Comparison of the egg recovery rates and limit of detection for soil-transmitted helminths using the Kato-Katz thick smear,faecal flotation and quantitative real-time PCR in human stool

BackgroundMonitoring the success of soil-transmitted helminth (STH) control programs relies on accurate diagnosis and quantitative assessment of infection prevalence and intensity. As preventative chemotherapeutic program coverage for STH expands, the necessity of gaining insights into the relative or comparative sensitivities, in terms of limits of detection (LOD) and egg-recovery-rates (ERR) for microscopy and quantitative polymerase chain reaction qPCR-based diagnostic techniques becomes imperative to inform suitability for their intended use for large scale STH monitoring and treatment efficacy studies.Methodology/Principal findingsThe diagnostic performance in terms of ERR and LOD of the Kato-Katz (KK) thick smear technique, sodium nitrate (NaNO₃) faecal floatation (FF) and qPCR for the accurate detection and enumeration of STH eggs were calculated and expressed in eggs per gram (EPG), by experimentally seeding parasite-free human faeces with Ascaris spp., Trichuris spp. and Necator americanus eggs representing low, medium and high intensity infections. The efficiency of NaNO₃ flotation was also calculated over a range of specific gravities (SpGr) for the optimum recovery of STH eggs. FF of SpGr 1.30 recovered 62.7%, 11% and 8.7% more Trichuris spp., Necator americanus and Ascaris spp. eggs respectively, than the recommended SpGr of 1.20. All diagnostic methods demonstrated strong direct correlation to the intensity of seeded EPG. KK and FF (SpGr 1.30) resulted in significant lower ERRs compared to qPCR (p <0.05). qPCR demonstrated significantly (p <0.05) greater sensitivity with an ability to detect as little as 5 EPG for all three STH, compared to 50 EPG by KK and FF (SpGr 1.30).Conclusions/SignificanceThis study compares the diagnostic parameters in terms of LOD and ERRs of STHs for the KK, FF and qPCR. These results indicate that the diagnostic performance of qPCR assays should be considered by control programs in the phase that aims to seek confirmation of transmission break and cessation of preventive chemotherapy in low-transmission settings, in line with the control targets of the WHO neglected tropical diseases 2030 Roadmap.     

P-selectin-mediated adhesion impairs endothelium-dependent arteriolar dilation in hypercholesterolemic mice

Hypercholesterolemia is associated with an attenuation of endothelium-dependent dilation in arterioles and an increase in leukocyte and platelet adhesion in venules. The proximity of closely paired arterioles and venules is thought to facilitate heat and mass transport between the two and could be involved in transport of inflammatory and/or vasoactive mediators from venule to arteriole. In the current study, we tested the hypothesis that the impaired arteriolar dilation associated with hypercholesterolemia might be dependent on P-selectin-dependent blood cell adhesion in the closely paired venules. Leukocyte and platelet recruitment in venules and the endothelium-dependent response to bradykinin in second-order arterioles were observed in the mouse intestinal submucosa using intravital microscopy. Four weeks of a high-cholesterol diet decreased bradykinin-induced arteriolar dilation more dramatically in closely paired arterioles than in distantly paired arterioles. The dysfunctional arteriolar dilation of closely paired arterioles in hypercholesterolemic mice was significantly improved when the experiments were repeated in P-selectin-deficient mice (given the high-cholesterol diet) or in hypercholesterolemic mice injected with a P-selectin monoclonal antibody. A similar improvement in dilation of closely paired arterioles was attained in hypercholesterolemic mice given the superoxide dismutase mimetic Tempol. These findings indicate that hypercholesterolemia-induced increases in venular leukocyte and platelet adhesion might contribute to the impaired endothelium-dependent dilation of closely paired arterioles via a mechanism that is distance limited and dependent on P-selectin and superoxide.     

High-Resolution Gene Flow Model for Assessing Environmental Impacts of Transgene Escape Based on Biological Parameters and Wind Speed

Environmental impacts caused by transgene flow from genetically engineered (GE) crops to their wild relatives mediated by pollination are longstanding biosafety concerns worldwide. Mathematical modeling provides a useful tool for estimating frequencies of pollen-mediated gene flow (PMGF) that are critical for assessing such environmental impacts. However, most PMGF models are impractical for this purpose because their parameterization requires actual data from field experiments. In addition, most of these models are usually too general and ignored the important biological characteristics of concerned plant species; and therefore cannot provide accurate prediction for PMGF frequencies. It is necessary to develop more accurate PMGF models based on biological and climatic parameters that can be easily measured in situ. Here, we present a quasi-mechanistic PMGF model that only requires the input of biological and wind speed parameters without actual data from field experiments. Validation of the quasi-mechanistic model based on five sets of published data from field experiments showed significant correlations between the model-simulated and field experimental-generated PMGF frequencies. These results suggest accurate prediction for PMGF frequencies using this model, provided that the necessary biological parameters and wind speed data are available. This model can largely facilitate the assessment and management of environmental impacts caused by transgene flow, such as determining transgene flow frequencies at a particular spatial distance, and establishing spatial isolation between a GE crop and its coexisting non-GE counterparts and wild relatives.     

Stress proteins are not induced in mammalian cells exposed to radiofrequency or microwave radiation

The induction of stress proteins in HeLa and CHO cells was investigated following a 2 h exposure to radiofrequency (RF) or microwave radiation. Cells were exposed or sham exposed in vitro under isothermal (37 ± 0.2 °C) conditions. HeLa cells were exposed to 27- or 2450 MHz continuous wave (CW) radiation at a specific absorption rate (SAR) of 25 W/kg. CHO cells were exposed to CW 27 MHz radiation at a SAR of 100 W/kg. Parallel positive control studies included 2 h exposure of HeLa or CHO cells to 40 °C or to 45 μM cadmium sulfate. Stress protein induction was assayed 24 h after treatment by electrophoresis of whole-cell extracted protein labeled with [³⁵S]-methionine. Both cell types exhibited well-characterized responses to the positive control stresses. Under these exposure conditions, neither microwave nor RF radiation had a detectable effect on stress protein induction as determined by either comparison of RF-exposed cells with sham-exposed cells or comparison with heat-stressed or Cd⁺⁺ positive control cells. Bioelectromagnetics 18:499–505, 1997. © 1997 Wiley-Liss, Inc.     

mdfw:A Deafness Susceptibility Locus That Interacts with Deaf Waddler (dfw)

The deaf waddler (dfw) mutation is a model system to study the biology of neuroepithelial hearing defects in mice. Here we describe the identification and characterization of a new allele of deaf waddler (dfw^2J) and present evidence for a hearing susceptibility locus (mdfw) that interacts withdfw.We found that CBy-dfw^2J/dfw^2Jhomozygotes exhibit no discernible auditory brainstem responses (ABR) to sound pressure level stimuli up to 100 dB, indicating a profound deafness. Interestingly, the ABR in CBy-dfw^2J/+ heterozygotes is also abnormal, showing age-dependent elevated thresholds characteristic of a progressive hearing loss. When outcrossed onto the CAST/Ei strain, only 24% of the F2 CBy/CAST-dfw^2J/+ heterozygotes displayed increased ABR thresholds, suggesting that a second locus, controlling hearing function indfw^2J/+ heterozygotes, was segregating in the CBy/CAST-dfw^2Jintercross. By linkage analysis, we localized this locus (mdfw) to Chromosome 10, between markersD10Mit127andD10Mit185,within a 4.0 ± 1.1 cM genetic interval. All CBy/CAST-dfw^2J/+ heterozygotes that develop hearing loss are homozygous for the CBy-derived recessive allele (mdfw^C). In contrast, CBy/CAST-dfw^2J/+ heterozygotes expressing even a single copy of the CAST/Ei-derivedmdfwallele (Mdfw) retain their normal hearing function. Our results reveal an epistatic relationship between themdfwand thedfwgenes and provide a model system to study nonsyndromic hearing loss in mice.     

Genetic Heterogeneity in Bacillus sporothermodurans as Demonstrated by Ribotyping and Repetitive Extragenic Palindromic-PCR Fingerprinting

Thirty-eight strains of Bacillus sporothermodurans isolated from ultra-high-temperature (UHT)-treated milk or sterilized milk (UHT isolates) and from animal feed or raw milk (farm isolates) were characterized by automated ribotyping and by repetitive extragenic palindromic (REP)-PCR fingerprinting. By investigating the genetic relationships among isolates from these various sources, the relative importance of different contamination sources could be evaluated. The results of the separate clustering analyses of the PvuII and EcoRI ribopatterns and the REP-PCR patterns were largely consistent with each other and revealed the existence of two main clusters; there was one homogeneous group containing all (REP-PCR) or most (ribotyping) of the UHT isolates, and there was a second more diverse group comprising the farm isolates. A combined three-dimensional analysis of all data showed that three German UHT isolates did not belong to the compact group containing the majority of the UHT isolates. These results demonstrate that B. sporothermodurans is more heterogeneous than previously assumed and that most of the UHT isolates form a genetically distinct subgroup and are capable of producing highly heat-resistant spores. The close genetic relationship of these UHT isolates suggests a clonal origin of a few predominant strains of B. sporothermodurans that can be found in UHT-treated or sterilized milk products.     

Dynamics of escape mutants

We use multi-type Galton-Watson branching processes to model the evolution of populations that, due to a small reproductive ratio of the individuals, are doomed to extinction. Yet, mutations occurring during the reproduction process, may lead to the appearance of new types of individuals that are able to escape extinction. We provide examples of such populations in medical, biological and environmental contexts and give results on (i) the probability of escape/extinction, (ii) the distribution of the waiting time to produce the first individual whose lineage does not get extinct and (iii) the distribution of the time it takes for the number of mutants to reach a high level. Special attention is dedicated to the case where the probability of mutation is very small and approximations for (i)-(iii) are derived.     

Stochasticity in the adaptive dynamics of evolution: the bare bones

First a population model with one single type of individuals is considered. Individuals reproduce asexually by splitting into two, with a population-size-dependent probability. Population extinction, growth and persistence are studied. Subsequently the results are extended to such a population with two competing morphs and are applied to a simple model, where morphs arise through mutation. The movement in the trait space of a monomorphic population and its possible branching into polymorphism are discussed. This is a first report. It purports to display the basic conceptual structure of a simple exact probabilistic formulation of adaptive dynamics.