Performance evaluation of Baermann techniques: The quest for developing a microscopy reference standard for the diagnosis of Strongyloides stercoralis

BackgroundSoil-transmitted helminths (STH) are common in low and middle income countries where there is lack of access to clean water and sanitation. Effective diagnosis and treatment are essential for the control of STH infections. However, among STH parasites, Strongyloides stercoralis is the most neglected species, both in diagnostics and control strategies. Diagnostic methods cover different approaches, each with different sensitivities and specificities, such as serology, molecular techniques and microscopy based techniques. Of the later, the Baermann technique is the most commonly used procedure. In the literature, several ways have been described to perform the Baermann method, which illustrates the overall lack of a ‘(gold) reference standard’ method for the diagnosis of S. stercoralis infection. In this study we have evaluated the performance of three Baermann techniques in order to improve the reference standard for the microscopic diagnosis of S. stercoralis infection thereby facilitating individual case detection, mapping of the disease and proper evaluation of treatment responses.Methods/Principal findingsA community based cross sectional study was conducted at Zenzelima, Bahir Dar Zuria Ethiopia. A total of 437 stool samples were collected and analyzed by the following procedures: conventional Baermann (CB), modified Baermann (MB), and modified Baermann with charcoal pre-incubation (MBCI). The diagnostic sensitivity and Negative Predictive Value (NPV) of each technique was calculated using the combination of all the three techniques as a composite reference standard. Our result indicated that larvae of S. stercoralis were detected in 151 (34.6%) stool samples. The prevalence of S. stercoralis infection based on the three diagnostic methods was 9.6%, 8.0%, and 31.3% by CB, MB, and MBCI respectively. The sensitivity and NPV for CB, MB, and MBCI were 26.7% and 70.8%, 22.1% and 69.6%, and 87.0% and 93.2%, respectively. The MBCI showed significant difference (P- value = <0.001) in the sensitivity and NPV values when compared with CB and MB values. The agreement between CB, MB, and MBCI with the composite reference standard was 31.8%, 26.7%, 89.6%, respectively.Conclusion/SignificanceOur results suggest the superior performance of MBCI. It is relatively easy to implement, simple to perform and comparatively cheaper. The CB is by far the commonly used method in routine diagnostic although this technique significantly underestimates the true burden of the disease and thereby contributing to the exclusion of S. stercoralis from the control strategies. Therefore, MBCI is recommended as a routine microscopy-based diagnostic test for S. stercoralis infection, particularly in settings where molecular procedures are not available.     

Gene Expression-Based Classifiers Identify Staphylococcus aureus Infection in Mice and Humans

Staphylococcus aureus causes a spectrum of human infection. Diagnostic delays and uncertainty lead to treatment delays and inappropriate antibiotic use. A growing literature suggests the host’s inflammatory response to the pathogen represents a potential tool to improve upon current diagnostics. The hypothesis of this study is that the host responds differently to S. aureus than to E. coli infection in a quantifiable way, providing a new diagnostic avenue. This study uses Bayesian sparse factor modeling and penalized binary regression to define peripheral blood gene-expression classifiers of murine and human S. aureus infection. The murine-derived classifier distinguished S. aureus infection from healthy controls and Escherichia coli-infected mice across a range of conditions (mouse and bacterial strain, time post infection) and was validated in outbred mice (AUC>0.97). A S. aureus classifier derived from a cohort of 94 human subjects distinguished S. aureus blood stream infection (BSI) from healthy subjects (AUC 0.99) and E. coli BSI (AUC 0.84). Murine and human responses to S. aureus infection share common biological pathways, allowing the murine model to classify S. aureus BSI in humans (AUC 0.84). Both murine and human S. aureus classifiers were validated in an independent human cohort (AUC 0.95 and 0.92, respectively). The approach described here lends insight into the conserved and disparate pathways utilized by mice and humans in response to these infections. Furthermore, this study advances our understanding of S. aureus infection; the host response to it; and identifies new diagnostic and therapeutic avenues.     

Protein composition and biomechanical properties of in vivo-derived basement membranes

Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.     

The Effect of Aerobic Exercise on Intrahepatocellular and Intramyocellular Lipids in Healthy Subjects

Background

Intrahepatocellular (IHCL) and intramyocellular (IMCL) lipids are ectopic lipid stores. Aerobic exercise results in IMCL utilization in subjects over a broad range of exercise capacity. IMCL and IHCL have been related to impaired insulin action at the skeletal muscle and hepatic level, respectively. The acute effect of aerobic exercise on IHCL is unknown. Possible regulatory factors include exercise capacity, insulin sensitivity and fat availability subcutaneous and visceral fat mass).

Aim

To concomitantly investigate the effect of aerobic exercise on IHCL and IMCL in healthy subjects, using Magnetic Resonance spectroscopy.

Methods

Normal weight, healthy subjects were included. Visit 1 consisted of a determination of VO_2max on a treadmill. Visit 2 comprised the assessment of hepatic and peripheral insulin sensitivity by a two-step hyperinsulinaemic euglycaemic clamp. At Visit 3, subcutaneous and visceral fat mass were assessed by whole body MRI, IHCL and IMCL before and after a 2-hours aerobic exercise (50% of VO_2max) using ¹H-MR-spectroscopy.

Results

Eighteen volunteers (12M, 6F) were enrolled in the study (age, 37.6±3.2 years, mean±SEM; VO_2max, 53.4±2.9 mL/kg/min). Two hours aerobic exercise resulted in a significant decrease in IMCL (−22.6±3.3, % from baseline) and increase in IHCL (+34.9±7.6, % from baseline). There was no significant correlation between the exercise-induced changes in IMCL and IHCL and exercise capacity, subcutaneous and visceral fat mass and hepatic or peripheral insulin sensitivity.

Conclusions

IMCL and IHCL are flexible ectopic lipid stores that are acutely influenced by physical exercise, albeit in different directions.

Trial Registration

ClinicalTrial.gov {"type":"clinical-trial","attrs":{"text":"NCT00491582","term_id":"NCT00491582"}}NCT00491582     

F508del disturbs the dynamics of the nucleotide binding domains of CFTR before and after ATP hydrolysis

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is an ion channel responsible for chloride transport in epithelia and it belongs to the class of ABC transporters. The deletion of phenylalanine 508 (F508del) in CFTR is the most common mutation responsible for cystic fibrosis. Little is known about the effect of the mutation in the isolated nucleotide binding domains (NBDs), on dimer dynamics, ATP hydrolysis and even on nucleotide binding. Using molecular dynamics simulations of the human CFTR NBD dimer, we showed that F508del increases, in the prehydrolysis state, the inter-motif distance in both ATP binding sites (ABP) when ATP is bound. Additionally, a decrease in the number of catalytically competent conformations was observed in the presence of F508del. We used the subtraction technique to study the first 300 ps after ATP hydrolysis in the catalytic competent site and found that the F508del dimer evidences lower conformational changes than the wild type. Using longer simulation times, the magnitude of the conformational changes in both forms increases. Nonetheless, the F508del dimer shows lower C-α RMS values in comparison to the wild-type, on the F508del loop, on the residues surrounding the catalytic site and the portion of NBD2 adjacent to ABP1. These results provide evidence that F508del interferes with the NBD dynamics before and after ATP hydrolysis. These findings shed a new light on the effect of F508del on NBD dynamics and reveal a novel mechanism for the influence of F508del on CFTR.     

Adverse responses of Cabera pusaria caterpillars to high dietary manganese concentration

The adaptation of insects to environmental changes can constitute a crucial factor in their development and activity. The response of Cabera pusaria L. (Lepidoptera: Geometridae) caterpillars to high manganese (Mn) concentrations in the diet was studied. Birch leaves were treated by dipping in MnCl₂·4H₂O solutions, thereby achieving Mn contents of 370 (T0), 695 (T1), 3 198 (T2), and 6 302 mg kg⁻¹ (T3). The reactions were determined by observing caterpillar mortality, development time, food consumption, and pupal weight. Manganese concentrations in larval excrement, pupae, and food were determined. Manganese in the diet at unnaturally increased concentrations caused great stress for caterpillars. All individuals in the treatment with the highest Mn concentration (T3) died during rearing and successful pupation occurred in only four individuals in T2. Even in the case of caterpillars from T1 (twofold higher than T0) a negative reaction (increased food consumption and prolonged development) was recorded. We also determined significantly increased Mn concentration in pupae from T1 (T2 and T3 were not included in this evaluation due to mortality) and excrement (T1‒T3) compared with T0 having a natural Mn concentration. Caterpillars were seen to eliminate negatively acting dietary Mn by its translocation to excrement. However, the highest mortality rate in T2 and T3 and negative reactions of individuals in T1 very likely demonstrate energy insufficiency and the high energy requirements of Mn elimination mechanisms.     

Evolution under pH stress and high population densities leads to increased density-dependent fitness in the protist Tetrahymena thermophila

Abiotic stress is a major force of selection that organisms are constantly facing. While the evolutionary effects of various stressors have been broadly studied, it is only more recently that the relevance of interactions between evolution and underlying ecological conditions, that is, eco-evolutionary feedbacks, have been highlighted. Here, we experimentally investigated how populations adapt to pH-stress under high population densities. Using the protist species Tetrahymena thermophila, we studied how four different genotypes evolved in response to stressfully low pH conditions and high population densities. We found that genotypes underwent evolutionary changes, some shifting up and others shifting down their intrinsic rates of increase (r₀). Overall, evolution at low pH led to the convergence of r₀ and intraspecific competitive ability (α) across the four genotypes. Given the strong correlation between r₀ and α, we argue that this convergence was a consequence of selection for increased density-dependent fitness at low pH under the experienced high density conditions. Increased density-dependent fitness was either attained through increase in r₀, or decrease of α, depending on the genetic background. In conclusion, we show that demography can influence the direction of evolution under abiotic stress.     

Nicotinic acid and DP1 blockade: studies in mouse models of atherosclerosis

The use of nicotinic acid to treat dyslipidemia is limited by induction of a “flushing” response, mediated in part by the interaction of prostaglandin D₂ (PGD₂) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr^−/−ApoE^−/− mice versus ApoE^−/− mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr^−/−ApoE^−/− mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE^−/− mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr^−/− mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.