and the details of the ciguatoxin-specific r-RBA are not well-documented, which limit interlaboratory comparison and progress toward method validation. This work presents the development of an optimized r-RBA for ciguatoxins and provides guidance on its use and quality control checks for analysis of environmental samples. We focus on the analysis of critical parameters involved in determining assay acceptability. Calculation of toxin concentrations in fish samples is illustrated with four examples. Thus, this paper provides the detailed information required for a full validation of the r-RBA, a necessary step toward the development and implementation of a regulatory monitoring programme for ciguatoxins in seafood products using the r-RBA.

Detection of human papillomavirus in laryngeal squamous cell carcinoma: Systematic review and meta-analysis.

BACKGROUND: Recent studies have reported a human papillomavirus (HPV) prevalence of 20% to 30% in laryngeal squamous cell carcinoma (LSCC), although clinical data on HPV involvement remain largely inconsistent, ascribed by some to differences in HPV detection methods or in geographic origin of the studies. OBJECTIVE: To perform a systematic review and formal meta-analysis of the literature reporting on HPV detection in LSCC. METHODS: Literature was searched from January 1964 until March 2015. The effect size was calculated as event rates (95% confidence interval [CI]), with homogeneity testing using Cochran's Q and I(2) statistics. Meta-regression was used to test the impact of study-level covariates (HPV detection method, geographic origin) on effect size. Potential publication bias was estimated using funnel plot symmetry. RESULTS: One hundred seventy nine studies were eligible, comprising a sample size of 7,347 LSCCs from different geographic regions. Altogether, 1,830 (25%) cases tested HPV-positive considering all methods, with effect size of 0.269 (95% CI: 0.242 to 0.297; random-effects model). In meta-analysis stratified by the 1) HPV detection technique and 2) geographic study origin, the between-study heterogeneity was significant only for geographic origin (P = .0001). In meta-regression, the HPV detection method (P = .876) or geographic origin (P = .234) were not significant study-level covariates. Some evidence for publication bias was found only for studies from North America and those using non-polymerase chain reaction methods, with a marginal effect on adjusted point estimates for both. CONCLUSIONS: Variability in HPV detection rates in LSCC is explained by geographic origin of study but not by HPV detection method. However, they were not significant study-level covariates in formal meta-regression. LEVEL OF EVIDENCE: NA.

Evaluation of dental enamel caries assessment using Quantitative Light Induced Fluorescence and Optical Coherence Tomography.

An in vitro study of morphological alterations between sound dental structure and artificially induced white spot lesions in human teeth, was performed through the loss of fluorescence by Quantitative Light-Induced Fluorescence (QLF) and the alterations of the light attenuation coefficient by Optical Coherence Tomography (OCT). To analyze the OCT images using a commercially available system, a special algorithm was applied, whereas the QLF images were analyzed using the software available in the commercial system employed. When analyzing the sound region against white spot lesions region by QLF, a reduction in the fluorescence intensity was observed, whilst an increase of light attenuation by the OCT system occurred. Comparison of the percentage of alteration between optical properties of sound and artificial enamel caries regions showed that OCT processed images through the attenuation of light enhanced the tooth optical alterations more than fluorescence detected by QLF System. QLF versus OCT imaging of enamel caries: a photonics assessment.

Neutral and adaptive genomic signatures of rapid poleward range expansion.

Many species are expanding their range polewards, and this has been associated with rapid phenotypic change. Yet, it is unclear to what extent this reflects rapid genetic adaptation or neutral processes associated with range expansion, or selection linked to the new thermal conditions encountered. To disentangle these alternatives, we studied the genomic signature of range expansion in the damselfly Coenagrion scitulum using 4950 newly developed genomic SNPs and linked this to the rapidly evolved phenotypic differences between core and (newly established) edge populations. Most edge populations were genetically clearly differentiated from the core populations and all were differentiated from each other indicating independent range expansion events. In addition, evidence for genetic drift in the edge populations, and strong evidence for adaptive genetic variation in association with the range expansion was detected. We identified one SNP under consistent selection in four of the five edge populations and showed that the allele increasing in frequency is associated with increased flight performance. This indicates collateral, non-neutral evolutionary changes in independent edge populations driven by the range expansion process. We also detected a genomic signature of adaptation to the newly encountered thermal regimes, reflecting a pattern of countergradient variation. The latter signature was identified at a single SNP as well as in a set of covarying SNPs using a polygenic multilocus approach to detect selection. Overall, this study highlights how a strategic geographic sampling design and the integration of genomic, phenotypic and environmental data can identify and disentangle the neutral and adaptive processes that are simultaneously operating during range expansions.

Solitary bronchial squamous cell papilloma - another human papillomavirus (HPV)-associated benign tumor: systematic review and meta-analysis.

AIM OF THE STUDY: To perform a systematic review and formal meta-analysis of the literature reporting on HPV detection in bronchial squamous cell papillomas (SCP). MATERIAL AND METHODS: The literature was searched up to June 2012. The effect size was calculated as event rate (95% CI), with homogeneity testing using Cochran's Q and I(2) statistics. Meta-regression was used to test the impact of study-level covariates (HPV detection method, geographic origin) on effect size, and potential publication bias was estimated using funnel plot symmetry. RESULTS: Fifteen studies were eligible, covering 89 bronchial SCPs from different geographic regions. Altogether, 38 (42.7%) cases tested HPV-positive; effect size 0.422 (95% CI: 0.311-0.542; fixed effects model), and 0.495 (95% CI: 0.316-0.675; random effects model). In meta-analysis stratified by i) HPV detection technique and ii) geographic study origin, the between-study heterogeneity was not significant for either; p = 0.348, and p = 0.792, respectively. In maximum likelihood meta-regression, HPV detection method (p = 0.150) and geographic origin of the study (p = 0.164) were not significant study-level covariates. Some evidence for publication bias was found only among in situ hybridization (ISH)-based studies and among studies from Europe, but with a negligible effect on summary effect size estimates. In sensitivity analysis, the meta-analytic results were robust to all one-by-one study removals. CONCLUSIONS: In formal meta-regression, the variability in HPV detection rates reported in bronchial SCPs is not explained by the HPV detection method or geographic origin of the study.

Detecção de Listeria monocytogenes pela técnica de PCR em leite contaminado artificialmente / Detection of Listeria monocytogenes by PCR in artificially contaminated milk samples

Avaliou-se a técnica de PCR como opção para reduzir o tempo de detecção de Listeria monocytogenes no leite. Para tanto, amostras de leite desnatado esterilizado e de leite cru integral - com baixa, média e alta contagem de microrganismos aeróbios mesófilos - foram inoculadas experimentalmente com diversas concentrações de L. monocytogenes. Os resultados da reação de PCR foram comparados com os da cultura da amostra empregando-se metodologia padronizada tradicional. Não se detectou L. monocytogenes pela reação de PCR quando esta foi realizada a partir do caldo de enriquecimento de Listeria (LEB) após 24 horas de incubação, nem no leite desnatado esterilizado, nem no leite cru integral. Após 48 horas de enriquecimento em LEB, a bactéria foi detectada por PCR nas amostras de leite desnatado esterilizado, com a sensibilidade de 1UFC/mL, mas não nas amostras de leite cru integral. Pela metodologia tradicional, a bactéria foi recuperada de todos os ensaios. Entretanto, nas amostras de leite cru com altas contagens de aeróbios mesófilos, a sensibilidade da metodologia tradicional foi reduzida (a partir de 7UFC/mL). Melhores resultados foram obtidos quando a reação de PCR foi feita utilizando-se DNA obtido diretamente da colônia suspeita em meio sólido (Oxford e Palcam). Foi possível substituir os testes fenotípicos de identificação de L. monocytogenes pela técnica de PCR reduzindo-se o tempo de identificação da bactéria de vários dias para algumas horas.

The polymerase chain reaction (PCR) was used to detect Listeria monocytogenes in inoculated milk samples after selective enrichment. Samples of sterile skim milk and raw whole milk (with low, intermediate, and high counts of aerobic mesophilic microorganisms) were inoculated with several concentrations of L. monocytogenes. The results of PCR assays were compared to the results of culturing the samples using a standardized traditional method for isolation of L. monocytogenes. The pathogen was detected by PCR in Listeria Enrichment Broth (LEB) after 48h-incubation (sensitivity of 1CFU/mL) but not after 24h-incubation from the samples prepared with sterile skim milk. L. monocytogenes was not detected by PCR in LEB after 24 and 48h-incubation from the samples prepared with raw whole milk. Using the traditional method, the pathogen was detected in all experiments. However, sensitivity decreased in raw whole milk with high counts of aerobic mesophilic microorganisms (up to 7CFU/mL). Best results were obtained when PCR was done to identify presumptive L. monocytogenes colonies directly from Palcam and Oxford media, after the enrichment step. This procedure allowed reducing to a few hours the period of several days usually needed to obtain the final identification of L. monocytogenes using phenotypic tests.

Detecção de Listeria monocytogenes pela técnica de PCR em leite contaminado artificialmente / Detection of Listeria monocytogenes by PCR in artificially contaminated milk samples

Avaliou-se a técnica de PCR como opção para reduzir o tempo de detecção de Listeria monocytogenes no leite. Para tanto, amostras de leite desnatado esterilizado e de leite cru integral - com baixa, média e alta contagem de microrganismos aeróbios mesófilos - foram inoculadas experimentalmente com diversas concentrações de L. monocytogenes. Os resultados da reação de PCR foram comparados com os da cultura da amostra empregando-se metodologia padronizada tradicional. Não se detectou L. monocytogenes pela reação de PCR quando esta foi realizada a partir do caldo de enriquecimento de Listeria (LEB) após 24 horas de incubação, nem no leite desnatado esterilizado, nem no leite cru integral. Após 48 horas de enriquecimento em LEB, a bactéria foi detectada por PCR nas amostras de leite desnatado esterilizado, com a sensibilidade de 1UFC/mL, mas não nas amostras de leite cru integral. Pela metodologia tradicional, a bactéria foi recuperada de todos os ensaios. Entretanto, nas amostras de leite cru com altas contagens de aeróbios mesófilos, a sensibilidade da metodologia tradicional foi reduzida (a partir de 7UFC/mL). Melhores resultados foram obtidos quando a reação de PCR foi feita utilizando-se DNA obtido diretamente da colônia suspeita em meio sólido (Oxford e Palcam). Foi possível substituir os testes fenotípicos de identificação de L. monocytogenes pela técnica de PCR reduzindo-se o tempo de identificação da bactéria de vários dias para algumas horas.(AU)

The polymerase chain reaction (PCR) was used to detect Listeria monocytogenes in inoculated milk samples after selective enrichment. Samples of sterile skim milk and raw whole milk (with low, intermediate, and high counts of aerobic mesophilic microorganisms) were inoculated with several concentrations of L. monocytogenes. The results of PCR assays were compared to the results of culturing the samples using a standardized traditional method for isolation of L. monocytogenes. The pathogen was detected by PCR in Listeria Enrichment Broth (LEB) after 48h-incubation (sensitivity of 1CFU/mL) but not after 24h-incubation from the samples prepared with sterile skim milk. L. monocytogenes was not detected by PCR in LEB after 24 and 48h-incubation from the samples prepared with raw whole milk. Using the traditional method, the pathogen was detected in all experiments. However, sensitivity decreased in raw whole milk with high counts of aerobic mesophilic microorganisms (up to 7CFU/mL). Best results were obtained when PCR was done to identify presumptive L. monocytogenes colonies directly from Palcam and Oxford media, after the enrichment step. This procedure allowed reducing to a few hours the period of several days usually needed to obtain the final identification of L. monocytogenes using phenotypic tests.(AU)