Baculoviridae, a family of invertebrate viruses that have evolved to modify the feeding behaviour of their lepidopteran hosts and liquefy their cadavers as part of the course of infection.Niche construction has received increasing attention in recent years as a vital force in evolution and examples of niche construction have been identified in a wide variety of taxa, but viruses are conspicuously absent. In this study we explore how niche construction can lead to viruses engineering their hosts  with feedback on selective pressures for viral transmission and virulence. To illustrate this concept we focus on We present a mathematical model showing how niche construction leads to feedback from the behavioural manipulation to the liquefaction of the host, linking the evolution of both of these traits, and show how this association arises from the action of niche construction. Model results show that niche construction is plausible in this system and delineates the conditions under which niche construction will occur. Niche construction in this system is also shown to be sensitive to parameter values that reflect ecological forces.Baculoviridae via the niche constructing effects on the host. These results show the potential for niche construction theory to provide new insights into viral evolution.Our model demonstrates that niche construction can be a potent force in viral evolution and can lead to the acquisition and maintenance of the behavioural manipulation and liquefaction traits in  Niche construction, defined as the “process whereby organisms, through their metabolism, their activities, and their choices, modify their own and/or each other’s niches” , has recIn a fashion similar to other parasites , host maBaculoviridae. Baculoviruses are rod-shaped, enveloped dsDNA viruses with large genomes that range in size from 90 kb to 180 kb [Baculoviridae into four genera [Alphabaculovirus, lepidopteran-specific NPVs, Betabaculovirus, lepidopteran-specific GVs, Gammabaculovirus, the hymenopteran-specific NPVs, and Deltabaculovirus, the dipteran-specific NPVs. Baculoviruses are evolutionarily old, with molecular estimates placing their origin around 310 million years ago during the Carboniferous period of the Paleozoic era [In this study, we illustrate the operation of viral niche construction with an example drawn from the family of invertebrate viruses known as egt) in LdMNPV (host: Lymantra dispar) will disrupt the climbing behaviour and reinstating the gene also restores the climbing behaviour. Other genes, such as the viral gene for the baculovirus protein tyrosine phosphatase (ptp) of BmNPV (host: Bombyx mori), are occasionally implicated in the behavioural manipulation as well [egt, chitinase, and cathepsin are not necessarily the only causes for these traits, and given the constraints on viral genomes it is unsurprising that they may also have other functions . As par as well . Importa as well ,16), tho as well . Genes srulence: ,19).L. dispar, in the absence of the virus caterpillars will hide in bark crevices or climb down the tree during the day to avoid predation from birds; this suggests that climbing behaviours without liquefaction would harm the transmission of the virus. Similarly, liquefaction of the host may have a much greater effect on transmission if it occurs at the top of the host plant / tree, as the virus is spread into the path of feeding conspecific by mechanical factors such as wind and rain. In contrast, liquefaction at the bottom of the host vegetation or in crevices may lead to sequestration of the virus in the soil and bark reservoir that leads to next season’s infections but reduce transmission in the current season, as virus left unprotected will degrade quickly from UV exposure or be removed by other mechanical means [Despite the advances in understanding the mechanistic basis for host manipulation and liquefaction, insight into the evolutionary consequences and origins of these baculovirus characteristics remains speculative. It is thought that tree-top disease combined with liquefaction enhances transmission of the virus to conspecifics by shedding virus directly into the path of feeding conspecifics . However, this leaves open the question of how such a conjunction of behaviours could arise to begin with. As Hoover et al.  note aboal means . Thus, eIn this study we aim to contribute to the broadening of the domain of niche construction to viral evolution. To do so, we apply a niche construction perspective to make a general evolutionary argument about the acquisition and maintenance of tree-top disease and liquefaction in baculoviruses infecting Lepidopteran hosts. We construct an epizootic model of baculovirus transmission to link viral fitness to genotypes with each combination of features and demonstrate that there are valid evolutionary paths from a hypothetical virus with neither feature to one such as LdMNPV with both Figure .S) that have not been exposed to the virus, exposed individuals (E) that have ingested the virus, intact cadavers (C) that spread the virus at a low rate, liquefied cadavers (L) that spread virus at a higher rate, and a reservoir (V) that harbours virus for the next epizootic outbreak . Epizootics are assumed to begin when some small proportion of the recently hatched larvae come in contact with virus from the previous season’s reservoir and become infected (E(0) ≈ 1.0 × 10-5). Susceptible individuals enter the exposed class by coming in contact with intact or liquefied cadavers, after which they die at some rate ν.To study the effects of baculovirus behavioural manipulation and liquefaction on viral fitness, we begin with a compartmental epizootic model similar to a Susceptible-Exposed-Infectious-Recovered (SEIR) model . Our modβC, are liquefied at a rate λ, and shed virus directly into the reservoir at a rate θ. The egt gene also increases the virion yield by deactivating moulting hormone and causing infected larvae to grow larger and die later than uninfected larvae. This potentially niche constructing effect can be incorporated into the transmission parameters βL and βC, as an increased virion yield would increase transmission.Intact cadavers infect susceptibles at a rate μ). However, we do allow for excess mortality as a consequence of viral infection. For instance, exposed hosts suffer from reduced mobility and behavioural changes that may increase their chance of being predated (e.g. [δ is higher as the disease progresses (δ > μ). Intact cadavers that die are liquefied internally until their cuticle bursts in a matter of hours; during this time, they can be removed by predation [δ rate as intact cadavers, after which they transmit to susceptibles at a rate βL, and contribute to the reservoir at a rate τ. Virus that enters the reservoir degrades at some small rate ε. Note that this model includes a protected reservoir that will cause future outbreaks sensu[As we are tracking the infection for a single season, we presume a host in which eggs overwinter and hatch in spring; thus, we neglect births in the model, and track only baseline mortality from natural causes and predation ed e.g. ,23) or c or cμ). ed  so that the first m = 3 contain infected individuals. Let x = t,xi ≥ 0 be the corresponding number of individuals in each disease compartment. Let i, and i. The difference xi.We order the compartments of the model as , and i,j ≤ m.where F and V are Using equation (7), and R0 of the model is the spectral radius of the next generation matrix which is given by FV-1, which simplifies to The basic reproduction number We can clarify the contribution of each model component by rewriting this expression as δ + θ + λ)-1 time units. It includes the factor ν/(ν + μ) which is the proportion of exposed individuals that die. The second component corresponds to the contribution of the liquefied state and includes both the proportion entering the cadaver state from the exposed state and the proportion entering the liquefied state from the cadaver state, given by λ/(λ + δ + θ).The first term above gives the number of new infections produced by an intact cadaver, over  gene. G and g are gooey and non-gooey alleles, corresponding to the presence of the liquefying genes cathepsin and chitinase. This gives us zg, Zg, zG, ZG as the set of genotypes in the system, each with its own basic reproduction number Ri0, where i denotes the genotype .To trace the selective effects of the behavioural manipulation and liquefaction features of baculovirus, we define four genotypes from two hypothetical loci with two alleles each. For the purposes of our model we adopt a simplified view of y Figureῠ; furtherith genotype, βLi, τi, λi, and θi. Note that we replace the generic symbols introduced in the section above describing the ecology of disease spread with new notation given in Table γ, which represents the advantage or disadvantage to each mode of transmission . Relative to the zg genotype ZG increases horizontal transmission by liquefying ‘tree-top’  larvae and increasing spread of occluded virus over foliage that conspecific larvae will eat . However, this reduces contribution to the reservoir (lowering τZG) because exposed virus will degrade quickly, especially as a result of UV light . Occluded NPVs in forest settings have been shown to persist as long as 41 years after a natural outbreak [γ decreases βL,zG and reduces τzG relative to the zg genotype. Since they do not liquefy their host , it is assumed that the Zg and zg genotypes are neutral with respect to transmission of the virus (relative to the protovirus zg). However, the zombie allele would still modify contribution to the reservoir by the same argument as above, so we can represent this by modifying the values of θi; we represent this as a low value of θi, θl for the Z allele and a high value θh for the z allele (θh > θl).As shown in Table V light . Thus, n Figure . If ZG dur Table .βL and βC were set to 1 and 0.5 per day respectively, to provide a large transmission advantage to liquefied cadavers, following baculovirus pathology and ecology [τ (set to 0.2) and θ (set to 0.25), is expected to be low . For the remainder of this study we neglect mortality to susceptible and exposed hosts (μ) and clearance from the reservoir (ε) as being unimportant for the niche construction aspect of the model and so set them to zero, but deaths from predation may have an effect on viral fitness and so we set a low value of δ = 0.05 as a baseline. Finally, we take the average time from exposure to death as being 5–10 days  to 1/7 = 0.14. In exploring the effect of model parameters on niche construction, we varied βL from 0.5 to 2, βC from 0.1 to 1, θl from 0.01 to 0.5, λ from 0.1 to 2, δ from 0 to 0.5, and τ from 0.01 to 0.5.For numerical analyses of the model, we set the baseline parameters as described in Table  ecology . Contribfections ), and soC(0)→1×10-8,E(0) → 0,L( 0) → 0,V( 0) → 0, and S (0) →1 -(E(0) + C(0) + L(0) + V(0)). Calculation and plotting of genotype fitness and fitness orderings over parameter space was done using R [We numerically solved equations (1)-(5) using Mathematica . Initial using R .L. dispar . Lar. LarL. deks each .R0) as a function of genotype and the advantage parameter γ is shown in Figure γ (less than 0.3) lead to evolution proceeding from the protovirus with neither trait (zg) and acquiring either trait first before arriving at the zombie-gooey genotype ZG via the niche constructive feedback on the virus’s selective pressures. At higher values of γ (greater than 0.5), we see a fitness ordering that only allows niche construction to proceed by acquiring the zombie trait first (ZG).Viral fitness , combined with the rate at which the virus is shed into the reservoir  from either source affect the sequence of trait acquisition that is most profitable for the virus, such that low / high transmission from liquefied and intact cadavers respectively restricts the virus to acquiring the zombie trait to increase its fitness when the contribution to the reservoir is high  and transmission is low, no niche construction occurs, but high transmission rescues the niche constructive pathway. When the effect of Z inverts, high rates of transmission still allow niche construction to acquire both traits (green region), but only through the gooey trait. And at low rates of transmission, niche construction may maintain the two traits but is unlikely to lead to their acquisition (blue region), as going from zg to ZG now requires acquiring both traits simultaneously.Figure 25 Table , the verBaculoviridae family. Results from our modelling suggest that the feedback from manipulating the host is sufficient to acquire and maintain both the behavioural manipulation (zombie) and liquefaction (gooey) traits. Niche construction provides a strong explanation for the acquisition of these traits by virus, because the ecological inheritance from manipulating the host via the expression of the egt gene changes the selective pressures over successive viral generations until liquefaction changes from neutral or slightly advantageous to highly advantageous  and favouring ZG. On the other hand, larvae of crop pests like Anticarsia gemmatalis (velvetbean caterpillars) that feed on soybean and other legume plants have a dispersed spatial distribution [G allele, which may account for the lack of liquefaction in this NPV [Our model results are sensitive to parameter values that reflect ecological forces. For instance, differences in host ecology across species such as the choice of host plant and density of hosts per plant could affect transmission to conspecifics or increase contribution to the reservoir, which would change the predictions of our model as to whether niche construction would favour ribution  and are this NPV , and wheBaculoviridae, this allows us to explain why the virus would manipulate its host’s behaviour in such a dramatic fashion; the resulting change in the host creates conditions that modify the effect of the liquefaction trait on viral fitness and couple the two traits together. The niche construction explanation for viral manipulation of hosts can also provide testable predictions for empirical work; our model predicts that, when niche construction is favoured, disrupting the link between between the zombie and gooey traits by blocking the manipulation inspired by genes like egt should render both (nearly) neutral and thus subject to silencing or removal. This may also help to explain why in vitro cultures of NPVs often lose their egt genes quickly and show much greater levels of virulence [We argue that niche construction provides a more complete view of the benefits of host manipulation to viruses by providing a clear link between traits that, together, enhance fitness through the action on the host. Alternative explanations, such as standard natural selection with epistasis between the zombie and gooey traits, are not sufficient because they do not account for the fitnesses changing over time in this system; the zombie trait creates an ecological inheritance that builds up and cannot affect the fitness of the gooey trait until the behavioural manipulation has run its course. For irulence ,19, and irulence , though irulence ,42.egt and chitinase/cathepsin deletions have been created (separately) for LdMNPV, and the effects of the deletion on host behaviour / transmission studied  and compare their effects on transmission in the lab and in the field, using the methodology of D’Amico et al. [ZG (wildtype) viruses are prevented from experiencing the behavioral effects of tree-top disease (i.e. climbing to the top of the host plant), then over multiple generations we expect rapid loss of egt and (or) chitinase or cathepsin in treatment strains but not control strains that are free to manipulate the host.Empirically, the family Baculoviridae has been extensively studied on a molecular level, both for its use as a vector for protein expression and as a possible biopesticide. In addition to this, sufficient basic research on the properties of its pathology and ecological inheritance is available to make studying niche construction in this system possible; all that remains is to put the pieces together appropriately. For example, recombinant strains with .g. egt: , and chi.g. egt: ). The neo et al.  (confineBaculoviridae.With over 600 viruses described in the literature  and mixeNiche construction provides a new lens through which to study viral manipulations of the host. The analysis of the mathematical model developed in this study has shown that niche construction provides a plausible explanation for the behavioural manipulation and liquefaction of Lepidopteran hosts by baculoviruses. Understanding the evolutionary forces behind these manipulations provides not only new insights into viral evolution, but can form the basis for new empirical work on baculoviruses and research on the use of these viruses as biocontrol agents.Both authors declare that they have no competing interests.SH and MT conceived and designed the study, SH produced the data and analysed it with MT. SH and MT wrote the manuscript. Both authors read and approved the final manuscript.


Advances in lab-on-a-chip systems have strong potential for multiplexed detection of a wide range of analytes with reduced sample and reagent volume; lower costs and shorter analysis times. The completion of high-fidelity multiplexed and multiclass assays remains a challenge for the medical microdevice field; as it struggles to achieve and expand upon at the point-of-care the quality of results that are achieved now routinely in remote laboratory settings. This review article serves to explore for the first time the key intersection of multiplexed bead-based detection systems with integrated microfluidic structures alongside porous capture elements together with biomarker validation studies. These strategically important elements are evaluated here in the context of platform generation as suitable for near-patient testing. Essential issues related to the scalability of these modular sensor ensembles are explored as are attempts to move such multiplexed and multiclass platforms into large-scale clinical trials. Recent efforts in these bead sensors have shown advantages over planar microarrays in terms of their capacity to generate multiplexed test results with shorter analysis times. Through high surface-to-volume ratios and encoding capabilities; porous bead-based ensembles; when combined with microfluidic elements; allow for high-throughput testing for enzymatic assays; general chemistries; protein; antibody and oligonucleotide applications. Here,lycomics –6. Unforlycomics ,8. FurthTraditional approaches to clinical analysis involve a well-appointed centralized laboratory, three degrees of separation from the patient. This hierarchy introduces a number of critical junctures in which errors may be introduced and delays incurred. To simplify and offer assay results immediately, research into devices that give results at the POC, specifically bedside, ambulance or remote location, currently flourishes—a situation advantageous to both patients and healthcare providers –13. POC In the past few decades, significant advances in medical microdevice technologies have afforded new sensor ensembles capable of multiplexed detection of a wide range of analytes –23, inclThrough the miniaturization of macro-components, micro total analysis system (μTAS) and lab on a chip (LOC) devices have ushered in a new generation of high-throughput testing modalities that promise new options for biomarker measurements ,42–44. FThere now is a strong potential to leverage these medical microdevice discoveries for a broad impact in diagnostics for IVD and global heath applications using such chip-based approaches. Unfortunately, to date very few complete workable POC clinical devices have emerged despite tremendous progress in LOC, microfabrication, microfluidics, and related areas ,54. Indei.e., lab confined) sample processing. The emergence of high-density planar microarrays has enabled parallelized testing for clinical testing and validation. With modest sample volume requirements, these microarrays have afforded multiplexed testing of hundreds to thousands of analytes simultaneously for both proteomic and genomic applications [In addition to key work in the LOC area, which includes on-chip sample processing, significant progress has also been made recently in the area of protein, antibody and oligonucleotide planar microarray technologies with off-chip , the final signal was found to be similar. However, the incorporation of higher amounts of beads increased the total surface area for capture. In contrast, the total surface area for binding on microwells remained constant. By increasing the amount of beads per test by a factor of 4, the signals on microbeads were two times more than those of microwells. Moreover, nucleic acid hybridization kinetics performed much faster on beads than on microwells.When microfluidics is integrated with bead-based capture ensembles, immunoassays can be performed over much shorter timeframes. For example, Sato er plate . Becausend beads . When th2.2.i.e., several years) until they are ready for use [Further, beads exhibit a high degree of practicality with respect to their production and incorporation into microfluidic devices. The synthesis of beads can be completed in a very scalable manner. Beads can be produced in bulk quantities on the order of millions to billions of beads per batch. Once made, the surfaces (and interior regions in the case of porous media) of such beads allow for the functionalization of a variety of a capture probes for both genomic and proteomic applications. The beads produced and functionalized in these batches exhibit high reproducibility for both size and chemistry considerations. Moreover, beads produced in such large numbers benefit from the economics of scale with lower cost associated to each sensor. Once processed, these beads can be stored for long durations  allows for rapid sample processing with rates in the thousands of measurements per second . This plr minute . Here, tr minute . For exar minute , cytokinr minute , and nucr minute .2.4.In multiplexed assays in planar microarrays, the identities of capture probes are determined from their positions in the array. This positional encoding affords microarrays the ability to perform thousands of tests simultaneously. Similarly, methods to attach a code to each bead (encode) allows for its differentiation (decode) from other bead types and permits parallel screening of multiple analytes in a single sample.One of the most common methods to encode beads is to employ a fluorophore. These luminescent dyes with different spectral characteristics and concentration values allow for a set of uniquely distinguishable codes. These spectrally encoded beads are commonly used in flow cytometers. Luminex Corp, one of most well-established bead-based instrument suppliers, uses three fluorophores to encode a panel of up to 500 different 5.5 μm beads. Each bead type is matched to a specific capture probe. Using a 2-laser system, beads delivered through a suspension array are quickly decoded and their intensities are measured . SimilarSimilarly, Illumina developed a high-density optical fiber microwell array ,102–104.et al. used an array of polyacrylamide gel pads to form pillars to trap different bead types. Because of the natural immobilization of beads to the polymeric matrix, beads with similar probes are easily anchored in gaps between the micropillars. A second set of beads with a different probe set, spotted onto different positions of the array, allow for the differentiation of bead types. This process can be repeated to attach different beads [et al. immobilized microbeads in hydrogel supports to allow for position-based, addressable decoding [One limitation of these encoding schemes is the complication of possible overlap between spectral encoders or reporters. This complication limits the potential amount of simultaneous tests that can be performed in a single assay to about 100 different groups. The positionally-addressable identification of bead types, analogous to location-based encoding in microarrays, may extend the limit of different tests performed in a single assay. For example, Ng nt beads ,107. Simdecoding . Here, fdecoding .3.COMMAND QUALS [Cheap, Obvious, Miniaturized, Multiplexed, Automated, Nonperishable, Dependable, Quick, Unobtusive, Adaptable, Limited (volume), and Self-contained.The advantages of high-throughput multiplex testing through high surface-to-volume ratios of solid support beads have allowed for shorter analysis times with low sample and reagent requirements. Nonetheless, the timeframes to complete these tests are often still not consistent with the POC ,110. ForND QUALS . LikewisImprovements in mass transport and high-efficiency signaling are crucial here to achieve the ideal timeframes and high-fidelity analyte detection using simple instrumentation as is necessary in POC usage. Advantages of enhanced mass transport in porous mediums, such as gel pads and hydrogels, have demonstrated faster timeframes over planar microarrays. With the ability to functionalize a range of different capture probes, these porous networks have the ability to capture a wide range of analytes. The development of new microstructure concepts with engineered active transport through and within porous reactive particles serves as a promising new method for rapid yet high-efficiency capture within minisensor ensembles –114. TheIn the supported porous bead array area, prior efforts have led to the development of a microfluidic bead-based platform termed the Programmable Bio-Nano-Chip (p-BNC). This p-BNC platform is based on the concept that multiplex assays for a variety of disease applications could be achieved on a modular sensor suite that combines an application specific cartridge in conjunction with a universal analyzer. The approach uses a sensing platform that employs an array of 280 μm porous, agarose beads situated in an array combining individual flow-through wells. As shown in Likewise, porous beads, when combined with new LOC concepts, afford an opportunity to complete high-performance testing using simple instrumentation that is compatible with POC instrumentation. Shown in 4.In contrast to a 2-dimensional planar surface of a typical microfluidic structure, high surface-to-volume ratios of porous substrates allow for enhanced sensitivities and lowers limit of detection values for delivered analytes ,119. DueLikewise, the p-BNC method that utilizes the porous bead as an immunosensor meets and often exceeds analytical characteristics, such as test dynamic range and limit of detection  of maturi.e., seaweed). The same beads are already made in large quantities to support immunochromatographic applications that are dedicated to applications such as the purification of proteins. In addition to its tunable porosities (see below), this polymerized sugar matrix exhibits ultra-low nonspecific binding characteristics and the medium is index matched with water. The latter optical characteristics (unlike paper) make the material ideal not only for separation, but also as an environment  for optical detection.The strong analytical performance of the p-BNCs can be linked directly to the porosity and 3-dimensionality of the agarose bead capture elements. The choice of agarose is based in part on the potential for scalability, as it is derived from inexpensive sources (Other advantages of the agarose bead sensors include (a) a capacity to be tailored so as to accommodate the specifications  of the targeted analytes, (b) a capacity to be implemented for both two-site immunometric as well as competitive assays, (c) a capacity to be mass produced for widespread clinical purposes, (d) a capacity to be stabilized so as to withstand extreme storage conditions, (e) similar to immunochromagraphic applications, a capacity to be recycled for successive assay runs, as needed, and (f) a capacity to support fluorescence-, colorimetry-, and electrochemistry- based signal transduction .4.1.i.e., water), the high open surface area on the agarose fibers within the porous matrix with is ideal for the capture of large biomolecules.Agarose beads are typically produced in bulk using standard emulsion polymerization methods . By varyWhile planar microarrays have the capacity for high-throughput testing, limited surface areas do not afford the flexibility for planar microarrays to overcome differences in timeframes and differences in concentration response curves for various analytes that need to be measured in the same setting. This limitation has resulted in slow progress in the area of protein and antibody arrays . The tunet al. has reported that the typical capture efficiency of planar microarrays, under a flow of 5 μL/min through a channel with cross-sectional area of 50 × 500 μm2 and a detection zone of 1mm, is only 7%. Likewise, this diffusion-limited transport leads to a 93% loss in analyte capture [Due to the small-scale dimensions of microfluidic channels, transport typically exists in the laminar flow regime. The domination of viscous forces over inertia reduces irregularities from turbulent flow . As such capture . Further4.2.The hydrophilic fibers within the porous medium allow for the functionalization of a variety of capture probes. For example, several groups have successfully demonstrated the functionalization of hydrogel for the detection of a range of analytes that include proteins, nucleotides, and cells –126. Moret al. developed a model in an attempt to understand the effects of analyte diffusion coefficient, flow rate, and capture probe density on the kinetics on bead surfaces [Transport into the bead matrix is dependent on external flow rate and the pore size of the dense fibrous matrix . Carefulsurfaces . Furthersurfaces .et al. implemented a breathing bead methodology to expand and compress porous beads to accelerate mass transfer within such beads to increase signal intensity by a factor of ∼2.5 [Prior work here has revealed the existence of internal convection within porous beads. The unique design employed by the p-BNC, consisting of individual beads in flow-through microcontainers, allows for pressure-driven convective transport of analytes within the porous matrix . This pr of ∼2.5 . Within  of ∼2.5 .Further, modification of the pore size through control of agarose content in the beads allows for the increase of internal transport. In contrast to lateral flow designs, the flow-through design redirects fluids to create a high-pressure gradient that enhances internal mass transport. An adaptation of the Koreny-Carman equation shows that fluid velocity in a porous medium is proportional to applied pressure gradient and square of the pore diameter . Previouet al. observed intra-particle fluid velocities in superporous beads to be as high as 17% of the interstitial velocity in a chromatography column [Superporous beads have shown promise to enhance the mass transport of analytes that include proteins and cells into the internal bead matrix –142. In y column . Moreovey column . Large c4.3.et al. revealed that while the mean binding distances between antibodies in porous hydrogels is an order of magnitude higher than that of flat surfaces, fibrous 3D surfaces offer capacities that are two to three orders of magnitude higher than those available on surfaces [The capacity of binding of capture probes on porous medium is two to three orders of magnitude higher than that of planar surfaces . For exasurfaces . FurtherAs revealed by simulations, et al. witnessed limited signal increases for capturing antibody concentrations higher than 0.5 mg/mL [Furthermore, the density of binding is nonlinear to the concentration of capturing antibody used for conjugation. While porous medium offers high capacities for binding, no further benefits in signal occurs as higher concentrations of probes are used for immobilization due to saturation of available sites for antibody immobilization. For example, the high, medium, low densities of capture probes shown in .5 mg/mL . While t5.Having clearly articulated the analytical performance advantages of using porous beads for biomarker capture with microstructures, it is now essential to explore the next critical steps required to use these mini-sensor ensembles in real-world clinical practice. Indeed, despite large investments in translational research programs, most bioscience research efforts remain largely decoupled from broad-scale clinical practice, both for the biomarkers as well as for the devices that measure them . This geet al. [A case in point for the dismal rate of translation of new medical tests into real-world practice is extracted from a recent analysis by Schully et al.  of the FIn an attempt to move the medical microdevices and porous bead sensor systems into broad-scale clinical practice, a number of clinical trials and pilot studies have been initiated. Likewise, the laboratory version of the p-BNC bead-based methodology described above along with a related membrane-based system not covered here are now involved in six clinical trials and two pilot studies, respectively, involving over 5,000 patients, including over 10 clinical sites for diseases in the areas of cardiac heart disease, ovarian cancer, prostate cancer and drugs of abuse See  147–149149.High-impact diseases and conditions, such as cardiovascular disease and various cancers, including ovarian cancer and prostate cancer are the targets of these p-BNC development and biomarker validation trials. The ability to use validated biomarkers in a common platform affords interesting synergies with respect to opening up new and more efficient treatment methods for management of patients ,8. The sThe capacity of the p-BNCs to multiplex, or simultaneously measure multiple analytes within a single assay run, is not only economically beneficial, but it also allows to test for biomarkers that collectively provide a more comprehensive look into the overall well-being of a person, while at the same time derive a more specific look to specific stages of the disease process.Case in point, to date there are no global methods to define in an efficient way the entire cardiovascular health of patients. Today cardiac heart disease is the number one killer globally . While tFor example, biomarkers for all aspects of care regarding cardiovascular disease, including diagnosis of acute myocardial infarction, prognosis or risk for secondary cardiac events, monitoring, risk stratification and guidance for therapeutic interventions of cardiac patients, were developed as multiplexed panels . Figure vs. diseased. Likewise, in order for these chip-based tests to have clinical relevance, they must not only respond on a timeframe consistent with near-patient usage, but they must meet or exceed the analytical, and, thereby, clinical performance of the gold standards or reference methods, that are for the most part limited to the laboratory setting  for the two-site immunometric and competitive assays have been established as reported in From 6.The combination of porous support beads and tailored microstructures with active transport features allows for enhanced mass transport of analytes. Short analysis timeframes of these highly sensitive sensing elements reduce analysis times achieving what would be consistent with the point-of-care.7.As mentioned above, in order for the medical microdevices area to reach their full potential, it will be necessary for these mini-test ensembles and their associated measurement platforms to be fully validated with results that exhibit strong performance characteristics that rival those achieved in traditional laboratory settings . FurtherThe pathway to scalability would be expected to occur with modular assay formats that encompass major diagnostic classes and with the establishment of standard testing procedures . It is iUnfortunately, today healthcare costs are increasing at an alarming rate with only modest increases in the quality of patient care . Today oIt might be expected, therefore, that the development of scalable high-performance diagnostic platforms alongside Moore's law-like goals could have potential to lead to transformative changes in healthcare. For the purpose of this graph, it is assumed that all the information content obtained from microarray discovery data is used in the clinical decision making process. However, more typically only a small number of sequences contribute to the differential disease diagnosis and thus these specific signatures serve as a small portion of the acquired information. In the future it might be expected that as these devices move closer to widespread clinical practice, a stronger focus on key information from the selected sequences derived from more efficient capture methods will evolve. Integrated LOC systems with improved efficiencies in sample processing are expected to play key roles in extracting the key information that is required to manage patient care in cost effective and efficient ways moving forward.8.In many ways, the convergence of microfluidics, biomarker validation, and porous bead ensembles serve to overcome some of the significant challenges that the medical microdevices have, to date, faced with respect to scalability and performance. When combined with new concepts for noninvasive sampling , there iFurther, the power of scalability through reduced time per test could lead the way to a new generation of diagnostic and detection tools for health care applications. These devices, with the ability to quickly provide robust, affordable, and accurate results, could potentially diagnose diseases at early stages, monitor health risks, manage illness and provide appropriate treatment options . The abi


Xenotropic murine leukemia virus-related virus (XMRV) was first identified in 2005 in a study of human prostate cancer patients with genetic variants of the antiviral enzyme, RNase L. Subsequent investigations in North America, Europe and Asia have either observed or failed to detect XMRV in patients  or normal, healthy control individuals. Among the confounding factors are the potential for lab contamination with similar or identical viruses or viral sequences originating in mice. In some studies, relatively contamination-resistant methods  suggest that either XMRV or a similar type of virus is present in some patients. Evidence for and against genuine infections of humans with this intriguing virus (and/or related viruses) will be discussed.


Neurotropic virus disease is often misdiagnosed as Japanese encephalitis. Coltivirus. The type species, Colorado tick fever virus (from North America), has been isolated from patients with flulike syndromes, meningitis, encephalitis, and other severe complications. Another coltivirus, Eyach virus, has been isolated from ticks in France and Germany and incriminated in febrile illnesses and neurologic syndromes. Seadornaviruses are endemic in Southeast Asia, particularly Indonesia and China. The prototype virus of the genus, Banna virus (BAV), has been isolated from many mosquito species, humans with encephalitis, pigs, and cattle. Two other seadornaviruses, Kadipiro and Liao Ning, were isolated only from mosquitoes. The epidemiology of seadornaviruses remains poorly documented. Evidence suggests that BAV is responsible for encephalitis in humans. Infection with BAV may be underreported because it circulates in regions with a high incidence of Japanese encephalitis and could be misdiagnosed as this disease.Coltiviruses are tickborne viruses of the genus  Reoviridae and having 12-segmented dsRNA genomes at one time were classified in the genus Coltivirus (Colorado tick virus [CTFV]). At the time the genus was created, it included tickborne and mosquitoborne viruses. Presently, the genus Coltivirus contains only CTFV, California hare coltivirus (CTFV-Ca), which is considered a serotype of CTFV, Eyach virus (EYAV), which is distinct from CTFV, and Salmon River virus (SRV), which may be a serotype of both CTFV and CTFV-Ca. Alternatively, analysis of sequence data and antigenic properties of the mosquitoborne viruses led to their reassignment to a new genus designated Seadornavirus (Southeast Asian dodeca RNA virus)  in northern California, and SRV isolated from a person in Idaho. Many mosquitoborne viruses were also considered tentative species in this genus, including Banna virus (BAV) from humans. The tickborne viruses in this genus have many distinctive features and sequence data have led to a reevaluation of their taxonomic status.Tickborne vertebrate viruses include CTFV isolated from humans and ticks, EYAV isolated from ticks, CTFV-Ca isolated from a hare  (CTF is found in the Rocky Mountain region of the United States and in Canada. The virus distribution closely matches that of its vector Ixodes ricinus ticks (EYAV-Gr) and in 1981 from I. ricinus (EYAV-Fr578) and I. ventalloi (EYAV-Fr577) (EYAV was isolated in Europe in 1976 from Oryctolagus cunniculus), mice, mountain goats, domestic goats, sheep, and deer . Prolonged viremia observed in humans and rodents is due to the intraerythrocytic location of virions, which protects them from immune clearance  or with organic solvents (such as Freon 113 or its ozone-friendly substitute Vertrel XF), but viral infectivity is abolished by treatment with sodium deoxycholate or sodium dodecyl sulfate  of CTFV is homologous to segment 7 (VP7) of EYAV . The amiAntigenic variation between CTFV strains is low, especially between strains from humans  or hemorrhagic fever, pericarditis, myocarditis, and orchitis have been rarely observed, mainly in children. Severity is sufficient to result in hospitalization of ≈20% of patients. Offspring of mice experimentally infected with CTFV showed teratogenic effects , tularemia, relapsing fever, and Lyme disease. However, rash and leukocytosis distinguish Rocky Mountain spotted fever from CTF. Signs in the CNS confuse CTF with other causes of viral meningitis and encephalitis, including St. Louis encephalitis virus, Western equine encephalitis virus, and enteroviruses.CTFV can be isolated from blood because it is present in circulating erythrocytes for as long as 4 months .BAV is the type species of the genus Culex vishnui, Cx. fuscocephalus, Anopheles vagus, An. aconitus, An. subpictus, and Aedes dorsalis. BAV, KDV, and LNV are found in tropical and subtropical regions where other mosquitoborne viral diseases, especially JE and dengue, are endemic. Several cases of encephalitis in China have been diagnosed as Japanese encephalitis (JE) without any detection of JEV or specific anti-JEV antibodies. Recently, 89 paired serum samples from these patients were tested by ELISA for anti-BAV IgG antibodies. At least a 4-fold (up to 16-fold) increase in IgG antibodies was observed in 7 cases. An additional 1,141 serum specimens of patients from a large number of health institutes in China, who supposedly had JE or viral encephalitis, were tested for anti-BAV IgM antibodies; 130 samples were positive , A20 (Ae. aegypti), and Aw-albus . More than 40% of the virus particles are released into the culture medium before cell death and massive CPE (fusiform cells). Infected cells are not lysed, and the virus leaves cells by budding, thus acquiring a temporary envelope , BGM, Vero, BHK-21, L-929, and MRC-5 (human lung fibroblasts). Infection with LNV results in a massive lytic effect and also kills adult mice.Antigenic relationships between seadornaviruses were investigated by using mouse immune sera. BAV from southern China and Indonesia and KDV from Indonesia are classified as distinct species   with thoavirus A .The only seadornavirus isolated from humans and associated with human disease is BAV. Persons infected with BAV exhibited flulike symptoms, myalgia, arthralgia, fever, and encephalitis (Reoviridae. In infections with CTFV or BAV, symptomatic treatment includes acetaminophen for relief of fever and pain. Patients infected with CTFV show long-lasting immunity. An experimental vaccine was developed in the 1960s and produced long-lasting immunity, but production was stopped in the 1970s. Patients infected with BAV show a strong immunologic response (,No specific treatment exists for infections caused by any members of the family


Drugs could treat neuropathic pain more effectively if they simultaneously targeted two or more types of ion channel. Related research article Ratté S, Zhu Y, Lee KY, Prescott SA. 2014. Criticality and degeneracy in injury-induced changes in primary afferent excitability and the implications for neuropathic pain. eLife3:e02370. doi: 10.7554/eLife.02370Image Changes in the sodium  and potassium  conductances modify the firing behaviour of neuronsComplex biological systems are able to use multiple different solutions to produce similar phenotypes or outputs. This property is called degeneracy  and it meLife, Steven Prescott and colleagues at the University of Toronto and the University of Pittsburgh—including Stéphanie Ratté as first author—show that degeneracy may also underlie the robustness of pathological states against pharmacological treatments  and sensory stimuli (dysesthesia and allodynia; Ratté et al. studied how the degeneracy involved in generating action potentials may affect the development of the hyperexcitability associated with neuropathic pain. First, they showed that the change in neuron behaviour from normal physiological states to pathological states of excitability occurs when a tipping point is crossed . Using pThis finding sheds light on the surprising results obtained in transgenic animals lacking two types of sodium ion channels called Nav1.7 and Nav1.8 . While bRatté et al. also demonstrate that simultaneously increasing the sodium conductance and decreasing the potassium conductance is the most efficient way to trigger the hyperexcitability associated with neuropathic pain, whilst the opposite conductance changes are the most efficient way of relieving it . As a coAltogether, the work of Ratté et al. demonstrates that excitability disorders such as neuropathic pain rely on the degeneracy of excitability, with numerous ion channels defining the excitability profile of the neuron. These results call for our principles of drug design for excitability disorders to be reconsidered, to take into account how the degeneracy of biological systems helps develop pathological robustness and resistance to pharmacology.


Despite global eradication efforts, female genital cutting is still deep routed practice in some parts of Asia and East Africa. The crude and unscientific natures of the practice lead to many complications. Epidermoid inclusion cysts of the vulva are one of the late complications of female genital cutting and typically present as painless cystic swellings. But clinical presentation as ‘stone’ containing, hard vulvar mass is reported only once in the literature and presentation with acute vulvar pain has never been documented.A 21 yrs old, Ethiopian, unmarried, lady presented with sever acute vulvar pain, discharge, and a stony hard vulvar swelling 13 years after type-III female genital cutting (infibulation). Surgical excision of the cyst, which contained two ‘stones’ inside it, and defibulation were done. Histopathology confirmed calcified, keratinizing epidermoid inclusion cyst of the vulva.Clinicians, in areas where female genital cutting is prevalent, should be aware of such unusual late vulvar complication of the practice which might be misdiagnosed for other solid vulvar swellings and be familiar with the appropriate management. Female genital cutting is deep rooted practice in East Asia and north-east of Africa. It is associated with distressing immediate and late complications. Epidermoid inclusion cysts of the vulva are one of the commonest late complications of female genital cutting. These cysts occur as a result of the implantation of the epidermal keratinized squamous epithelial cells and sebaceous glands into the dermis in the line of circumcision scars and typically present as painless cystic vulvar swellings. Calcifications inside these cysts very rarely lead to formation of ‘vulvar stones’ which are the result of delayed presentation of patients. Extraction of the stones and complete excision of the cysts is the optimal management. We report, here, a case of stone containing post circumcision inclusion cyst of the vulva presenting with unusual acute vulvar pain in a lady who had type III female genital cutting during her childhood.A 21 yrs old single, nulligravid lady presented with excruciating vulvar pain and swelling of 3 days associated with itching and difficulty of walking. She had also thick discharge from the site of the swelling, dysuria and frequency.The lady noticed a progressively increasing vulvar swelling since 4 years before her current presentation which initially was painless. But since a year back she had had recurrent episodes of sudden increases in size of the swelling with itching and pain which were subsiding after spontaneous ulceration followed by extrusion of thick ‘sand-like’ discharge from the swelling. She used to take non prescription antibiotics from pharmacies.The lady underwent circumcision (infibulation) at the age of 8 years. Her menstrual cycles were unremarkable. She had no abdominal pain or vaginal discharge. She had no history of genital trauma or any similar swellings in other body parts. She had no known medical illnesses.On physical examination she was acutely sick looking with difficulty of walking. Her vital signs were stable. The remarkable findings were on genital examination which revealed swollen & glistening vulva with scattered areas of dark tiny old scars on the skin. The labiae were completely fused having only a small (1 cm) opening just above the posterior fourchette. There was a palpable 4 cm by 3 cm sized, irregular, hard mass located in the midline along the fused labiae with a thick non offensive discharge extruding through pin pointed defect on the overlying skin at the cranial end.th post operative day visit, with healed labial wound  infiltration, incision was made on the mass which revealed thick walled biloculated cyst containing two discrete stones Figures  and 2. Ed Figure . The hisFemale genital cutting is gender based violence against women and a violation of basic human rights. Female genital cutting comprises all procedures involving partial or total removal of the external female genitalia or other injury to the female genital organs for non-medical reasons ,2. AccorThe World Health Organization has classified female genital cutting into four types: the first 3 including procedures of increasing invasiveness and the last one being a general category including unclassified genital injuries . Type- IImmediate complications of female genital cutting include: severe pain, hemorrhage, infection, urine retention, and injury to nearby genital tissue. The long-term consequences include recurrent urinary tract infections, infertility, psychosexual disturbance, an increased risk of childbirth complications and epidermal inclusion cysts ,6.Epidermoid cysts are intradermal keratinizing cysts lined by squamous epithelium. The cysts result from ingrowths or implantation of epidermal tissues underneath other tissues. The accumulation of epidermal desquamations, secretions and other debris in a closed space leads to the formation of a cystic and often painless swelling that gradually increases in size over time . The comThough histopathologically similar, the pathogenesis of epidermoid cysts of the vulva differ from that of cysts arising from hairy (acne prone) areas such as the face, neck which arise from the damage to the pilosebaceous unit ,8. But OThe frequency and delay from circumcision to development of inclusion cyst are extremely variable. The delayed development of inclusion cysts after circumcision is postulated to be secondary to unopposed estrogenic stimulation of the embedded epidermal tissue and sebaceous glands during anovulatory menstrual cycles of adolescence . This waThe clinical presentation of patients is variable . Most EpComplication of epidermoid inclusion cysts include rupture & release of keratin leading to intense inflammatory reaction, infection, hematoma and rarely carcinomas ,11. The Diagnosis of epidermoid inclusion cysts is confirmed by Histologic examination which was done also in our case and revealed the typical finding of cyst lined by keratinized stratified squamous epithelium .The Management of inclusion cysts is surgical excision which was done in our patient with concurrent correction of the infibulation (defibulation), which resulted in satisfactory clinical and cosmetic outcomes.Post circumcision epidermoid inclusion cyst of the vulva containing stones and presenting with acute vulvar pain is extremely rare. Health professional in areas where female genital cutting is prevalent, should be aware of such unusual late vulvar complications of the practice, which might be misdiagnosed for other solid vulvar dermatologic tumours and be familiar with the appropriate management.Verbal explanation was given to the case (the participant) and an informed written consent was obtained both for the writing up and taking the pictures. I assure that I will produce the consent form at any time upon request.The author does not have any competing interests with respect to the research, authorship and publication of this article.All the activities related to the case report were done by the author.WJG is an Obstetrician and Gynecologist at Departement of Obstetrics and Gynecology, Karamara regional referral hospital, Somali regional state, Jijiga, Ethiopia.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6874/14/2/prepub


To estimate the incremental costs of visual inspection with acetic acid (VIA) and cryotherapy at cervical cancer screening facilities in Ghana; to explore determinants of costs through modelling; and to estimate national scale-up and annual programme costs.Resource-use data were collected at four out of six active VIA screening centres, and unit costs were ascertained to estimate the costs per woman of VIA and cryotherapy. Modelling and sensitivity analysis were used to explore the influence of observed differences between screening facilities on estimated costs and to calculate national costs.Incremental economic costs per woman screened with VIA ranged from 4.93 US$ to 14.75 US$, and costs of cryotherapy were between 47.26 US$ and 84.48 US$ at surveyed facilities. Under base case assumptions, our model estimated the costs of VIA to be 6.12 US$ per woman and those of cryotherapy to be 27.96 US$. Sensitivity analysis showed that the number of women screened per provider and treated per facility was the most important determinants of costs. National annual programme costs were estimated to be between 0.6 and 4.0 million US$ depending on assumed coverage and adopted screening strategy.When choosing between different cervical cancer prevention strategies, the feasibility of increasing uptake to achieve economies of scale should be a major concern. Cervical cancer is the second most frequent cancer in women worldwide and is responsible for an estimated 493 000 cases and 274 000 deaths annually , and in In developing countries, logistical barriers in implementing screening programmes using cytology on Papanicolaou-stained cervical smears to detect precursor cervical lesions have led to failures in reducing cervical cancer incidence and mortality . These sCareHPV™) that have shown higher sensitivity and specificity than VIA  estimate the costs of screening using VIA and treatment with cryotherapy at existing screening facilities in Ghana; (ii) explore the most important determinants of costs through modelling and (iii) estimate national scale-up and annual programme costs under varying assumptions for the level of coverage and the frequency of screening.Incremental economic costs of VIA and cryotherapy were estimated in two steps using an ingredients approach: (i) quantities of resources used were measured, and (ii) unit costs or prices were assigned to resources consumed . As VIA Data on resource requirements were collected at four of the six active VIA screening facilities in Ghana – three in the Kumasi area , and one in Accra . Providers were observed during performance of their regular activities for between one and 3 days per facility. Providers generally followed the VIA and cryotherapy protocol that had been established during the initial set-up of the VIA pilot-study in Ghana . HoweverInformation was collected on capital  and recurrent  resources used for VIA and cryotherapy. For VIA, resource use was collected only for activities directly related to the performance of VIA, i.e. we did not include resources used for evaluation of women referred to physicians for apparent cancer during visual inspection. For cryotherapy, the entire treatment process was included, i.e. extra counselling after a positive VIA test, treatment with cryotherapy, post-treatment counselling and one follow-up visit after 2 weeks.To estimate unit costs of labour, staff were asked to estimate the percentage of effective working time, which was defined as the time during which they were seeing patients, and the percentage of daily working time dedicated to cervical cancer-related activities. Further information was obtained from actors involved in developing the VIA pilot sites and in attempts to scale-up VIA in rural areas. Resource requirements for mobilization/recruitment of women were estimated on the basis of information on mobilization/recruitment activities at Kumasi South hospital. Costs attributable to maintenance and utilities were calculated based on results of a prior costing study from Ghana .Unit costs were collated from multiple sources including the Kumasi University of Science and Technology development office for building construction costs, provider salary slips for personnel costs and market prices for equipment and supplies (a list of unit costs and prices can be obtained from the authors). For personnel, unit costs were calculated by dividing monthly salaries by the estimated effective working time per month. If necessary, costs were adjusted to the year 2009 using the country-specific GDP deflator from IMF (2009) . CalculaA model was constructed to test the influence of observed differences between surveyed facilities and providers on estimated costs. The costing model made assumptions for costs of inputs, number of women screened and treated per provider, effective working time of capital and staff, costs of training and duration of screening/management per woman . The modBase Case assumptions usually reflect findings from observed facilities and considerations about the availability of infrastructure in the country . High-CoUnivariate and multivariate SA were carried out using the alternative ‘High-Cost’ and ‘Low-Cost’ assumptions. For the univariate sensitivity analysis, one model parameter was varied at a time with all other parameters kept constant. For the multivariate SA, all variables were simultaneously set to the ‘Low-Cost’ assumptions or ‘High-Cost’ assumptions, respectively, to generate the largest possible range of costs in an analysis of extremes  were estimated using ‘Base Case’ model parameters , and assAs scale-up costs were estimated separately, national annual VIA/cryotherapy programme costs were calculated for two different scenarios: one scenario excluded start-up costs ; the other scenario included annuitized start-up costs. To do so, modelled incremental economic costs per woman under Base Case assumptions  were multiplied with assumed numbers of women requiring screening in the year 2009 in Ghana. Incremental economic costs per woman treated with cryotherapy are presented in A single-visit approach consisting of VIA followed by immediate cryotherapy for all eligible women remains one of the internationally recommended screening strategies for developing countries . Our stuResults from surveyed facilities showed high variability of VIA and cryotherapy costs in Ghana. VIA costs lie closer to figures previously published for South Africa 10.63 US$) than to those estimated for Kenya (1.31 US$)  are becoming available, cost-effectiveness of VIA compared with new HPV tests will need to be reevaluated.According to estimates by When interpreting our findings, important data and methodological limitations need to be considered. First, some data were not available during our observations of providers’ activities, which make the information less reliable: the percentage of effective working time, resources used for training or for recruitment/mobilization of women and the life-expectancy of cryotherapy equipment had to be estimated based on provider accounts. In addition, providers were unable to relate life-expectancy of cryotherapy equipment to the volume of treated patients. Therefore, our analysis assumed that life-expectancy was not significantly dependent upon the number of patients treated per year. Furthermore, this study was unable to relate costs to relevant outcomes for cervical cancer prevention, as no information was available on the effectiveness of providers in Ghana to detect and treat precancerous lesions. While evaluations comparing providers’ performance quality with that of a ‘Master Trainer’ have confirmed quality was maintained beyond the pilot project  systems costs (such as administrative and overhead costs) were excluded. Yet, these cost categories are important because insufficient support and attention to management can contribute to programmes failing during implementation; (ii) the ingredients approach may underestimate wastage and losses, which would have been accounted for by using a step down methodology based on total hospital usage . Both types of limitations may have led to under-estimation of resource requirements to implement a national cervical cancer screening programme. Furthermore, a national screening programme would result in additional costs for the treatment of detected cervical cancer patients, and for (re)screening of women for whom VIA could not be completed .Third, model assumptions are always open to criticism. Our model calculated national programme costs based on assumptions derived from observations at only three screening facilities in the country. In addition, it assumed that mobilization/recruitment costs remained constant for every woman recruited for screening. Yet, it is likely that the costs per woman depend on specific characteristics of the screening programme, such as geographic accessibility of facilities and the duration of the programme. Another limitation is that we calculated national scale-up and annual programme costs under the assumption of constant returns to scale. However, our results suggest the presence of economies of scale in conducting VIA and cryotherapy, which would imply lower average costs per woman screened when screening higher numbers of women. Yet, our sample was too small to identify optimal scale of operation. Furthermore, results would have been different, if alternative High-Cost or Low-Cost estimates would have been considered for calculating annual programme costs.Despite these caveats, our research has major implications for programme managers and researchers. First, costs of VIA and cryotherapy are highly context-dependent, even within the public sector of one country, showing large variability between facilities. Second, volume effects are an important determinant of costs per woman screened and treated with cryotherapy.Economies of scale have not been considered in prior cost-effectiveness studies of cervical cancer prevention strategies in developing countries . Yet, ecAs scale is an important determinant of costs, policy-makers in Ghana and elsewhere should aim to increase the number of women screened per facility. To do so, demand-side barriers such as user-fees, which were introduced at Ghanaian screening facilities after discontinuation of external support, should be removed and replaced by continued public funding. Availability of services needs to be assured as non-functional cryotherapy machines are discouraging both, providers and clients. Investing in recruitment/mobilization campaigns becomes important to make efficient use of resources. Approaches to integrate screening in family planning clinics appear useful, as they allow easy recruitment of women attending such services, and at the same time, women receiving screening can benefit from treatment of vaginal infections detected during visual inspection.Policy-makers should be aware that trade-offs may exist: improving geographic accessibility is likely to result in higher costs of screening and treatment per woman. However, concentrating screening at urban centres could exacerbate inequalities between urban and rural populations. Planning for scale-up of cervical cancer screening becomes more difficult when having to consider economies of scale because assuming a simple linear relationship between inputs (employed resources) and outputs (number of women screened/treated) appears to be inadequate. When choosing between different cervical cancer prevention strategies, including vaccination, low-cost HPV tests and VIA/cryotherapy alone or in combination, the feasibility of increasing uptake of screening to achieve economies of scale should be a major concern.In conclusion, scaling-up screening in developing countries remains essential to achieve reductions in female cancer mortality. In doing so, policy-makers should pay particular attention to the most important determinants of costs of VIA and cryotherapy identified in our study. Research should focus more attention on the determinants of costs and consider that economies of scale are likely to exist when embarking on necessary future cost-effectiveness analyses of alternative cervical cancer prevention strategies.


Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level.The C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied.Protein extracts obtained via the formic acid extraction method of 164 C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established.The obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this species complex in the clinical laboratory. The obtained mass spectra provide further evidence that the major molecular types warrant variety or even species status.MALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic  Cryptococcus neoformans or C. gattiiC. neoformans var. grubii (serotype A), C. neoformans var. neoformans (serotype D) and the well-recognized hybrid AD is mainly an opportunistic infection of HIV patients C. gattii (serotypes B and C) is mainly affecting immunocompetent hosts, involving humans, domestic and wild animals C. neoformans var. grubii, VNIII/AFLP3 for the AD hybrid, VNIV/AFLP2 for C. neoformans var. neoformans and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5 and VGIV/AFLP7 for C. gattii).Cryptococcosis is a life-threatening infection caused for the inhalation of infectious propagules of the encapsulated yeasts C. neoformans/C. gattii species complex differ also in their mating type distribution, virulence factors, disease characteristics, geographical distribution, epidemiology and ecological niche Strains of the eight major molecular types of the C. neoformans and C. gattiiRecently as a substitute for phenotypic and genotypic methodologies used in clinical and applied laboratories, new, convenient and highly standardized technologies for the diagnosis and identification of infectious microorganisms to the subspecies level have been described. Among them, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is being well renowned as an important tool for the rapid recognition of both bacteria C. neoformans and C. gattii to the species/sub-species (major molecular types) level. This is the first time that this technology is used to identify and generate a library of the protein masses of clinical, environmental and veterinary isolates of the C. neoformans/C. gattii species complex representing all eight major molecular types previously determined by molecular methods.Therefore, the aim of this study was to evaluate the use and suitability of MALDI-TOF MS for a fast and reliable identification of strains of the two currently recognized pathogenic yeasts C. neoformans/C. gattii species complex by MALDI-TOF MS, resulted in a library of 960 individual mass spectra and 160 combined mass spectra, which allowed for the correct identification of all isolates according to the two currently recognized species C. neoformans and C. gattii, with identification scores ranging between 1.882 and 2.711 (C. neoformans and the two strains of C. gattii that are currently included in the MALDI Biotyper BDAL MSP library (2011)  see . In geneC. neoformans/C. gattii species complex. Representative spectra obtained from the reference strains for the seven haploid major molecular types are presented along with a phylogram generated from randomly selected strains of those major molecular types based on the concatenated sequences of the ISHAM MLST consensus loci  C. neoformans and VGI to VGIV for C. gattii, respectively. The isolates belonging to each molecular type produced distinct mass spectral signatures that were distinguishable from each other . In addition, when analyzing the spectra of the proteins extracted from one or three colonies and incubated at 24 or 48 hours, no remarkable changes in the obtained spectra were found. The spectra obtained from one or three colonies or after 24 or 48 hours incubation presented peaks in the same mass range and intensity. No spectra were obtained when intact cells were used directly on the MALDI-TOF plate.C. neoformans and C. gattii isolates at the species or subspecies level is important, mainly because of the differences in epidemiology, virulence, and antifungal drug susceptibility and subsequent disease outcome of the different strains C. neoformans and C. gattii, as well as the detection of four newly recognized hybrid strains C. neoformans/C. gattii species complex. URA5 RFLP, which is currently the most commonly used method to determine the major molecular types, e.g. includes not only culturing the isolates, but also to carry out DNA extraction, URA5 gene amplification and restriction enzyme digestion, followed by additional electrophoresis steps for the two latest procedures, with the whole process taking at least 3 days The distinction and characterization of pathogenic C. neoformans and C. gattii, although in previous studies, the isolates of both pathogenic Cryptococcus species were only identified to the species level The herein obtained results are broadening the findings from recent studies, which reported that mass spectrometry-based identification proved to be a fast, reliable and sensitive alternative for the correct identification of a broad range of human pathogenic filamentous fungi and yeast Cryptococcus isolates, the MALDI-TOF technique also allowed for a clearly separation of the major molecular types of the C. neoformans/C. gattii species complex , in which separate or combined sequence analyses of all four or seven loci, respectively, revealed well-defined groups with significant support for each major molecular type, suggesting that the major molecular types are cryptic species and as such deserve either variety or species status C. neoformans than within C. gattii. In C. neoformans, the two varieties, C. neoformans var. grubii and C. neoformans var. neoformans, were clearly separated, which is in accordance with the grouping of the serotypes A and D, respectively C. gattii, despite the fact that four different genotypes have been well identified by several molecular methods, they have not yet been interpreted as distinct taxa C. gattii (C. neoformans var. grubii (VNI/VNII) separated from C. neoformans var. neoformans (VNIV) 24, VNI and VNII 4.7 and the C. gattii major molecular types VGIII and VGI 8.5, VGIV 11.7 and VGII 12.5 million years ago Additionally to the species differentiation among clinical  complex . These f. gattii . C. neofMycobacterium isolates, at both the species and strain level Candida species Pseudallescheria/Scedosporium species complex C. neoformans/C. gattii species complex was greatly enhanced through the addition of 160 new spectra to the MALDI Biotyper BDAL MSP library (2011), containing currently only five C. neoformans and two C. gattii spectra. From now on, it will be possible to use the mass spectra obtained for the recognition of the two currently recognized pathogenic Cryptococcus species, C. neoformans and C. gattii, and the differentiation of the major molecular types. This method can be used as a single identification technique or to complement traditional morphological or molecular identification/typing techniques like PCR fingerprinting, RFLP, AFLP, and MLST MALDI-TOF MS proved once again its potential to be very useful for species differentiation, as has been already reported for the discrimination of e.g. C. neoformans/C. gattii species complex, MALDI-TOF MS permitted the detection of recently characterized inter-species hybrid strains C. neoformans/C. gattii species complex but did not group within any of the eight major molecular types. In all cases they grouped closely to one of the parental major molecular type, VNI. It is currently unclear why they did not group in between both of the parental major molecular types. This could reflect different contribution of each parent strain to the gene composite of the hybrids or different expression levels of the proteins involved. A clear grouping of the well-known AD hybrid, VNIII/AFLP3 was obtained. Further studies with more proven hybrid strains for each molecular type combination are needed to assess the usability of the MALDI-TOF MS for the identification of hybrids and the parental origins.As well as the identification of the currently recognized species and the major molecular types within the C. neoformans/C. gattii species complex are also characterized by different single nucleotide polymorphisms (SNPs) Considering that different strains within the Candida albicans, for which it was reported that it is important to consider parameters that could affect mass signature reproducibility, like the number of cells used, the cell incubation conditions, the matrix compound used, or even the sample spotting technique CandidaCryptococcus isolates, it is necessary to use ethanol in the protein extraction method, not only to avoid cellular aggregations, but also to inactivate this pathogen. Additionally, the alcohol fixation step has been proven to be critical to obtain high-quality mass spectra from yeast cells, and to be essential in the success of the technique for the identification of fungal species with the same accurate results as for bacterial species, for which no prior treatment or extraction method is needed In opposite to reports for other fungal species such as C. neoformans and C. gattii. The comprehensive in-house library of the obtained mass spectra created in this study is substantially improving the MALDI Biotyper BDAL MSP library (2011), provided by Bruker, allowing for its use for the identification of the currently recognized two pathogenic Cryptococcus species. MALDI-TOF MS analysis provides for the first time a clear, rapid and highly reproducible method for the separation/identification of the major molecular types within the C. neoformans/C. gattii species complex and the detection of hybrids in the clinical diagnostic laboratory setting. Although the use of MALDI-TOF MS has until now been relatively limited for fungal identification compared with its application for bacterial identification, it is a cost-effective, rapid, sensitive and easy-to-handle tool for the early identification of these clinically important human pathogens. The diversity among the mass spectra obtained from isolates of the major molecular types, which is comparable with those obtained from other well defined yeast species, provides further evidence that the seven major haploid molecular types are cryptic species, which deserve separate species status.In conclusion, MALDI-TOF MS is an effective tool for the inter- and intra-specific differentiation of strains of the human pathogens C. neoformans/C. gattii species complex was investigated using 116 clinical, 32 environmental and 16 veterinary C. neoformans and C. gattii isolates , WM 626, WM 628  and WM 629  and for C. gattii, WM 179 , WM 178 , WM 175  and WM 779  were studied initially to generate reference spectra for each major molecular type. Subsequently, 20 isolates per molecular type were chosen randomly from over 3000 global cryptococcal strains to evaluate the ability of MALDI-TOF to distinguish between the eight major C. neoformans/C. gattii molecular types.The usability of MALDI-TOF MS for the classification of the major molecular types within the isolates  [6], [8]URA5 RFLP analysis and Luminex suspension array After analyzing all the spectra obtained from the 140 isolates with haploid genotypes  and 20 AD hybrids (VNIII), 4 additional inter-species hybrids strains were analyzed to assess the ability of the MALDI-TOF to detect potential hybrids. The strains tested included one VNI/VGI and three VNI/VGII hybrids previously identified by (URA5) via double digestion with the enzymes Sau96I and HhaI as previously reported The major molecular type of all isolates was identified by RFLP analysis of the orotidine monophosphate pyrophosphorylase gene C. neoformans and C. gattii were studied by MLST using the ISHAM consensus MLST typing scheme for the C. neoformans/C. gattii species complex, which includes the following seven genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5, and IGS1 C. neoformans major molecular type VNI were: WM 05.406, WM 05.473, WM 05.475, WM 05.479, WM 05.496, WM 05.553, WM 05.557, WM 09.170, WM 148, for VNII were: WM 05.483, WM 05.484, WM 05.485, WM 05.486, WM 05.490, WM 05.491, WM 05.523, WM 1816, WM 626 and for VNIV were: WM 01.126, WM 01.127, WM 02.142, WM 04.171, WM 04.172, WM 04.174, WM 05.469, WM 2242, WM 629. The strains for the haploid C. gattii major molecular type VGI were: WM 01.34, WM 02.103, WM 02.204, WM 05.410, WM 05.449, WM 08.108, WM 1009, WM 11.115, WM 179, for VGII were: WM 03.312, WM 03.697, WM 04.75, WM 04.78, WM 04.84, WM 05.272, WM 05.274, WM 05.275, WM 05.339, for VGIII were: WM 02.138, WM 02.139, WM 06.38, WM 09.43, WM 09.44, WM 09.45, WM 09.47, WM 11.10, WM 11.106 and for VGIV were: WM 04.20, WM 1220, WM 2363, WM 2579, WM 779, WM 780, WM 08.314, WM 1434, WM 2570.Nine strains of each of the seven haploid major molecular types of C. neoformans/C. gattii databases accessible at mlst.mycologylab.org.A phylogram of the studied strains was obtained with PAUP* 4.0b10 . The obtIsolates were cultured on Sabouraud dextrose agar and incubated for 48 h at 27°C. For the protein extraction one colony of each isolate was transferred separately into a 1.5 ml tube (Eppendorf) containing 300 µL of double-distilled water and mixed thoroughly for 1 minute. Subsequently, 900 µL of absolute ethanol was added in each tube and mixed for 1 minute. The samples were centrifuged for 2 minutes at 13000 rpm and the supernatant was removed. The pellet was dried at room temperature and according to the size of the pellet, 30 µL  or 50 µL (big pellet) of formic acid (70%) was added. In addition, an equivalent volume of acetonitrile was added and the mix was centrifuge for 2 minutes at 13000 rpm. After cleaning the MALDI target plate, 1 µL of the supernatant was place in each well, dried at room temperature (5 minutes), and overlaid with 1 µL of the matrix containing a saturated solution of α-cyano-4-hydroxy-cinnamic acid in 50% acetonitrile/2.5% trifluoroacetic acid. The plate was let to dry at room temperature before placing in the MALDI-TOF reader. Each obtained extract was placed in 6 different spots on the plate to generate 6 combined mass spectra (MSP) per isolate. Mass spectra were generated using the MALDI Biotyper Automation Control software version 2.0.43.8 .The proteins of the reference strains were extracted twice per isolate to assess the reproducibility of the results. To evaluate the effect of the number of colonies and the growth conditions of the isolates in the mass spectra obtained, one and three colonies of the reference strains incubated for 24 and 48 hours, were studied. Intact cells of the references strains were also placed directly in the MALDI-TOF plate in order to evaluate the extraction method.C. flavus (DSM70227T), C. laurentii (VML), C. macerans (DSM70822T), C. albidus (DSM 70197 PAH) and C. uniguttulatus , two of C. gattii (formerly classified as C. bacillisporus (RV490)) and five of C. neoformans  were used as reference spectra for the identification of the isolates, considering that they are the references for Cryptococcus species included in the All spectra generated were added to the in-house supplementary MALDI Biotyper BDAL MSP library (2011).The MALDI Biotyper software 3.0  was used to identify the isolates, to visualize the mass spectra and to generate the MSP dendrograms. The scores for identification were expressed as log(score) values, indicating highly probable species identification (2.300–3.000), secure genus identification with probable species identification (2.000–2.299), and probable genus identification (1.700–1.999). The dendrograms were generated with the following settings: seuclidean as distance measure, ward linkage and 1 (one) as the maximum number of top levels nodes. One strain of each Table S1Cryptococcus neoformans and C. gattii isolates studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).(DOC)Click here for additional data file.


Viruses have developed unique and oftentimes complex molecular mechanisms to ensure efficient egress of mature virions from infected cells. Over the years, virologists have begun to unravel and appreciate the intricate roles of both viral and host proteins in this process, and particularly the specific recruitment of host factors to promote efficient budding of infectious virus. A better understanding of these virus-host interactions and the mechanisms of virus budding not only will provide fundamental insights into the functions of both viral and host proteins, but also will lead to the emergence of novel strategies to inhibit virion egress and spread. In this special issue on virus budding/host interactions, we have invited both review articles to summarize recent findings in the field as well as original research articles to provide new insights into this late stage of virus replication. The first paper of this special issue is a review article that focuses on the current knowledge of arenavirus budding and the critical role that the small RING finger Z protein plays in this process. S. Urata and J. C. de la Torre present a comprehensive discussion on the structure and function of Z and highlight the various L-domain motifs conserved in arenavirus Z proteins and in the matrix proteins of other emerging RNA viruses. Viral L-domains are of particular interest since they mediate recruitment of host proteins to promote virus budding and, as such, constitute attractive and potentially broad-spectrum targets for antiviral drugs designed to inhibit virus egress. Indeed, the authors discuss several strategies and ongoing efforts to identify and screen candidate budding inhibitors. in silico screening) of L-domain/host interactions for their ability to inhibit particle release.  The viral L-domain/host interaction theme continues in the second paper, where Y. Liu et al. have utilized a bimolecular complementation (BiMC) approach to detect, localize, and follow filovirus VP40-host complexes in live mammalian cells in real time. The authors postulate that adaptation of the BiMC approach to the study of virus-host interactions and budding may help address gaps in our understanding of the dynamics, kinetics, and trafficking patterns of virus-host complexes involved in the budding process. In addition, the BiMC approach was used in conjunction with the more established filovirus VP40 virus-like particle (VLP) budding assay to test candidate small molecule inhibitors  and neuraminidase (NA) glycoproteins of influenza virus, along with the critical roles of M1 and M2 proteins in orchestrating virus assembly and in membrane bending/particle scission, respectively.  Little is known about the role of human respiratory syncytial virus (HRSV) glycoproteins during assembly and budding of mature virions. In the fourth paper, M. Batonick and G. W. Wertz address this gap by engineering recombinant viruses and using microscopic and biochemical analyses to demonstrate that deletion of the G and F proteins affected the incorporation of other viral proteins into budding virions; however, their absence did not directly affect the efficiency of virion egress. Thus, this study ascribes a novel role for the G and F glycoproteins during the late stage of HRSV replication.Sulfolobus turreted icosahedral virus (STIV) and Sulfolobus islandicus rod-shaped virus 2 (SIRV2). The authors demonstrate that the STIV c92 protein forms unique pyramid-like structures on the cell surface through which newly assembled virions are released during cell lysis. The authors speculate that this new lysis system may be common within other archaeal viral populations present in acidic hot springs.  In the fifth paper, Snyder et al. describe a novel and intriguing cell lysis system utilized by two archaeal viruses, Ronald N. HartyAnthony P. SchmittFadila BouamrCarolina B. LopezClaude Krummenacher