Recently Zika virus (ZIKV) has been the focus of much attention, primarily because of its association with an increased incidence in microcephaly. Although ZIKV was initially discovered in a sentinel rhesus monkey in Uganda , its ranOne of the components necessary for the rapid advance of ZIKV research is the availability of a small animal model that faithfully recapitulates the disease process seen in humans. Such a model was developed using mice that lacked type I interferon signaling. Female mice that were Ifnar1−/− were crossed with WT males to produce fetuses that were heterozygous (Ifnar1+/−) . PregnanAnother recent paper reported the development of a useful mouse model of ZIKV infection and also demonstrated that the virus could be vertically transmitted. The mouse strain that was utilized for infection was WT SJL, rather than a strain defective in immunological response, and the virus used was the Brazilian isolate (ZIKABR) which has been associated with the greatly increased incidence of microcephaly . In thisAround the same time, another group demonstrated vertical transmission of ZIKV wherein they infected 10 week old C57 mice either via intra peritoneal or in utero injection. These mice showed infection of radial glia cells of dorsal ventricular zone of fetuses and marked reduction in cortex founder cells . In anotTaken together these reports demonstrate substantial progress in research focused on ZIKV and microcephaly. The reports described above represent significant advances towards developing appropriate vaccines and/or therapeutics for this affliction. In addition to providing animal models, these studies have provided further evidence for the role of interferons in ZIKV resistance. However, many questions remain. For example, the exact mechanism(s) by which ZIKV causes microcephaly remain to be determined, as does the time during gestation when the fetus is most susceptible to the effects of the virus. Determination of these factors, as well as the elucidation of other aspects of ZIKV-induced microcephaly will facilitate further progress in this research area.


LRR-RLK families has been investigated in some eudicot and monocot plants, no comprehensive evolutionary studies have been performed for these genes in basal angiosperms like Amborella trichopoda. In this study, we identified 94 LRR-RLK genes in the genome of A. trichopoda. The number of LRR-RLK genes in the genome of A. trichopoda is only 17–50% of that of several eudicot and monocot species. Tandem duplication and whole-genome duplication have made limited contributions to the expansion of LRR-RLK genes in A. trichopoda. According to the phylogenetic analysis, all A. trichopoda LRR-RLK genes can be organized into 18 subfamilies, which roughly correspond to the LRR-RLK subfamilies defined in Arabidopsis thaliana. Most LRR-RLK subfamilies are characterized by highly conserved protein structures, motif compositions, and gene structures. The unique gene structure, protein structures, and protein motif compositions of each subfamily provide evidence for functional divergence among LRR-RLK subfamilies. Moreover, the expression data of LRR-RLK genes provided further evidence for the functional diversification of them. In addition, selection analyses showed that most LRR-RLK protein sites are subject to purifying selection. Our results contribute to a better understanding of the evolution of LRR-RLK gene family in angiosperm and provide a framework for further functional investigation on A. trichopoda LRR-RLKs.Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases, which are one of the largest protein superfamilies in plants, and play crucial roles in development and stress responses. Although the evolution of  Arabidopsis thaliana were divided into more than 50 families. The largest group is the leucine-rich repeat RLK family (LRR-RLK).All living organisms sense and conduct signals through cell surface receptors. In plants, cellular signal transduction is mainly mediated by receptor-like kinases (RLKs), a protein superfamily. RLKs contain three functional domains: a ligand-binding extracellular domain, a membrane-spanning domain, and an intracellular serine/threonine kinase domain  in their extracellular domain  as far as we know   against the A. trichopoda protein databases available on Phytozome v11.0  were downloaded from Phytozome v11.0. Identical and defective sequences were identified and eliminated by manual inspection in BioEdit. Next, potential kinase sequences were analyzed with CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)   was used to produce the schematic diagrams of physical locations of LRR-RLK genes in scaffolds. As previous literature, tandem duplication cluster in this study was defined as a region containing two or more genes within 200 kb   Edgar, . For bot) Edgar, . The bes) Edgar, . The staA. thaliana, the members of each subfamily usually have the same number of LRRs. To explore patterns in the number of LRRs in the identified LRR-RLK genes, we analyzed the genes with CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)  .The KD domain contained 12 subdomains, which usually included some conserved amino acids that play important roles in the activity and regulation of kinases. Although the KD domain is relatively well conserved, divergence was found. To elucidate the evolution of the KD domain, conserved motifs were identified with Multiple Expectation Maximization for Motif Elicitation (MEME) v.4.10.2.  v. 2.0 (http://gsds.cbi.pku.edu.cn/)  is an effective method for detecting positive or purifying selection on protein-coding genes  with high bootstrap support and sequence number greater than four. The ω value was estimated using the codeml program in the PAMLX software  assumes sites with ω ≤ 1, whereas the positive selection model (M2) adds a third class of sites with ω > 1 to M1. The beta model (M7) assumes a beta distribution for the ratio over sites, whereas the beta&ω model (M8) adds an extra class of sites with ω > 1 to M7. These two pairs of nested models  were compared to test for evidence of sites under positive selection.Comparison between non-synonymous and synonymous substitution rates . Reads were filtered to obtain high quality clean reads using trimmomatic v. 0.32 .For LRR-RLK gene expression analysis, Illumina RNA-seq data from whole plant, apical meristem and young leaves (AMYL), and premeiotic female buds were download from the NCBI SRA database . This finding is similar to the results of a phylogenetic analysis of the kinase domains of LRR-RLK genes in other organisms . Protein structures were mapped to each protein in the phylogenetic tree : M1 to M15  and M11 (subdomain VIII)  do not correspond to the known subdomains. M8 are shared by all members of subfamilies XIII-1 and XIII-2, and most members of subfamilies I and XI*. Motif M9 is subfamily-specific, and appeared only in subfamily II. Motif M14 were absent from several members of a few subfamilies and shared by most LRR-RLK proteins. Motif M13 together with M3 (subdomain III), and M11 (subdomain VIII) were absent from a few LRR-RLK subfamilies. For example, M3 (subdomain III) was absent from all LRR-RLK genes of subfamilies V and VI-2, as well as most of those of subfamilies III, VI-1, and X*. Motif M11 (subdomain VIII) was not observed in any LRR-RLK genes of subfamilies VI-1 and VI-2, as well as most genes of subfamily III  Table , Table 1http://pfam.xfam.org/). The results showed that three members in subfamily I and all members in subfamily III each harbored an extracellular malectin-like (ML) domain.Besides, to find all possible domains in LRR-RLK proteins, all proteins identified in this study were analyzed with Pfam , and pre-meiotic female flower buds. As shown in the heat map  is 150. However, in the present study, we only identified 94 LRR-RLK genes in A. trichopoda. The number of LRR-RLK genes in the last common ancestor of eudicots and monocots is 1.60 times (150/94) that of A. trichopoda, and the number of LRR-RLK genes in eudicots and monocots is roughly 2–6 times that of A. trichopoda,. The difference in the numbers of LRR-RLK genes between A. trichopoda and the ancestor of eudicots/monocots, and between A. trichopoda and eudicots and monocots, suggests a relatively greater degree of lineage-specific expansion of this gene family in the lineages leading to the ancestor of eudicots/monocots and to eudicots and monocots. Indeed, Fischer et al. (LRR-RLK genes are very dynamic in angiosperm (eudicots + monocots) and LRR-RLK genes showed some degree of expansion in most species. When we compared the copy number of each subfamily between A. trichopoda and A. thaliana, in consistent with previous studies  were located on the retention regions after genome duplication  usually have relatively similar functions. According to the functional studies, we know different subfamily members of LRR-RLK gene in A. thaliana usually have different functions. For example, A. thaliana FRK gene in subfamily I is involved in defense signaling  in LRR-RLK subfamily II contain a shorter LRR, 4 LRRs. Moreover, LRR domains interaction with different substrates  showed different LRR numbers and arrangements  domain before a short stretch of LRR repeats in three members of subfamily I and all members of subfamily VIII-1. According to the previous study, the malectin-like domain is likely involved in carbohydrate binding . Therefore, for all of the analyzed subfamilies, most sites were under negative or relaxed purifying selection. This finding was largely consistent with the results of selection testing of the LRR-RLK subfamilies of O. sativa  and the National Natural Science Foundation of China (31500178).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We present here the full-length genome sequences of two hepatitis E virus genotype 3 (HEV-3) isolates from a human stool sample from a patient with acute hepatitis and a raw sausage containing pig liver. Sequence analysis implies that Swiss HEV isolates may form a novel subgroup of HEV-3 viruses. Hepeviridae. Genotypes 1 and 2  circulate within the human population and are a major health issue in developing countries. HEV-3 and HEV-4 are zoonotic viruses that are highly prevalent in porcine species and may be transmitted to humans by the consumption of pig liver and meat (Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide. It is a small nonenveloped or quasienveloped virus  with a pand meat , no furtHere, we present the full-genome sequences of two HEV isolates from a human and a food sample. The stool sample originated in a 78-year-old male hospitalized in October 2016 in Lugano  with acute hepatitis and was collected within 10 days from symptom onset. Total RNA was extracted using the QIAamp viral RNA minikit, according to the manufacturer’s instructions . The food sample was a traditional raw dry-cured pork sausage containing pork liver (“mortadella di fegato crudo”), sold by a local butcher shop. For the RNA extraction, a combination of TRI reagent  and the NucliSENS easyMAG system  was used. Both RNAs were shown to be HEV positive by a commercial quantitative real-time reverse transcription-PCR (RT-PCR) . To prepare the RNA samples for next-generation sequencing (NGS), sequence-independent single-primer amplification was performed (MF346772 and MF346773.Both sequences are deposited in GenBank under the accession numbers


This MA shows unknown behavior that the easy magnetization axis rotates by 90° at an energy of 0.2 eV below the Fermi level in LSMO. We attribute this phenomenon to the transition between the eg and t2g bands at the LSMO interface. Our finding and approach to understanding the energy dependence of the MA demonstrate a new possibility of efficient control of the interfacial magnetic properties by controlling the band structures of oxide heterostructures.Understanding and controlling the interfacial magnetic properties of ferromagnetic thin films are crucial for spintronic device applications. However, using conventional magnetometry, it is difficult to detect them separately from the bulk properties. Here, by utilizing tunneling anisotropic magnetoresistance in a single-barrier heterostructure composed of La For efficient control of MA and for developing materials that are suitable for the MA control, it is necessary to understand the MA of the magnetic thin films over a wide energy range; however, there are few studies from this point of view. In ferromagnetic (FM) materials, the MA energy is related to the magnetization-direction dependence of the density of states (DOS) via the spin orbit interaction5. Tunneling anisotropic magnetoresistance (TAMR) is a phenomenon observed in tunnel diodes composed of ferromagnetic (FM) layer/tunnel barrier/nonmagnetic (NM) electrode. TAMR is defined as the change of the tunnel resistance or conductance dI/dV, which is proportional to the DOS of the electrodes, when rotating the magnetization of the FM layer10. Thus, TAMR is useful to understand the magnetic-field direction dependence of the DOS. By measuring TAMR at various bias voltages, one can obtain a high-resolution carrier-energy-resolved map of MA of the FM layer10.Control of magnetic anisotropy (MA) is crucial for low-power magnetization reversal in magnetic thin films. From the perspectives of energy efficiency and scalability, gate-voltage control of the MA via modulation of the carrier density and thus, the Fermi level, is highly desirabletunneling interface of the FM layer, and thus it provides a sensitive probe of the interfacial magnetic properties. Thin film interfaces present both problems and opportunities for exploring new functional devices. As a good example, the “dead layer” at the interface of the perovskite oxide Lax1−SrxMnO3 , which is one of the most promising materials due to its intriguing magnetic and electrical properties such as the colossal magnetoresistance12, half-metallic band structure14, and high Curie temperature (TC ~ 370 K)11, is a serious problem for its device applications. For the formation of the dead layer, various possible origins have been proposed, such as intermixing of atoms16, oxygen vacancies17, lattice distortion20, and MnO6 oxygen octahedral rotations (OOR)23, which induce orbital, charge, and spin reconstruction at the interfaces of LSMO. These studies on dead layers, however, suggest new ways for controlling the interfacial properties at an atomic level, which are not available in the bulk. To this end, the characterization of the interfacial magnetic properties is highly demanded, but it is difficult with conventional magnetometry because the interfacial properties are usually concealed by the dominant signals from the bulk. Here, by utilizing TAMR in an LSMO/LAO/Nb:STO junction, we obtain the carrier-energy dependence of MA of LSMO for the first time. We also reveal a peculiar strong two-fold symmetry component of MA at the LSMO/LAO interface, which is not observed in bulk LSMO. Moreover, this interfacial MA shows unknown behavior that the symmetry axis of this interface MA rotates by 90° at an energy of 0.2 eV below the Fermi level in LSMO. We attribute this phenomenon to the transition between the eg and t2g bands at the LSMO interface. Our results suggest that controlling the band structure at interfaces will pave a new way for efficient control of the magnetization of FM thin films, which is essential for devices with low-power consumption.An equally important aspect of TAMR is that it reflects the DOS at the 3  grown on a TiO2-terminated Nb-doped SrTiO3 (001) substrate  by molecular beam epitaxy (MBE)  patterns in the [100] direction of the 4-u.c. LAO and 40-u.c. LSMO layers show streaky patterns, and especially LSMO exhibits a bright pattern o reflections of the LSMO epilayer  o peaks of LSMO have nearly the same out-of-plane reciprocal lattice vector Q⊥, indicating that the (260)o and (620)o atomic plane spacings are equal. This is consistent with the common reports on LSMO thin films grown under tensile strain, indicating that the strain effect in LSMO is accommodated equally between the [100]c and [010]c directions22.The heterostructure used in this study consists of LSMO (40 unit cell (u.c.) = 15.6 nm)/LaAlOern Fig. , indicatern Fig. . In the ly) Fig. . These r2 mesas were formed by standard photolithography and Ar ion milling. The bias polarity is defined so that the current flows from the LSMO layer to the Nb:STO substrate in the positive bias.For tunneling transport measurements, 600 × 700 μmV. The Fermi level EF is located at 10–20 meV above the CB bottom of STO due to the Nb doping 26, while EF lies in the CB formed by the Mn 3d-eg states in LSMO. The LAO layer serves as a tunnel barrier with a height of ~2.4 eV for STO and ~2 eV for LSMO27. TAMR measurements were conducted as follows: dI/dV − V curves were measured at 4 K while applying a strong external magnetic field of 1 T, which aligned the magnetization direction parallel to the magnetic field, in various in-plane directions with an angle step of 10°. The change in dI/dV when rotating the external magnetic field is attributed to the change in the DOS at the LSMO/LAO interface or the LAO/STO interface. As illustrated in Fig. V, electrons tunnel from Nb:STO to LSMO (from LSMO to Nb:STO), and thus dI/dV probes the DOS of unoccupied (occupied) states in LSMO. At each V and Φ, where Φ is the angle of the magnetization direction from the [100]c axis in the counter-clockwise direction in the film plane, we define Φ at V. As seen in the polar plots of Φ in Fig. V: At V = −0.1 V (left panel), the maximum is located at ~150°, between the [010]c and c axis) when V = −0.35 V (right panel). The whole picture of this behavior for all V is represented in Fig. Δ(dI/dV) is plotted as a function of Φ at V ranging from −0.5 to 0.5 V. This plot shows a peculiar behavior that the symmetry axis changes at V ~ −0.2 V. We fit the data at each bias V using the following equation:C4〈110〉 is the four-fold component with the symmetry axis of 〈110〉c, C2[100] and C2[110] are the two-fold components with the [100]c and [110]c axes, respectively. As shown in Fig. V are summarized in Fig. C2[100] or C2[110]) is stronger than the four-fold symmetry (C4〈110〉]) in almost all the bias region. The C4〈110〉 and C2[100] components show a similar V-dependence, stretching from −0.45 V to 0.4 V. The sign of C2[110] changes at V ~ −0.2 V, which indicates an opposite dependence of the DOS on the magnetization direction between the regions of V > −0.2 V and V < −0.2 V. Because the change in the DOS when changing the magnetization direction, i.e. Δ(dI/dV), is proportional to the MA energy5, the sign change of C2[110] indicates a 90°-shift of the magnetization direction where the MA energy becomes minimum, and consequently indicates a 90°-shift of the easy magnetization axis of the C2[110] component from the [110]c to the V ~ −0.2 V when decreasing V. This is the main origin of the symmetry axis rotation observed in Fig. Figure c axes and the uniaxial (two-fold) MA along the [100]c axis have been reported for LSMO films grown on STO (001) substrates30, and thus they are bulk-like properties. The biaxial component originates from the in-plane cubic symmetry of LSMO thin films grown on STO. The uniaxial MA along the [100]c axis has been attributed to various origins such as step edges29 or different OOR between the [100]c and [010]c directions30. In our study, this uniaxial MA along the [100]c axis may also be partially contributed from the high-mobility two dimensional electron gas possibly formed at the LAO/STO interface, where the magnetoconductance has been reported to possess the same two-fold symmetry31. On the other hand, the uniaxial MA component along the [110]c direction has never been reported for LSMO. Because the crystal structure of LSMO grown on a substrate with a cubic symmetry such as STO is equivalent between the [110]c and c direction is not a bulk property, and thus must be attributed to the LSMO/LAO interface.We discuss the origins of these anisotropy components. The biaxial (four-fold) MA along the 〈110〉c axis, we measured the planar Hall resistance (PHR) of a Hall bar with a size of 50 × 200 μm2 formed along the [100]c direction of a reference sample composed of LSMO (40 u.c.)/LAO (4 u.c.). This sample was grown on a non-doped STO (001) substrate under the same conditions as those for the sample used for the TAMR measurements. PHR is proportional to sin Φ cos Φ, where Φ is the angle between the magnetization and the current direction32. In Fig. θ is the angle between the magnetic field and the current flown in the [100]c axis. ΔR is defined as the PHR with respect to the one at a zero magnetic field. In contrast to the TAMR results, the PHRs of the reference sample measured when the magnetic field is parallel to the [110]c and i.e. four-fold like) except the opposite signs, showing no clue of the uniaxial MA along the [110]c axis. On the other hand, the PHRs clearly show a dominant uniaxial MA with the easy axis along the [100]c direction. Because PHR mainly reflects the bulk properties of LSMO, our results confirm that the C4〈110〉 and C2[100] components are the MA inherited from bulk LSMO, while the C2[110] component is the MA that appears only at the LSMO/LAO interface.To confirm the interface origin of the uniaxial MA component along the [110]C2[110] component is attributed to a symmetry breaking between the [110]c and C2[110]. Recently, it has been clarified that adjacent corner-sharing oxygen octahedra in LAO grown on STO (001) rotate in the opposite directions around the 34, and that the OOR in the underlayer is transferred to the first 3–4 u.c. layers of LSMO22 c plane of LSMO near the LSMO/LAO interface. We see that under the OOR around the c direction rotate in the same direction (see the green oxygen spheres around Mn1 and Mn3). This rotation direction is the opposite to that of the adjacent rows of Mn atoms (see Mn2 and Mn4). When Fig. c plane (dotted line). Because the vertical O–Mn1–O bonds are rotated in the same direction as the O–Mn3–O bonds, the hopping integral (t13) between Mn1 and Mn3 remains nearly unchanged from the value of the bulk. On the other hand, when Fig. c plane  between Mn2 and Mn4. The difference between t13 and t24 yields the anisotropic DOS between the [110]c and C2[110] at the LSMO/LAO interface.Because the O22 Fig. . Figure ane Fig. , one canC2[110] at V = −0.2 V  measurements for LSMO35. Therefore, the emergence of positive C2[110] below V = −0.2 V is likely associated with the t2g band, which is located below the eg band. Although the t2g state is located at 0.5–1 eV below EF in bulk LSMO35, it is thought to be largely pushed up by the polar mismatch at the LSMO/LAO interface36. Thus, we attribute the sign change of C2[110] to the transition from the eg band (V > −0.2 V) to the t2g band (V < −0.2 V) at the LSMO interface. As mentioned above, due to the OOR at the LSMO/LAO interface, the DOSs of both the eg and t2g bands in the [110]c direction are larger than those in the eg orbitals enhances the double exchange interaction and strengthens the ferromagnetism, while the one between the t2g orbitals enhances the super-exchange interaction and weakens the ferromagnetism37. Therefore, the enhancement of DOS in the [110]c direction relative to that in the c axis the easy magnetization direction in the case of the eg orbitals, while hard magnetization direction in the case of the t2g orbitals. The transition between these two bands at V = −0.2 V thus leads to the opposite magnetization direction-dependence of the DOS, as consequently observed by TAMR5.The most striking feature found in our study is the sign reversal of 2 V Fig. . This bec and the uniaxial MA along [100]c, which originate from bulk LSMO, we found a peculiar uniaxial MA along the [110]c, which is attributed to the LSMO/LAO interface. The symmetry axis of this interface MA rotates by 90° at an energy of 0.2 eV below EF of LSMO, which is attributed to the transition from the eg band (>−0.2 eV) to the t2g band (<−0.2 eV). These findings hint an efficient way to control the magnetization at the LSMO thin film interfaces, as well as confirm the rich of hidden properties at thin film interfaces that can be revealed only by interface-sensitive probes. This work also suggests the use of the TAMR measurement as a simple but highly sensitive method for characterizing interfacial magnetic properties of magnetic tunnel junctions, which is important for developing spintronic devices.In summary, using TAMR measurements, we have successfully obtained a high-resolution map of the MA spectrum of LSMO for the first time. In addition to the biaxial MA along 〈100〉3  grown on a TiO2-terminated Nb-doped SrTiO3 (001) substrate  by molecular beam epitaxy (MBE) with a shuttered growth technique25. The fluxes of La, Sr, Mn, and Al were supplied by Knudsen cells. The LAO and LSMO layers were grown at 730 °C with a background pressure of 2 × 10−4 Pa of a mixture of oxygen (80%) and ozone (20%). After the growth, the sample was further annealed at 600 °C in ambient atmosphere for 1 hour to reduce the density of oxygen vacancies.The heterostructure used in this study consists of LSMO (40 unit cell (u.c.) = 15.6 nm)/LaAlO2 mesas were then formed by standard photolithography and Ar ion milling. Au wires were bonded to the Au electrode and the backside of the Nb:STO substrate by indium.For tunneling transport measurements, a 50-nm-thick Au film was deposited on top of the sample, and 600 × 700 μmI/dV-V characteristics were numerically obtained from the I-V data with a differential interval of 10 mV. See Supplementary Information for more details on how to extract the The dentclass1pt{minimaThe datasets of the current study are available from the corresponding author on reasonable request.Supplementary Information


This label-free method holds potential for point-of-care diagnostics, as it is low-cost, fast, sensitive, and only requires a standard laboratory microscope for detection.Disease diagnostics requires detection and quantification of nano-sized bioparticles including DNA, proteins, viruses, and exosomes. Here, a fluorescent label-free method for sensitive detection of bioparticles is explored using a pillar array with micrometer-sized features in a deterministic lateral displacement (DLD) device. The method relies on measuring changes in size and/or electrostatic charges of 1 µm polymer beads due to the capture of target bioparticles on the surface. These changes can be sensitively detected through the lateral displacement of the beads in the DLD array, wherein the lateral shifts in the output translates to a quantitative measurement of bioparticles bound to the bead. The detection of albumin protein and nano-sized polymer vesicles with a concentration as low as 10 ng mL The detection and quantification of bioparticles is important to a range of applications including disease diagnostics. Here, the authors present a fluorescent label-free detection method using deterministic lateral displacement and demonstrate its use in detecting a range of proteins and vesicles. However, these methods use fluorescent labels that increase detection cost and complexity by reliance on expensive optical systems and involvement of multiple sample processing steps requiring minimum sample volumes. Thus, fluorescent label-free bioparticle detection gains traction as an alternative means in disease diagnosis.Disease diagnosis requires identification and quantification of various bioparticles such as DNA, RNA, proteins, virus, exosomes, and bacteria. Current clinical laboratories use well-established sandwich assay, PCR, gel electrophoresis, and flow-cytometry methods for detection of these bioparticles3, surface-enhanced Raman scattering (SERS)4, surface plasmon resonance (SPR)7, magnetic beads8, electrochemical detection9, and quartz crystal microbalance10 provide real-time information on bioparticle interactions, resulting in greater understanding of biochemical functions, drug interactions, and sensitive quantification of these bioparticles. The biosensor tracks changes in biophysical interactions of binding events, mass changes, refractive index or chemical reactions, and transduces the information as mechanical, electrical, or optical signals, and have shown detection of proteins down to femtomolar levels. However, these techniques often require precision engineering of nano-features, complex optical setups, secondary antibodies in sandwich assays, novel nanoprobes  or additional amplification step such as aggregation of nanoparticles to reduce the limit of detection (LOD)11.Technological advancement in label-free methods using microcantilever15. For a fixed critical DLD cut-off size (Dc), larger particles get displaced laterally relative to particles smaller than the Dc17. To separate nano-sized particles, it is challenging and costly to operate due to nanofabrication, precision injection of sample, and low throughput due to the small gap size18. So far, DLD research has mainly been focused on bioparticle separation and potential use of this technique for detection has not been extensively explored20.Deterministic lateral displacement (DLD) pillar array platforms have been used for size-sensitive separation of circulating tumour cells to bioparticles such as DNA and exosomes−1 (150 pM) and 3.75 μg mL−1, respectively is demonstrated. This work sets the precedent for sensitive detection and quantification of biological particles via sensitive changes in size or electrostatic interactions of the microbead carrier on DLD. We pushed the boundaries of DLD and applied our model for fluorescent label-free detection of nano-sized proteins and polymer vesicles, which open opportunities for low-cost medical diagnosis, liquid biopsy, and detection of biologically relevant DNA, RNA, exosomes, viruses, and proteins.Here, a fluorescent label-free method for sensitive detection of nano-sized proteins and polymer vesicles using a DLD pillar array with micrometer-sized features is demonstrated. Bioparticles of interest are captured or adsorbed onto polymer microbeads with specific ligands and detected quantitatively based on lateral displacement of the microbeads in the pillar array. Two domains exist for this bioparticle detection phenomenon: for small bioparticles, electrostatic interactions dominate, and for large bioparticles, physical particle size increase has a dominant role. The detection is performed through lateral displacement changes as a result from the modulation of microbead surface charge or size induced by the adsorption of bioparticles. The extent of the lateral displacement can be correlated to the amount of bioparticles in the sample. Using this bioparticle-on-bead method, changes in lateral shift correlating to the bioparticle concentrations can be sensitively discriminated. The detection of albumin proteins and nano-sized polymer vesicles with a concentration as low as 10 ng mL21  surface with 1 µm polystyrene (PS) National Institute of Standards and Technology (NIST) beads and NaCl solutions of different concentrations  and dynamic light scattering (DLS)  would alter the electrostatic interactions by increasing the mean Dapp to + 342 nm compared with pure native PDMS in NaCl solution. The use of HCl on the contrary reduces the electrostatic influence of the highly-charged plasma activated PDMS surface25. This is expected as the association of H+ to the surface of the DLD device would result in reduction of negatively charged groups, thus reducing electrostatic interactions to + 135 nm mean Dapp. It is important to note that the surface charge of plasma-treated PDMS is still negative as it is still beyond its isoelectric point.Figure Dapp shift of + 167 nm, which is similar to plain PS beads at + 164 nm. Further measurements of colloidal zeta-potential suggest similar colloidal stability and potential of – 21.4 mV for PS and – 29.5 mV for PS-COOH in deionised (DI) water. To induce a positively charged bead, the PS-COOH bead was coated with positively charged PAH which would physically adsorb to the negatively charged bead. The zeta potential measurement shows the PAH coating results in the + 43 mV surface zeta potential in DI water. The beads were flowed into the DLD setup and as expected, the positively charged beads attracted to the negatively charged DLD device surface at the entrance of the reservoir and could not enter the DLD pillar region -, and poly-allylamine hydrochloride (PAH)-coated beads were also tested. Using the same plasma-activated surface, COOH beads displayed a mean Dapp shift. The amount of Dapp shift is hypothesized to correlate to the amount of proteins on the bead surface. Native albumin protein has a hydrodynamic radius of 3.7 nm and a protein coated bead would increase the theoretical hydrodynamic size of the bead by at most 10 nm28. This size difference is lower than the resolution of our device . Figure −1 albumin protein-coated COOH beads in alkaline NaOH solution of concentration 2.5–10.0 mM (pH 11.4–12). At 2.5 µg mL−1 of albumin, the protein coat formed on COOH beads have a mean Dapp difference of 18 nm. However, at 5.0 µg mL−1, the mean difference has increased to 44 nm, which shows difference in peaks across all alkaline pH. The most significant observable difference is at 10 µg mL−1 protein concentration where there is a relatively large increase of 127 nm in Dapp of coated beads compared to the non-coated beads in alkaline pH 12 NaOH solution  buffer and lower concentrations of the beads. This, combined with the use of pH 12 NaOH solution for DLD separation, results in a significant decrease in the limit of albumin detection concentration Fig. . Four in−1, than the physical adsorption of albumin on 1 µm bead. Similarly, n = 3 independent sets of readings were performed using three PDMS device and the corresponding −1 of HSA protein were 771–842 nm, respectively  antibody were used to detect the presence of HSA Fig. . The binely Fig. . The \do37 Furthermore, integral proteins present in the membrane of EVs have significant roles in mediating these communications and have emerged as biomarkers for exosomes in various pathological and normal conditions.43 Thus, the detection of EVs and its membrane proteins is crucial for disease diagnosis. Owing to advantages of mechanical stability and membrane tunability, polymer vesicles were chosen over lipid vesicles for incorporating the membrane proteins for the current study.46 BD21 vesicles with a TEM size range of 132 ± 31 nm were prepared and characterised for size, shape, and surface charge  have importance in intercellular communication, regeneration, and transport.−1 under 0.1 × phosphate buffered saline (PBS) media . It is important to note that the reconstituted vesicles are smaller compared to the original size of vesicles which could be due to a faster rate of detergent removal  assay . As most EVs contain surface markers and proteins, Aqp1 was reconstituted onto polymer vesicles to demonstrate the detection by antibody coated beads in DLD device based on change in bead size. Aqp1 is a membrane pore protein which allows the permeability of water and was used as a model protein for detection of nano-vesicles.−1 down to 3.75 µg mL−1 , its cells type, pathological status , and purpose.The binding specificity of the BD21 nano-vesicles to the antibody-conjugated beads was confirmed using fluorescent probes Fig. . InteresL−1 Fig. . Four seL−1 Fig. . The det18. The attachment of bioparticles on microbeads also significantly reduces the effect of diffusion. Specific ligands can be immobilized on different microbeads to capture different bioparticles of interest. Moreover, the electrostatic interactions between surface proteins and DLD pillars could be modulated in real-time using different buffer ionic concentrations and the resultant lateral shift of the microbeads can be used to detect different amounts of proteins21. The detection of HSA proteins on beads via electrostatics dominant change in DLD has a LOD of 10 ng mL−1, whereas detection of polymer vesicles based on particle size change has a detection limit of 3.75 µg mL−1. The vesicle membrane protein detection by antibody-coated beads in DLD device can be further extended to specific detection of EVs such as exosomes based on their respective membrane proteins.Using albumin and polymer vesicle as proof of concept, we demonstrated a fluorescent label-free method for detection of nano-sized albumin proteins and vesicles using a PDMS DLD pillar array with micrometer-sized features. The advantages of this method are multi-fold. Fabrication of such a pillar array with micrometer-sized features is much less challenging as compared with the devices that require nanofabrication. The fluid flow does not require high pressures and the detection can be easily performed using standard bright-field bench-top microscopesWe have demonstrated that the fluorescence label-free method for nano-sized bioparticle detection is inexpensive, sensitive and only requires a standard laboratory microscope for the measurement of bead lateral position in the DLD PDMS device. Furthermore, with a 50 fps capture framerate, it is possible to integrate the detection onto portable imaging solutions and hold great potential for use in point of care diagnostics.Dapp and DLD System 2 (DLD-S2) was designed for high resolution displacement. DLD-S1 has a 100 nm Dapp increment measurement and DLD-S2 is 50 nm. The two systems also have different sensitivity for separation of 1 µm bead substrates in ionic buffer. DLD system 1 (DLD-S1) has dynamic range from 1 µM to 3 mM, whereas DLD system 2 (DLD-S2) has greater range of 0.5–150 mM. These DLD devices have 14 DLD segments connected in series with gap sizes fixed at 4 µm for DLD-S1 and 2 µm for DLD-S2. This results in a particle size resolvable quasi-resolution of 34 nm for DLD-S1 and 17 nm for DLD-S2. The input sample stream is sandwiched by two buffer streams to a width of a single input channel.Using Eq. 13. This technique uses pillar array with certain gap which is tilted in an angle and generates unique number of streamline between the gap that can laterally displace particle above the critical diameter. This Dc of the separation is influenced by the gap between pillars and the row shift fraction. Davis et al.53 proposed an empirical formula for DLD array.54DLD is a robust label-free microfluidics particle separation technique pioneered by Huang et alDc will displaced laterally, whereas particles smaller than Dc flow through the array without any lateral displacement. Therefore, for lateral displacement to occur, the particle diameter (Dp) must be greater than Dc.Where 21. Using our DLD device, we can measure the effect of electrostatic forces by changes in the apparent size of the particles  and ionic concentration (c), temperature (T), Boltzmann constant e) and Avogadro number  of 50–200 nm to the microbead surface increases the overall size of the bead from a diameter (Dp) of 1 µm to Dapp of ~ 1.05–1.4 µm. It is hypothesized that the Dapp is increased as the amount of adsorbed vesicles increases. At lower vesicle concentration, the increase in Dapp is small due to random adsorption of small amount of vesicles on beads, whereas at higher concentration when the bead surface is fully adsorbed with the vesicles, the Dapp reaches saturation, and hence plateaus off. The usage of antibody specific to the membrane protein increases the sensitivity of the vesicle binding, and hence results in lower LOD of the vesicles.In contrast, the size-based change is dominant for larger bioparticles coat such as vesicles. The adsorption of the vesicles with a diameter  was used to develop the DLD device negative mould on a 4-inch silicon wafer at a height of 3 µm. The SU-8 was patterned using a hard chrome glass mask  on a SUSS-MA8 lithography mask aligner. The final device was fabricated using PDMS cured on the silicon SU-8 mould. The input and output holes were punched and the PDMS device was bonded onto a glass slide using a oxygen plasma treatment for 2 min in the March PX-250 plasma machine. All devices used in this work were fabricated from the same mould to ensure consistency in results.Three types of bead samples were used: 1 µm NIST PS beads , 1 µm amine beads , and 1 µm carboxylated beads . These beads were subsequently diluted to the required concentration of 0.1% (w/v) for all separation experiment. Sodium chloride , sodium hydroxide (Sigma S2770), and hydrogen chloride (Sigma H9892) were prepared as a stock solution of 1 M. Non-ionic Pluronic F-127 (Sigma P2443) was prepared at 1% (w/v), whereas albumin solution (Sigma A9576) was purchased as 30% (v/v) stock solution. The 50 mM MES buffer pH 5 solution was prepared for the optimized protein adsorption on beads. The 1 × PBS solution (Thermofisher 10010023) was used as the stock PBS solution for vesicle detection. All solutions were diluted subsequently in 18.2 MOhm cm Millipore ultra-pure DI water to the required concentration.–1, and 10, 7.5, 5, and 2.5 µg mL−1, and incubated for 15 min at room temperature with DI water as a buffer for the physical adsorption of albumin to the beads. Subsequently, the beads were washed with centrifugation for three times at 6000 r.p.m. for 3 min each, to get rid of the remaining unabsorbed albumin in the solution. The beads then were diluted to 15 µL for the experiment. The PAH (Sigma 283223) coating were performed by putting 3 µL beads to 0.01% (w/v) PAH in 0.5 M NaCl buffer for 5 min and the solution were washed 3 × to remove unabsorbed PAH solution. The optimized protein adsorption for more sensitive detection was performed using MES buffer and smaller volume of beads. Total of 0.5 µL of 2.64% (w/v) carboxylated beads were put into 1 mL of albumin solution in 50 mM MES buffer at pH 5.5 with concentration of 1, 0.75, 0.5, 0.25, and 0.1 µg mL−1, and incubated for 1 h at room temperature for the physical adsorption of albumin to the beads. Subsequently, the beads were washed with centrifugation for three times at 6000 r.p.m. for 3 min each, to get rid of the remaining unabsorbed albumin in the solution. The beads then were diluted to 3 µL for the experiment.Total of 3 µL of 2.64% (w/v) carboxylated beads were put into 1 mL of albumin solution with concentration of 1, 0.5, and 0.1 mg mlN-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride  and N-hydroxysuccinimide  coupling. Briefly, EDC and NHS was added to PS-COOH beads to activate the carboxyl groups and mixed under vortex for 1 h at 1650 r.p.m., at 4 °C. After activation, the beads were washed three times by centrifugation at 6000 r.p.m. for 5 mins. Before adding the antibody, the bead solution was subjected to probe sonication for 1 min, to ensure uniform dispersion of the beads. The antibody-beads solution was incubated for 3 h at 1650 r.p.m. vortexing at 4 °C.Polyclonal rabbit anti-HSA  was diluted 80 × and conjugated on 1 µm PS-COOH beads surface via −1 for 1 h at room temperature and washed by centrifugation at 6000 r.p.m. for 3 min before putting in the sample inlet for DLD separation with 10 mM NaOH buffer. The device used was plasma-treated PDMS, which has been treated with pluronic F-127 for 30 min to prevent particle adhesion. The number of particles in the sub-channels were counted and plotted as the particle output distribution. The mean of the distribution would correspond to the apparent particle size in the DLD device. The LOD of the vesicle was then determined from the apparent diameter difference with the uncoated beads.The conjugated beads were mixed with different concentration of HSA (Abcam) of 0.075, 0.05, 0.025, and 0.01 µg mL) was obtained to prepare vesicles, referred here as BD21, by film hydration method. Briefly, 5 mg of polymer was dissolved in 200 µl of chloroform, which was evaporated slowly by using stream of nitrogen in fume hood to make a thin film of polymer. To prepare dye labelled vesicles, Rhodamine B octadecyl ester perchlorate  dye was added in the polymer solution in chloroform before making the thin film. Polymer thin films (with and without RBOE) was vacuum dried for 4 h. To this, 1 mL of 1 × PBS (pH 7.2) was added and stirred overnight on magnetic stirrers  at 400 r.p.m. to make vesicles that was downsized by extrusion (6 times through 0.45 µm and 6 times through 0.22 µm filter) and dialysed to remove the free RBOE. Both BD21 and BD21-RBOE vesicles were characterized for size and surface charge by ZetaSizer  and transmission electron microscopy . To confirm the incorporation of RBOE dye in vesicles, fluorescence intensity spectra was measured by fluorescence spectroscopy at excitation of 533 nm.Poly(butadiene-b-ethylene oxide) (PBd(1200)-PEO(600)) di-block co-polymer  was added in 1:1 weight ratio to vesicle and incubated for 1 h at 4 °C. After this, 200 mg of Bio-Beads SM-2 was added for detergent removal accompanied with incorporation of Aqp1 in polymer vesicles. The Aqp1 reconstituted vesicle was characterized for vesicle size and fluorescence intensity by DLS and microplate reader, respectively. Furthermore, confirmation for the presence, functionality, and quantity of Aqp1 in vesicle after reconstitution was done by immunoassay by attaching them on PS micro-beads, osmotic permeability assay, and BCA assay, respectively.As prepared RBOE-BD21 vesicles were mixed with 1 µm-size PS beads and incubated at 1650 r.p.m. for 1 h. After this, the beads were washed three times by centrifugation at 6000 r.p.m. for 5 mins. The vesicles attachment on to the beads was characterized by the fluorescence intensity measurement at 533 nm excitation by Infinite M200 PRO multimode microplate reader (Tecan) and bright-field and fluorescence microscopy imaging.Polyclonal rabbit anti-Aqp1 antibody against Aqp1 membrane protein  was diluted 20 × and conjugated on 1 µm PS-COOH beads surface via EDC/NHS coupling. Briefly, EDC/NHS was added to PS-COOH beads to activate the carboxyl groups and mixed under vortexing for 1 h at 1650 r.p.m., at 4 °C. After activation, the beads were washed three times by centrifugation at 6000 r.p.m. for 5 min. Before adding the antibody, the bead solution was subjected to probe sonication for 1 minute to ensure uniform dispersion of the beads. The antibody beads solution was incubated for 3 h at 1650 r.p.m. vortexing, at 4 °C. The conjugation of antibody to beads was confirmed by binding of mouse anti-rabbit antibody against rabbit anti-Aqp1 bound to Aqp1 protein.Before adding the Aqp1-vesicles for detection, antibody conjugated beads were incubated with blocker solution , at 700 r.p.m. for 2 h, room temperature to reduce the nonspecific binding. After blocking, antibody beads were washed thrice at 6000 r.p.m. for 5 min at 4 °C and mixed with vesicles with and without Aqp1 for incubation at 700 r.p.m. for 1 h, room temperature. Next, the beads were washed thrice at 6000 r.p.m. for 5 min and mixed with primary antibody  at 700 r.p.m., 2 h, room temperature), washed thrice (6000 r.p.m. for 5 min at 4 °C) and mixed with secondary antibody . Finally, the beads were washed thrice at 6000 r.p.m. for 5 min at 4 °C before characterization using bright-field and fluorescence microscopy imaging.The conjugated antibody is mixed with different concentration of vesicles with Aqp1 membrane protein and put in the sample inlet for separation with 0.1 × PBS as the buffer. The DLD device used was native PDMS device which has been treated with pluronic F-127 for 30 min to prevent particle adhesion. The number of particles in the sub-channels were counted and plotted as the particle output distribution. The mean of the distribution would correspond to the apparent particle size in the DLD device. The LOD of the vesicle is then determined from the apparent diameter difference with the uncoated beads.The fluid flow in the microfluidic device was driven by output fluid extraction using a Chemyx syringe pump and a Hamilton 100 µl glass syringe. The input sample and buffer reservoirs were exposed to atmospheric pressure. This method facilitates rapid washing and change of buffer solutions. The experiment was visualised using an upright microscope and the particle flows for input and output regions of the device were captured using high speed Phantom M310 camera. The frame rates used were 50fps for the detection zone at × 100 magnification and 1000 fps for the high-speed imaging of individual bead motion within the DLD pillars at × 600 magnification. The number of particles flowing at different outlet sub-channels were counted and plotted as the particle output distribution for data analysis


Bst DNA polymerase following real-time fluorescence reverse transcriptase using genomic Zika virus standard strain (MR766), was conducted through fluorescent signaling. Among the six pairs of primers that we designate here, NS5 was the most efficient with a high sensitivity of up to 3.3 ng/μl and reproducible specificity on eight pathogen samples that were used as negative controls. The real-time fluorescence reverse transcription LAMP detection process can be completed within 35 min. Our study demonstrated that real-time fluorescence reverse transcription LAMP could be highly beneficial and convenient clinical application to detect Zika virus due to its high specificity and stability.To detect Zika virus more rapidly and accurately, we developed a novel method that utilized a real-time fluorescence reverse transcription loop-mediated isothermal amplification (LAMP) technique. The NS5 gene was amplified by a set of six specific primers that recognized six distinct sequences. The amplification process, including 60 min of thermostatic reaction with  Aedes aegypti mosquito in tropical regions, which must feed on blood to lay eggs  is a novel technology that has been developed in recent years , backward outer primer (B3), forward internal primer (FIP), backward internal primer (BIP) and loop backward primer (LB) were designed by Guangzhou Deaou Biotechnology Company Limited and synthesized by Tiangen Biochemical Technology (Beijing) Company Limited. The sequences of the primers used in this study are listed in Tables , backwarBacterial genomic DNA extraction kit was purchased from Tiangen Biochemical Technology Company Limited. The LAMP reaction system was prepared according to the manual for the LAMP amplification kit.Four sets of primers were designed: NS5-1, NS5-2, NS5-3, and NS5-4. Each of the working fluids to Zika virus RNA served as a template. The reaction procedure was set to 63 °C 15 s, 63 °C 45 s as one cycle for 60 cycles, 95 °C 15 s to terminate the reaction, and 63 °C 45 s at the collection of fluorescence signals. A reaction tube was used for the dissolution curve detection and to compare the amplification efficiency of four sets of primers while screening out the highest amplification efficiency without a primer dimer.The RNA concentration of Zika virus was measured by adjusting the RNA template concentration with 1 μl of TE buffer to the Thermo Scientific Nanodrop 2000 microphotometer and 1 μl of RNA extract of Zika virus. 10 μl of RNA stock solution was diluted 10-fold with ultra-pure water four times to obtain five RNA template concentrations. This was added to the LAMP system reaction, and the amplification curve was observed to evaluate the sensitivity of the primers.The genomic DNA of Zika virus and other negative pathogens were extracted and amplified according to the reaction conditions of Real-LAMP. The specificity of the primers was evaluated. One of the positive strains and five negative strains were tested three times with LAMP. The same primer used for the repeat test was used to evaluate the reliability of LAMP.Four sets of primers amplified without the loop primer showed that the primers NS5-3 had a peak at about 39 min and that the NS5-1 primers had a peak at about 31 min. The efficiencies are both lower than those with the loop primers. The NS5-2 and NS5-4 primers had no peak (Figs. At approximately 15 min after the initiation of the reaction, the system of the primer NS5-3 peaked, and at approximately 21 min, the reaction of the NS5-1 primers peaked. The Bio-Rad CFX Manager detected the amplification signal of the NS5 gene, NS5-2 and NS5-4 without a peak (Fig. In this study, the clinical common pathogens were selected as negative controls, but no Zika virus RNA was amplified. The typical “S” curve showed that the specificity of Zika virus RNA was better. The LAMP primer NS5-1 has good specificity and does not cross-react with non-target bacteria (Fig. Measured by micro-spectrophotometer, the experiment’s Zika virus nucleic acid concentration is 33 ng/μl. Diluted by 4 gradients, the resulting concentrations were 3.3, 0.33 ng/μl, 33 and 3.3 pg/μl. The limit of the detection of this test can reach 3.3 ng/μl. Reliable test results are shown in Fig. Take Zika virus NS5 for three replicates, wherein two peaks were at 23 min and another peak was at 20 min. The difference in peak time is approximately 3 min. The repeatability of the tests is shown in Fig. Flaviviridae family and the Flavivirus genus and is thus related to the dengue fever, yellow fever, Japanese encephalitis, and West Nile viruses (Zuanazzi et al. The Zika virus belongs to the In this study, the NS5 gene was selected as the target gene and 8 clinical common pathogens were used as negative controls. The yellow fever virus was theoretically selected as the negative control.NS5 gene sequences were successfully amplified and displayed as a typical ‘S’ shape in Zika virus, except for other negative pathogens, which were used as negative controls, indicating that the NS5 gene sequence as a highly specific candidate could be used in Zika detection. The results showed that the ring primer could significantly shorten the reaction time. The reaction of the ring primer lasted approximately 22 min, and the peak time of the ring primer was less than 10 min. The entire reaction process can be completed within 35 min, which is significantly shorter than that for PCR and is conducive to the clinical diagnosis of Zika virus infection, the timely detection of Zika virus infection, and timely treatment.Traditional PCR methods are based on agarose gel electrophoresis, which are cumbersome to operate (Wong et al. LAMP detection technology has the following advantages compared with other methods. The primers used in the LAMP detection technique are based on six primers designed for the target sequence (Abdul-Ghani et al. However, there are limitations. For example, inevitable aerosol pollution may lead to false positive results (Asiello and Baeumner Our current study demonstrated that the application of LAMP to detect Zika virus was superior to traditional methods with a high sensitivity and specificity. As a rapid and efficient novel method, LAMP may be a beneficial tool for clinical application.


There is an increasing recognition that non communicable diseases impose large economic costs on households, societies and nations. However, not much is known about the magnitude of diabetes expenditure in African countries and to the best of our knowledge no systematic assessment of the literature on diabetes costs in Africa has been conducted. The aim of this paper is to capture the evidence on the cost of diabetes in Africa, review the methods used to calculate costs and identify areas for future research.A desk search was conducted in Pubmed, Medline, Embase, and Science direct as well as through other databases, namely Google Scholar. The following eligibility criteria were used: peer reviewed English articles published between 2006 and 2016, articles that reported original research findings on the cost of illness in diabetes, and studies that covered at least one African country. Information was extracted using two data extraction sheets and results organized in tables. Costs presented in the studies under review are converted to 2015 international dollars prices (I$).Twenty six articles are included in this review. Annual national direct costs of diabetes differed between countries and ranged from I$3.5 billion to I$4.5 billion per annum. Indirect costs per patient were generally higher than the direct costs per patient of diabetes. Outpatient costs varied by study design, data source, perspective and healthcare cost categories included in the total costs calculation. The most commonly included healthcare items were drug costs, followed by diagnostic costs, medical supply or disposable costs and consultation costs. In studies that reported both drug costs and total costs, drug costs took a significant portion of the total costs per patient. The highest burden due to the costs associated with diabetes was reported in individuals within the low income group.Estimation of the costs associated with diabetes is crucial to make progress towards meeting the targets laid out in Sustainable Development Goal 3 set for 2030. The studies included in this review show that the presence of diabetes leads to elevated costs of treatment which further increase in the presence of complications. The cost of drugs generally contributed the most to total direct costs of treatment. Various methods are used in the estimation of diabetes healthcare costs and the costs estimated between countries differ significantly. There is room to improve transparency and make the methodologies used standard in order to allow for cost comparisons across studies.The online version of this article (10.1186/s12992-017-0318-5) contains supplementary material, which is available to authorized users. Diabetes mellitus is now one of the major challenges for many of the health systems in Africa . For a lThe increase in prevalence and premature mortality due to diabetes imposes huge financial costs to households and governments  whist plIndividuals with diabetes are likely to experience one or more chronic illnesses such as heart disease, and kidney disease . As the The usefulness of the results presented in cost of illness (COI) studies has often been questioned due to the wide variation in methods used , 13. DesRecent reviews on costs of diabetes have almost exclusively focused on developed countries, the United States of America and European countries, with a small number of African countries included –17. ReviThis literature review was guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines . The guiA desk research was conducted in order to identify articles for inclusion. The literature search was undertaken in PubMed, EMBASE, Medline and Science Direct. Searches were also conducted in the WHO Global Health Library and IDF. Additional searches were also undertaken in Google Scholar and manual searches were also undertaken in Google in order to identify publications that may not be indexed in international databases. Our PubMed search strategy is provided in an additional file  to identify studies to include in the review. If based on the article title alone, it was unclear whether the study was relevant with regard to our research question, the article’s abstract was then screened. The third and final step involved assessing for eligibility via full text review to determine if the inclusion and exclusion criteria were satisfied. Articles were then downloaded for a full-text review. In circumstances where the reviewer or a library assistant failed to access the full text, emails were sent to the corresponding authors whose contact details could be obtained, requesting a copy of the missing studies. Reference lists of all eligible articles and reference lists of excluded reviews were also screened for additional relevant material. In every step of the selection process if there was any doubt regarding the inclusion/exclusion of an article another author was consulted.Eligibility criteria were determined by relevant elements of the PICOS guidance for undertaking literature reviews . Papers In order to extract information from studies, we developed two extraction tables. One table was used to extract details such as year published, research objectives, study design and types of costs estimated. To present information on cost data across studies, we grouped the costs as follows: outpatient costs, inpatient costs, cost of drugs and combined costs. The combined costs category included studies in which inpatient and outpatient costs per patient were not separated. This category also included indirect costs due to loss of income, disability and premature mortality.Two adjustments were made in the presentations of costs. First, in cases where relevant unit costs were not provided, these were calculated wherever possible using the information provided in the study. Second, in order to increase comparability of costs across studies we converted costs into international dollars (further denoted with the I$ sign) using the 2015 purchasing power parity (PPP) estimates. We followed the approach used by Seuring et al. . AdjustmThe second table was used to list the technical criteria applied in assessing the quality of each study. A number of check lists have been developed for the evaluation of health economics studies, most of which focus on full economic evaluations such as cost benefit analysis, cost-effectiveness analysis and cost utility analysis . AlthougThe initial search strategy yielded a total of 799 articles, from which 178 duplicates were excluded. Of the remaining 621 articles, 387 were removed during the first level of title screening and 177 removed during the second level of abstract screening leaving 57 articles for full text evaluation. Of the 57 studies that underwent full text review, 32 were excluded for one of the following reasons: studies were not in English, were conference presentations, full text of articles was not available, were not peer reviewed, was a literature review of diabetes (not costs) in Nigeria, did not provide patient specific costs or presented costs that had been calculated in another included study  followed by East Africa (n = 6), Southern Africa (n-5), Northern Africa (n = 2) and Central Africa (n = 2). One study estimated costs for three groups of countries within the WHO African Region.The general characteristics of these twenty six studies are provided in Table In conducting COI studies, previous literature reviews have suggested that it is important for the study to firstly define the illness, epidemiological sources, type of costs and study perspective. After which resource consumption and unit cost data can be collected and results presented along with sensitivity analysis , 30, 31.n = 11), nine studies considered the costs of both type 1 and type 2 diabetes, only one study considered the costs of type 1 diabetes, whilst five studies did not clearly define the type of diabetes assessed , followed by the health system perspective (n = 2), the combined health system and patient perspective (n = 2) and the government perspective (n = 1). Ten studies did not mention what sort of perspective was adopted, however based on reviewer interpretation we were able to classify these , the employer perspective (loss of productivity), health system perspective , government perspective  and societal perspective (loss of income while caring for the sick) . The socThe majority of the studies used medical centers or hospital data as their cost data sources (13 studies). In three studies cost data were obtained from patient interviews , 42, 43,The estimation of resource consumption can be prospectively or retrospectively performed . In prosAll studies included in the review provided sufficient information to calculate per capita costs and four studies extended the national costs of diabetes , 50, 56.The direct costs considered in the studies under review were medical and non-medical costs. Detailed costing of the studies under review is provided in Additional file One crucial challenge highlighted in some of the studies was the OOP expenses incurred by patients. This challenge has also been reported elsewhere , 59. OOPn = 12), transportation costs (n = 9), and consultation costs (n = 7). Less than four studies reported on the costs associated with each of the following healthcare components separately; surgery, insurance premiums, service costs, physiotherapy, disposables, personnel, capital and diabetic diet costs.The cost of drugs was reported separately in a majority of the studies (65%). In studies reporting both drug costs and total costs of treatment, drug costs often took up a significant portion of total costs of treatment costs, 14% - 90% in Nigeria, 64% in Ethiopia, 53% in Sudan, 14% in the Seychelles, 4% to 7% in South Africa and 5% in Uganda (see Table Three studies that separately provided the costs associated with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) show that the direct costs of T1DM were higher than T2DM , 55, 56.Five studies included in the review estimated the costs of specific types of complications , 51, 60.A few other studies investigated the cost of diabetes in relation to income levels, age and by comparing costs of public versus private healthcare providers. Studies found that the highest burden of diabetes was amongst individuals of low socio-economic status , 57. OkoMost studies presented and explained their results in a clear way, consistent with the methodology of the study. Presentation of results was generally in agreement with the study aim and the conclusions were made in line with the results presented. More than 70% of the included studies carefully described the epidemiological sources, activity data, and unit costs. A few studies (24%) included in this review did not discuss any limitations regarding the methodologies employed in calculating costs. A common weakness discussed was that studies that collected data from patient interviews were subject to recall and social desirability bias. Also, the use of one study site and small sample sizes meant that results were not applicable to other sites or national estimates. The studies by Boutayeb et al.  and KiriCosts were discounted in three studies and the discount rate chosen was 3%. However, the discount rate chosen was not explained , 39, 46.Based on the methodological points discussed above, Table There has been growing concern over the increase in the prevalence and burden of NCDs. Endorsements such as the Moscow Declaration on NCDs in May 2011 and the Political Declaration on the prevalence and control of NCDs in September 2011 have been instrumental in raising awareness on the urgent need to place NCDs on the government agendas , 62. Foln = 26). Seuring et al. find that direct costs are much higher than indirect costs mostly in high income countries [This study contributes to the understanding of the costs of diabetes in Africa and the methods used to estimate these costs. The first objective of this review was to identify and capture the evidence presented in the literature on the overall direct and indirect costs of diabetes in Africa that have been published since 2006. The literature search identified 26 studies that met the eligibility criteria of this review. Our findings suggested that the annual economic burden of diabetes in Africa was huge. Most of these healthcare costs in Africa are borne by the patients and this influences the attainment of proper care due to financial constraints . These countries .Similar to a systematic review conducted by Seuring et al. , we alsoLike other diabetes COI reviews drug costs, diagnostic costs and consultation costs are some of the most commonly reported items , 17, 65.Amongst the studies that reported on the cost of diabetes across various socioeconomic parameters such as income, the general consensus was that the economic burden of diabetes weighed more heavily upon those in the low income group  or low sOur second objective was to review the methods that have been used to calculate the costs of diabetes in Africa and assess the quality of the COI studies that have been included in the review. A majority of the studies were prevalence based which is considered to be the most suitable for measuring costs related to chronic diseases . This fiFrom this review it is clear that national estimates on the economic impact of diabetes in African countries are lacking. Therefore this review provides a fragmented picture of the economic impact of diabetes in Africa. In addition, a few studies estimated the indirect costs of diabetes. Although diabetes potentially affects the ability of individuals to participate in the labor market, evidence on labor markets effects is lacking. The estimates provided on the cost of diabetes did not take into account undiagnosed diabetes. As a result, it is highly likely that the aggregate costs associated with diabetes have been severely under estimated. Studies that provide national estimates of unmet diabetes are important for carefully estimating the national impact of the disease.The current review has some limitations. The exclusion of articles not written in English could have led to the omission of relevant articles in the area under study. The review also focused on peer reviewed articles and excluded grey literature such as academic thesis. The exclusion of this literature might introduce some bias into the review. A broader search without this restriction would yield different results. Like many other checklists, the results presented in the checklist may not be directly replicable since the results are a subjective assessment by the reviewer. In addition the checklist used in this review does not give weighting scores on the various items included in the list. As a result all items are given equal scoring although some items influence results more than others . Due to There has been a growing global focus on NCDs since the United Nations High Level Meeting on NCDSs in 2011. More recently, the specific inclusion of NCDs in the SDGs provides African countries with an opportunity to directly focus on and increase the momentum in tackling NCDs. SDG3 makes a commitment to reduce pre mature mortality in NCDs, to eliminate inequalities in healthcare costs and protect patients from diabetes induced catastrophic health expenditure, amongst others. In order to accelerate the progress in meeting SDG3 set for 2030 consideration must also be given to promoting early detection and monitoring the prevalence of NCD risk factors. The adoption of policies targeted at a reduction in harmful alcohol use, tobacco consumption and physical inactivity will contribute to a reduction in NCD prevalence and therefore healthcare costs.This review provides a snapshot of the costs and economic burden of diabetes in Africa. We found that many studies on the cost of diabetes were conducted in West Africa and focused on the direct costs. This provides the West African region sufficient information to act on establishing cost effective treatments that could influence a reduction in cost. Few studies estimated the economic impact of the disease using the societal perspective and few studies estimated the costs of diabetes at the national level. A common conclusion in the studies included under the review is that diabetes imposes a considerable economic burden on individuals and that amongst the cost components included in cost calculation, the cost of drugs imposed the largest burden on total costs incurred. These areas call for policies that focus on a reduction in the burden of diabetes on individuals and the promotion of prescriptions of diabetes drugs in their generic names. As the prevalence of diabetes in Africa is expected to rise, the economic burden on individuals will also continue to increase. COI studies are vital in providing information that supports economic evaluations and policy development. Future research should focus on increasing the transparency and methodological principles of COI studies.Additional file 1:Search strategy. (DOCX 14 kb)Additional file 2:PRISMA Checklist. (DOCX 18 kb)Additional file 3:Articles included in review in alphabetical order. (DOC 27 kb)Additional file 4: Table S1.Components of direct and indirect costs for diabetes mellitus and reflecting cost. (DOCX 52 kb)Additional file 5:Cost ratio for individuals with and without diabetes mellitus complications (DOCX 21 kb)


Zaire ebolavirus  exhibit separate phenotypes when challenged with a range of sodium hypochlorite (NaOCl) concentrations or 70% ethanol (EtOH) at average West African temperature. The time dependent killing of Ebola virus was evaluated by measuring infectious virus and viral RNA (vRNA), to determine if RNA detection is a viable method for decontamination measurement in areas without high containment laboratory access. Makona was less susceptible to weaker concentrations of NaOCl (0.05 and 0.1%) than Mayinga and Kikwit. At the recommended concentration of NaOCl (≥0.5%) all of the variants were inert after 5 minutes of contact time. Similarly, all variants were inactivated by 70% EtOH after 2.5 minutes, only Makona was detected at 1 minute. In multiple instances, high amounts of vRNA was detected in the absence of infectious virus, suggesting that it does not serve as an accurate measure of remaining infectivity after cleansing.The recent Ebola virus outbreak in West Africa has forced experts to re-evaluate their understanding of how to best disinfect areas contaminated with infectious bodily fluids. Recent research has found that Ebola virus remains viable in blood for 7–10 days making appropriate disinfection crucial to infection control. We sought to determine if the three most important outbreak variants of  The ZEBOV subtype has proved the most deadly to the population of Central and West Africa with three major outbreak variants. The 1976 outbreak variant Mayinga was responsible for 380 cases with 218 fatalities ), and the 1995 Kikwit variant caused 315 cases with 250 mortalities (81% CFR)34The Published environmental studies on Ebola have been rather limited. The severity of the West African outbreak has led to a surge in investigations. These efforts have uncovered that Ebola virus can remain viable for significantly lengthy periods of time on multiple surfaces found in the environment and as a component of personal protective equipment (PPE)678912Ebola virus can persist in dried human/non-human primate blood 7–10 days at West African climate conditions (28 °C/90% relative humidity (RH))714Environmental recovery of the Ebola virus uses two metrics, titer of infectious virus and quantity of vRNA to determine virus presence. Field studies predominately focus on measuring vRNA by quantitative RT-PCR (qRT-PCR) to detect Ebola-contaminated surfaces. Such a measure may be misleading as vRNA detection cannot be used to confirm recovery of infectious virus particles and time of disinfection to time of sampling varies greatly. In addition, a few studies have demonstrated that genetic material is recovered much more efficiently than infectious virus particles for Ebola8This study examined the disinfection of Makona compared to the other two major ZEBOV outbreak variants, Mayinga and Kikwit with common chemical disinfectants that are recommended by the World Health Organization (WHO) for contaminated-surface and hand cleaning during Ebola outbreaksTo identify if the Makona variant characteristically differs with respect to decontamination, Makona was compared with the other major Ebola virus outbreak variants. The time-controlled action of 70% ethanol (EtOH) and 0.05%, 0.1%, 0.5% and 1% v/v concentrations of sodium hypochlorite (NaOCl) was tested against dried mixtures of organic soil load and ZEBOV variants on stainless steel carriers at 27 °C/30% RH. The organic soil load mixture mimics bodily excreta, similar to a severe EVD patient1810 TCID50/mL for Makona. The disinfection of all variants followed similar trends. The efficacy of the disinfectants positively correlated to increased concentration and contact time , input virus per carrier was determined prior to the addition of disinfectants as 6.6 (+/−0.3) for Mayinga, 6.5 (+/−0.2) for Kikwit and 6.8 (+/−0.2) logact time . These a10 TCID50/mL at 1 min disinfection time. These differences were determined to be not significant by one-way ANOVA with Holm-Bonferroni post-hoc analysis. Infectious virus was not detected between 2.5 to 10 min of contact time with disinfectant (When treated with the single concentration of EtOH (70%) at contact times of 1 through 10 min, Mayinga and Kikwit variants were fully inactivated by 1 min of contact time . Howevernfectant .10 TCID50/mL of virus was recovered for each virus: 3.7 (Mayinga), 3.4 (Kikwit) and 4.5 (Makona) treated with 0.05% NaOCl , 5 min  and 10 min . This pattern was also evident for Makona versus Kikwit at 1 min , 2.5 min , 5 min , 7.5 min  and 10 min  , 2.5 min  and 5 min . Similar trends in higher post-treatment titres were observed at the time points of 1 min , 2.5 min , 5 min  and 7.5 min  in comparison of Makona to Kikwit (The weakest concentration of NaOCl (0.05%) revealed differences in Makona viability when compared to both Mayinga and Kikwit. Using a time-specific approach to compare the quantity of Makona to both Mayinga and Kikwit remaining after treatment, Makona had statistically significantly higher titres remaining after treatment, expressed as log< 0.001) . A strono Kikwit .0) to 10−7, and viral replication is assessed by the appearance of cytopathic effect (CPE). However, it is important to note that CPE caused by the variants were only visible at the 10−1 dilution, as the undiluted (100) samples which contained the combination of 0.5% or 1% NaOCl and its neutralizer (1% sodium thiosulfate) is cytotoxic to cell monolayers and this complicated the observation of CPE. The absence of virus from 10−1 to 10−7 would suggest a complete kill had occurred. Thus to confirm complete kill, flasks of VeroE6 cells were infected by pooling the triplicate undiluted (100) samples from each biological replicate, and incubated for 14 days to await CPE development. The absence of CPE indicated that the 100 dilution was negative for virus (defined as complete kill) while the presence of CPE indicated that 100 was indeed positive for virus , as the combination of 0.5% or 1% NaOCl with 1% sodium thiosulfate did not display any signs of cytotoxicity  resulted in drastic reductions in Ebola virus titres for all variants. When 0.5% NaOCl was applied, the titre differences were subtle: at 1 min  and 2.5 min  Makona versus Mayinga and, at 1 min  and 2.5 min  Makona against Kikwit. The infection of VeroE6 cells in flasks revealed that all of the variants were inactivated by 5 min of contact (10 (p < 0.001) and 0.6 log10 (not significant), respectively. At 2.5 min, virus culture in flasks of VeroE6 cells indicated that virus was present at 100 for Mayinga, Kikwit and Makona, calculated as 1.8 log10 but, not viable after 5 min of contact  and genome equivalents (GE)/mL.10/mL and 12 (+/−0.4) log10/mL (10 TCID50/mL for Mayinga and 6.8 (+/−0.2) log10 TCID50/mL for Makona. After treatment with 70% EtOH, vRNA was detected at 1 min (15 and 10 Ct), 2.5 (13 and 10 Ct), 5 min (15 and 11 Ct), 7.5 min (16 and 9 Ct) and 10 min (15 and 11 Ct) . These controls demonstrated an average Ct of 11 (+/−1) and 11 (+/−2), and an average GE of 12 (+/−0.3) loglog10/mL . These vd 11 Ct) . The 1 m) , 7.5 min (18 and 18 Ct) and 10 min (19 and 20 Ct)  and thes10/mL . Similar10/mL , resultiL (10/mL .Disinfection of most major Ebola virus outbreak variants was achieved after 1 min of contact with 70% EtOH. Makona was an exception and required 2.5 min. Strong solutions of NaOCl (≥0.5%) inactivated the variants by 5 min of contact whereas, weaker concentrations of NaOCl (0.05% and 0.1%) were not able to fully inactivate any of the variants. Comparing the variants revealed that Makona was less susceptible than Mayinga and Kikwit to 0.05%, 0.1% NaOCl and 70% EtOH, but differences were not observed with higher concentrations of disinfectant. The discovery of high amounts of vRNA remaining after 70% EtOH and ≥0.5% NaOCl treatment suggested that genetic detection may not be a suitable methodology for accurately measuring disinfection as infectious virus may no longer be present.The United States Centers for Disease Control and Prevention (CDC) provides instructions for the daily preparation of the WHO recommended 0.5% chlorine for surface cleaning and 0.05% chlorine for hand washing used during Ebola virus outbreaks2While the genomes of the ZEBOV variants are 97% identical at the nucleotide levelet al., endorse the use of qRT-PCR to demonstrate decontamination efficacy in Ebola treatment units. These authors describe the detection of Ebola vRNA from swabbing performed at 30 and 60 min after cleansing with 0.5% NaOCl, thereby suggesting a nosocomial risk. Although interesting, it is noteworthy that attempts to culture Ebola virus from these swabs was not possible due to lack of suitable facility and containment resources at the treatment units, and nearly one-third of the swab sample-replicates were actually positive for vRNA with Ct values ranging from 32–39 (40 Ct was considered negative)et al., describe virus isolation by culturing was not possible in patients after 12 days of symptom onset or with Ct > 35.5, although this was also the case in some patients that had Ct values < 35.52829The lack of connection between Ebola virus particles and the amount of vRNA quantified after disinfection highlights the issue of using vRNA as a sole measure of decontamination success. Youkee The data presented indicates that the major Ebola outbreak variants can be efficiently inactivated with 70% EtOH by 2.5 min and ≥0.5% NaOCl at 5 min. Concentrations of NaOCl that are suboptimal for complete inactivation demonstrated that Makona was more resistant to disinfection than Mayinga and Kikwit. Performing qRT-PCR on inactivated Ebola samples demonstrated that high amounts of vRNA remained despite complete killing of infectious particles. Evaluation of effective decontamination should be determined or corroborated by assays that quantify infectious virus and not solely by the detection of viral components.2. For virus culture, virus maintenance medium was the same as growth medium with FBS reduced to 2%.African green monkey kidney (VeroE6) cells (ATCC CRL-1586) were propagated in growth media consisting of Dulbecco’s modified eagle medium (DMEM) (HyClone) which included 10% Fetal Bovine Serum (FBS) (Gibco) and 1% antibiotics (Penicillin-Streptomycin) (Gibco) at 37 °C/5% COEbola virus variant stocks of 1976 Mayinga, 1995 Kikwit and 2014 Makona (C05) were cultivated in VeroE6 cells within the Containment Level 4 (CL-4) laboratory at the National Microbiology Laboratory (NML) in the Canadian Science Centre for Human and Animal Health (CSCHAH) located in Winnipeg, Canada. Virus-containing supernatants were harvested at approximately 80% CPE (10–12 days post-infection). Virus samples were clarified by centrifugation at 5000 × g for 10 min followed by centrifugation through a 20% sucrose cushion for 2 hours at 108,000 × g to pellet the virus. Virus pellets were resuspended in virus maintenance medium and stored at −80 °C.Sodium hypochlorite (NaOCl) from 10.8% stock concentration  or ethanol (EtOH) from 95% stock concentration  were diluted, volume/volume into hard water  prior to each assay. The neutralizers for the NaOCl and EtOH disinfectants were prepared prior to each assay as follows; 1% sodium thiosulfate (from 10% stock concentrations) mixed volume/volume with DMEM or solely DMEM, respectively.50) quantification. For 0.5% and 1% NaOCl, the other 500 μl from each triplicate sample were pooled together and used for tissue culture flask infection (complete kill confirmation) and for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Disinfection experiments were performed independently as triplicate experiments each with three technical repetitions.As summarized previously90) samples during virus absorption. To remedy this, the remaining 500 μl of undiluted sample from each replicate were pooled together and used to infect T150 cell culture flasks of VeroE6 cells. After virus adsorption for 1 hour at 37 °C, virus maintenance medium was added making a final volume of 30 mL, flasks were incubated at 37 °C. Monolayers were visually analyzed for signs of CPE and cytotoxicity on 7 and 14 days post-infection.The combination of 0.5% or 1% NaOCl concentrations with the 1% sodium thiosulfate neutralizer are toxic to VeroE6 cells only in undiluted  and 50 μl of each dilution was added to VeroE6 monolayers in 96-well culture plates in quintuplicate for 1 hour at 37 °C/5% COAfter virus elution from carriers treated with 70% EtOH and ≥0.5% NaOCl, the triplicate samples from a singular biological experiment were pooled together. The vRNA was extracted (Qiagen) from 140 μl from the pooled supernatants and was quantified by qRT-PCR using Light Cycler 480 RNA Master Hydrolysis (Roche) and with the following ZEBOV primer/probe set: 300 nm of ZEBOV LF (CAGCCAGCAATTTCTTCCAT) and ZEBOV LR (TTTCGGTTGCTGTTTCTGTG) primers mixed with 150 nm of ZEBOV LP1 (FAM-ATCATTGGCGTACTGGAGGAGCAG) and ZEBOV LP2 (FAM-TCATTGGCGTACTGGAGGAGCAGG) probe combination. Data analysis and genome equivalents were quantified from a standard curve obtained from Ebo-Rib (Ebola reverse genetics system) DNA plasmid50 assay were compared for Makona against Mayinga and Makona against Kikwit using one-way ANOVA and Holm-Bonferroni post-hoc analysis using Excel to determine P values of <0.05, <0.01 and <0.001.Infectious virus particles as measured by TCIDHow to cite this article: Cook, B. W. M. et al. The Disinfection Characteristics of Ebola Virus Outbreak Variants. Sci. Rep.6, 38293; doi: 10.1038/srep38293 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.