BackgroundBirds harbour an astonishing diversity of haemosporidian parasites. Renewed interest in avian haemosporidians as a model system has placed a greater emphasis on the development of screening protocols to estimate parasite prevalence and diversity. Prevalence estimates are often based on the molecular or blood-smear microscopy techniques. However, variation in diagnostic sensitivity among screening methodologies represents a potential source of bias that may lead to erroneous inference in comparisons of prevalence across studies. Here, we analyzed a suite of blood samples for the presence of parasites using four diagnostic tools and compared method-specific estimates of detection probability to assess the relative performance of screening strategies.MethodsWe screened a total of 394 bird blood samples collected in India (n = 203) and Sweden (n = 191) for the combined presence of Plasmodium, Haemoproteus and Leucocytozoon with three PCR assays: (i) qPCR; (ii) restriction enzyme-based assay; and (iii) nested protocol. In addition, we examined blood smears for estimates of parasite intensity which was further screened using qPCR method to evaluate if parasite intensity shows a relationship with qPCR (Ct values). Furthermore, we used single infected samples from parasite intensities: low, medium, high, very high to establish the reproducibility in qPCR.ResultsFor the combined data sets from India and Sweden, detection probability for submicroscopic and low intensity infections was highest for the qPCR method, followed by the nested protocol and the restriction enzyme-based assay. For high parasite intensities, the qPCR had high PCR reproducibility, with three out of three PCR replicates being positive and with consistent Ct values across all tenfold dilution series. For parasite intensities at very low and submicroscopic samples, the qPCR was reproducible in one out of the three replicates. The intensity of parasitemia estimated from smears showed inverse relationship with Ct values in both the Indian and Swedish data sets.ConclusionsOur study highlights the importance of accounting for methodological issues to better estimate infection in parasitological studies and illustrates how a wider deployment of diagnostic tools combined with statistical approaches is needed for each study, in order to provide adequate insight into the most appropriate approach to avoid erroneous inferences.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2066-z) contains supplementary material, which is available to authorized users.

BackgroundComputational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (α/β)8 TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts.MethodsComputational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics.ResultsExtensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a Tm of 44 °C and a Gibbs free energy of unfolding (ΔG°) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a Cm of 1.6 M and a ΔG° of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics.ConclusionsDespite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-015-0047-4) contains supplementary material, which is available to authorized users.

The Implementation of Infant Pain Practice (ImPaC) Resource is an eHealth tool designed to support infant pain practice change and ultimately enhance pain outcomes. The aim of this study was to determine users' perspectives on usability, acceptability, and feasibility of the ImPaC Resource. A descriptive prospective mixed‐methods quality improvement study was conducted at a pediatric hospital in Canada. Individual “think aloud” interviews were conducted in a nonclinical environment (Phase A); “near live” testing was conducted while users interacted with the Resource in clinical setting (Phase B); individual “think‐aloud” interviews were conducted in a nonclinical environment (Phase C). Outcomes included usability (System Usability Scale—SUS), acceptability (Acceptability E‐Scale—AES), and feasibility. Interview transcripts were coded per a priori themes using deductive content analysis to create a structured categorization matrix. In Phase A, 10 clinicians interacted with the Resource in individual sessions. Median SUS score was 73.75 (range 52.5‐92.5). In Phase B, four clinicians implemented the Resource in the neonatal intensive care unit (NICU) over 4 months. Median SUS score was 85 (82.5‐92.5), and median AES score was 24 (21‐24). In Phase C, an enhanced prototype was produced, and the same users from Phase B navigated the Resource in individual sessions. Median SUS score was 88.75 (85‐95), and median AES score was 27.5 (25‐29). Users considered the Resource as feasible for implementation, easy to navigate, engaging, intuitive, comprehensive, and evidence‐based. Users highlighted the potential transferability of the Resource to other contexts and settings. The enhanced version of the ImPaC Resource was usable, acceptable, feasible, and met users' expectations and requirements. Results lead the way for evaluation of the Resource in a nationwide cluster randomized trial including 18 NICUs. This knowledge‐rich platform is expected to enhance infant pain practices and outcomes in diverse clinical settings.

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Background: Laughingthrushes (family: Leiothrichidae) consists of diverse and widespread species found in the Indian subcontinent but there is a lack of information on their avian haemosporidians.  Methods: We sampled 231 laughingthrushes of 8 species in the western and eastern Himalaya in India. Using parasite morphology and cytochrome b sequences we describe 2 new Haemoproteus species harbored in 3 species of laughingthrushes and report a case of cryptic speciation. Results: First Haemoproteus lineage TROERY01 (GenBank: KY623720) found in Trochalopteron erythrocephalum (27.47%) and Trochalopteron variegatum (2.9%) in mid to high altitude tropical forests in the western and eastern Himalaya, was described as Haemoproteus (Parahaemoproteus) leiothrichus n. sp. (Haemosporida: Haemoproteidae). Second Haemoproteus lineage TROERY02 (GenBank: KY623721) described as Haemoproteus (Parahaemoproteus) homoleiothrichus n. sp. (Haemosporida: Haemoproteidae) was found in T. erythrocephalum (2.19%) and Trochalopteron lineatum (3.84%), albeit in low intensity, only in the western Himalaya. Both H. homoleiothrichus n. sp. and H. leiothrichus n. sp. showed no significant difference in morphological features in blood stages. A genetic divergence of 4.4% along with distinct phylogenetic position indicates that these 2 lineages represent cryptic species. Previously, T. erythrocephalum has been described as an additional host for a morphologically described Haemoproteus timalus in the oriental region. Our described species have several morphological features that are absent in H. timalus. These are, the presence of dumbbell-like shaped mature gametocytes, ‘arm’ like extensions of gametocytes and lateral displacement of nuclei of infected erythrocytes. Illustrations of blood stages of the new species are given, and phylogenetic analysis with morphologically described Haemoproteus species identifies parasites closely related to the 2 described parasites. Conclusions: The lineages described here have been recorded only in the laughingthrushes so far. These are the first parasites to be described with T. erythrocephalum as a type host from the western and eastern Himalaya in India.