Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by Loop-Mediated Isothermal Amplification (LAMP)

<p><b>Background:</b> The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 103 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay.</p> <p><b>Methodology/Principal Findings:</b> For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 103 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards.</p> <p><b>Conclusions/Significance:</b> This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.</p&gt

Biphasic survival analysis of trypanotolerance QTL in mice

A marker-assisted introgression (MAI) experiment was conducted to transfer trypanotolerance quantitative trait loci (QTL) from a donor mouse strain, C57BL/6, into a recipient mouse strain, A/J. The objective was to assess the effect of three previously identified chromosomal regions on mouse chromosomes 1 (MMU1), 5 (MMU5) and 17 (MMU17) in different genetic backgrounds on the survival pattern following infection with 'Trypanosoma congolense'. An exploratory data analysis revealed a biphasic pattern of time to death, with highly distinct early and late mortality phases. In this paper, we present survival analysis methods that account for the biphasic mortality pattern and results of reanalyzing the data from the MAI experiment. The analysis with a Weibull mixture model confirmed the biphasic pattern of time to death. Mortality phase, an unobserved variable, appears to be an important factor influencing survival time and is modeled as a binary outcome variable using logistic regression analysis. Accounting for this biphasic pattern in the analysis reveals that a previously observed sex effect on average survival is rather an effect on proportion of mice in the two mortality phases. The C57BL/6 (donor) QTL alleles on MMU1 and MMU17 act dominantly in the late mortality phase while the A/J (recipient) QTL allele on MMU17 acts dominantly in the early mortality phase. From this study, we found clear evidence for a biphasic survival pattern and provided models for its analysis. These models can also be used when studying defense mechanisms against other pathogens. Finally, these approaches provide further information on the nature of gene actions

Perception of Everyday Sounds: A Developmental Study of a Free Sorting Task

International audienceObjectives The analysis of categorization of everyday sounds is a crucial aspect of the perception of our surrounding world. However, it constitutes a poorly explored domain in developmental studies. The aim of our study was to understand the nature and the logic of the construction of auditory cognitive categories for natural sounds during development. We have developed an original approach based on a free sorting task (FST). Indeed, categorization is fundamental for structuring the world and cognitive skills related to, without having any need of the use of language. Our project explored the ability of children to structure their acoustic world, and to investigate how such structuration matures during normal development. We hypothesized that age affects the listening strategy and the category decision, as well as the number and the content of individual categories. Design Eighty-two French children (6–9 years), 20 teenagers (12–13 years), and 24 young adults participated in the study. Perception and categorization of everyday sounds was assessed based on a FST composed of 18 different sounds belonging to three a priori categories: non-linguistic human vocalizations, environmental sounds, and musical instruments. Results Children listened to the sounds more times than older participants, built significantly more classes than adults, and used a different strategy of classification. We can thus conclude that there is an age effect on how the participants accomplished the task. Analysis of the auditory categorization performed by 6-year-old children showed that this age constitutes a pivotal stage, in agreement with the progressive change from a non-logical reasoning based mainly on perceptive representations to the logical reasoning used by older children. In conclusion, our results suggest that the processing of auditory object categorization develops through different stages, while the intrinsic basis of the classification of sounds is already present in childhood