Chlorobium tepidum (two-domain architecture), the hyperthermophile Methanocaldococcus infernus (single-domain architecture) and the mesophile Klebsiella oxytoca (two-domain architecture), showing in silico characterization of their nitrogen fixation (nif) gene clusters, conserved NifB motifs, and functionality. C. tepidum and M. infernus NifB were able to complement an Azotobacter vinelandii (ΔnifB) mutant restoring the Nif+ phenotype and thus demonstrating their functionality in vivo. In addition, purified C. tepidum NifB exhibited activity in the in vitro NifB-dependent nitrogenase reconstitution assay. Intriguingly, changing the two-domain K. oxytoca NifB to single-domain by removal of the C-terminal NifX-like extension resulted in higher in vivo nitrogenase activity, demonstrating that this domain is not required for nitrogen fixation in mesophiles.One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases. Here we study the diversity and phylogeny of naturally occurring NifB proteins, their protein architecture and the functions of the distinct NifB domains in order to understand what defines a catalytically active NifB. Focus is on NifB from the thermophile  Due to the strength of its triple bound, N2 shows very little reactivity and is therefore not easily available to living organisms (2 fixing organisms (diazotrophs) capable of converting N2 into NH3, an accessible form of nitrogen, probably appeared in the primordial Earth when the levels of combined nitrogen gradually depleted  or anfG (Fe-only nitrogenase), and either FeV or FeFe cofactors at the active site. These cofactors are proposed to be identical to FeMo-co except for containing V or Fe in place of Mo -radical protein that converts [4Fe-4S] clusters into NifB-co, an [8Fe-9S-C] cluster that serves as precursor to FeMo-co, FeV-co and FeFe-co, thus catalyzing the first committed step in nitrogenase active-site cofactor biosynthesis  . NifB isynthesis   (K. oxytoca (NifBKo) (Clostridium acetobutylicum (NifBCa) (M. infernus (NifBMi) (Methanosarcina acetivorans (NifBMa) (Methanobacterium thermoautotrophicum (NifBMt) , a number of conserved motifs were identified in the SAM-radical domain including an HPC motif, the AdoMet motif (Cx3Cx2C) common to all SAM-radical proteins, an ExRP motif, an AGPG motif, a TxTxN motif and a Cx2CRxDAxG motif. Putative NifB proteins that did not present all these motifs were eliminated from the dataset, which was then reduced by 28% down to 289 sequences   are show(NifBAv) , K. oxyt(NifBKo) , Clostri(NifBCa) , M. infe(NifBMi) , Methano(NifBMa) , Methano(NifBMt) , and C. Figure 1A). This protein configuration accounted for 73% of NifB sequences in the Bacteria domain of the curated database . The functionality of this NifB subfamily has been demonstrated exclusively in vitro for M. infernus (NifBMi) (M. acetivorans (NifBMa) and M. thermoautotrophicum (NifBMt)  , M. acet(NifBMt) .Clostridium genus of the Firmicutes phylum is unique in that it contains all three NifB architectures. The curated NifB database contains 45 Firmicutes species likely to be diazotrophic organisms. Among these, 55% carry the stand-alone SAM-radical domain, 33% carry the two-domain architecture, and 13% carry the three-domain architecture .Importantly, the Figure 2A and Supplementary Table M. infernus and M. villosus) and the Methanobacteria (Methanobrevibacter smithii and Methanothermobacter thermautotrophicus). Bacteria diazotrophic species were distributed as follows: Aquaficae ; Bacteroidetes , which clade with Chorobi ; Actinobacteria ; Chloroflexi ; Cyanobacteria (Anabaena sp. and Cyanothece sp.); and Firmicutes , all found in the same clade. Finally, α-proteobacteria , β-proteobacteria (Azoarcus sp.), γ-proteobacteria , and δ-proteobacteria (Arcobacter nitrofigilis) were all in the same clade.Using the curated NifB database, 28 organisms representing the diversity of phylogenetic groups having diazotrophic members  were selFigures 2B,C) and a 64 contiguous amino acid sequence used to generate two different NifX-like domain trees .Because of the existence of three different NifB architectures, poorly aligned segments could potentially distort the phylogenetic tree analyses. Therefore, these regions were removed with Gblocks software  leaving M. infernus and is shown in Figure 2B. A derivative tree illustrating the distribution of NifB domain architecture is presented in Figure 2C. The Aquaficae, Actinobacteria, and Cyanobacteria did not clade with Firmicutes, as expected according to Figure 2A, but with Proteobacteria classes, leaving the Firmicutes as out-group to all of them. Interestingly, the γ-proteobacteria NifBKo was found as out-group to all proteobacteria in agreement with previous analysis . No Archaea NifB with a NifX-like domain has to our knowledge been found. Chloroflexi also lack this domain, suggesting acquisition from Archaea by a lateral gene transfer event (LGT) as previously suggested (M. infernus), Figure 2D uses NifX from Methanobacteriales (M. thermautotrophicus) and Figure 2E uses NifX from Methanosarcinales (M. acetivorans). The pattern of the first tree is similar to that of the SAM-radical domain tree, suggesting Chloroflexi as the bacterial ancestor from which the lineage emerged. The second tree, however, points to Firmicutes as the bacterial ancestor from which nif genes proliferated in Bacteria. This remains an interesting possibility given that Firmicutes present the three different NifB architectures known to date.The phylogenetic signal of the NifX-like domain of NifB was also analyzed , the hyperthermophile M. infernus (single-domain architecture), and the mesophile K. oxytoca (two-domain architecture). NifBKo and NifBMi have previously been purified and characterized in vitro , FeMo-co biosynthetic genes , and regulatory genes   strain under the control of the nifH promoter . A. vinelandii is a strict aerobe with optimum growth temperature of 30°C and is used here to provide an initial screen of NifB functionality that will be useful for further screening and implementation in Eukaryotic hosts. Strains UW418  and UW422  exhibited diazotrophic growth both in solid and liquid culture media , in contrast to the Nif- phenotype of the parental strain UW140 (ΔnifB). Calculated diazotrophic growth rates (ln2/td) were: 0.23 for the wild type, <0.001 for UW140, 0.015 for UW418, and 0.13 for UW422. This data shows that, although both NifBCt and NifBMi originate from strict anaerobic and thermophilic microbes, the proteins were functional and could complement the A. vinelandii ΔnifB mutant phenotype. However, whereas NifBCt supported similar growth rate at 30°C as the A. vinelandii wild type strain, the recombinant NifBMi did not, possibly explained by the almost 40°C difference in optimal growth temperature between C. tepidum .Genetic complementation analyses were performed by expressing synthetic codon-optimized um 48°C,  and M. ius 85°C, . No diffIn vivo nitrogenase activities determined by the acetylene reduction assay showed significant activity in UW422 in the 8 h period following nitrogenase derepression . No activity was detected in UW418 within this period of time, consistent with its significantly lower diazotrophic growth rate.Ct was expressed and purified from a recombinant A. vinelandii strain . The yield of pure NifBCt from A. vinelandii cells was 0.3 μg NifBCt per gram of cell, 15-fold higher than that of overexpressed NifBAv , in good agreement with theoretical mass determined by the amino acid sequence (46.8 kDa). As isolated NifBCt contained 3.05 Fe atoms per monomer. In vitro reconstitution of its [Fe–S] clusters under reducing conditions increased Fe contents to 10.1 ± 0.07 Fe atoms (n = 3). Consistently, features characteristic of [Fe–S] proteins  were more prominent in the reconstituted NifBCt UV-vis spectrum . Reconstituted NifBCt was active in the in vitro FeMo-co synthesis and nitrogenase activation assay: 5.2 ± 2.2 nmol ethylene formed⋅min-1⋅assay-1 (n = 2) compared to 8.2 ± 1.5 nmol ethylene formed⋅min-1⋅assay-1 (n = 2) when using pure NifB-co.NifBd NifBAv . Purity MinifB to complement the ΔnifB strain strongly suggests that the SAM-radical domain of NifB is the only one required for the synthesis of the FeMo-co precursor, but this could be a property specific to the stand-alone SAM-radical domain subfamily. To determine whether the NifX-like domain naturally present in the two-domain NifB architecture is required for NifB-co synthesis, a truncated NifBKo variant lacking the entire NifX-like domain (Ko-nifBΔC) was generated, introduced in K. oxytoca UC9 (ΔnifB) and expressed under the control of a tac promoter  were measured at 3 h intervals in a 24 h time course following derepression and compared to those of UC16 , a control strain expressing full-length NifBKo. Surprisingly, UC28 exhibited diazotrophic growth similar to UC16 and in vivo nitrogenase activity higher than UC16 . The UC9 parental strain did not exhibit nitrogenase activity or diazotrophic growth, confirming that the functionality of the expressed NifBKo variants and suggesting that the NifX-like extension of NifBKo is not required for NifB-co synthesis, at least under the growth conditions tested in this study, and that this could be a general rule for the two-domain family of NifB proteins.The capacity of in vivo in the model organisms A. vinelandii and K. oxytoca.To our knowledge, this work presents the largest compilation of NifB proteins described to date. The NifB database was stringently filtered to exclude faulty annotated proteins and the curated dataset provides insights about NifB origin, taxonomy and architecture that complement previous work . In this3Cx2C motif required for SAM-radical catalysis. In contrast, the NifX domain was identified in each one of them and we think that these faulty annotated NifB proteins are instead NifX. This confusion originates from the fact that the NifX domain is present in NifB, NafY, NifY as well as NifX proteins.A strict filter, based on motifs exhibited by experimentally confirmed NifB proteins, was applied to the initial database. As a result, 28% NifB sequences were excluded from further analysis. Although these criteria might be too strict, we reasoned that it was better to miss some true-positives than to risk including false-positives. Most excluded NifB proteins lacked the conserved Cxnif cluster via LGT to a Firmicutes ancestor that co-existed in the same ecological niche. Then, a fusion event happened that resulted in the nifB-nifX protein occurring in Firmicutes. It was later suggested that the wide spread of the nifB-nifX fusion protein in Bacteria was independent of the selective pressure associated with aerobic diazotrophy . It was also not clear whether this NifB family would function in a mesophilic and aerobic environment, which could prevent their use for plant nitrogenase engineering. Therefore, the Nif+ phenotype exhibited by the A. vinelandii ΔnifB strain complemented with MinifB presented in this study is convincing evidence of its in vivo functionality in a mesophilic and aerobic bacterium.Stand-alone SAM-radical domain NifB proteins catalyze NifB-co synthesis in vitro . However+ phenotype was achieved by complementation with NifBCt. C. tepidum is a mild thermophile with optimum growth temperature of 48°C and therefore much closer to the 30°C optimum of A. vinelandii. In addition, NifBCt has a two-domain NifB architecture similar to NifBAv. Interestingly, NifBCt was a monomer, similar to the archaeal single-domain NifB proteins and different from the NifBAv and NifBKo homodimers. Although constrained by the limited set of available experimental data, it appears that NifB monomers might be more stable and therefore favored in thermophilic organisms regardless of protein architecture. Importantly, both configurations are functional in vivo in a mesophilic host. The strong diazotrophic growth of UW418 in plates compared to liquid medium suggests that there are other factors limiting NifBMi activity A. vinelanii in addition to operational temperature. One possibility is that oxygen limitation during growth in plate has a positive effect on NifBMi that is not observed in liquid medium.As expected, stronger NifAv domain architecture  could be required to coordinate [Fe–S] cluster precursors prior to catalysis resulting in NifB-co synthesis  rates even higher than full-length NifB. It is thus reasonable to think that NifB catalysis only requires the SAM-radical domain, and that other domains may perform complementary functions that are beneficial but not essential for FeMo-co biosynthesis. A critical role in cofactor biosynthesis for alternative nitrogenases is not likely as this domain is absent in NifB from M. acetivorans, which carries all three types of nitrogenase  (K. oxytoca (NifBKo) (Clostridium pasteurianum (NifBCp) (M. infernus (NifBMi) (Methanosarcina acetivorans (NifBMa) (Methanobacterium thermoautotrophicum (NifBMt) (C. tepidum (this work) were aligned to determine conserved motifs. These NifB fingerprint motifs localized in the SAM-radical domain and included an HPC motif, the AdoMet Cx3Cx2C motif, an ExRP motif, an AGPG motif, a TxTxN motif, and a Cx2CRxDAxG motif . The full NifB dataset was then analyzed for the presence of these fingerprints, reducing the initial 390 sequences to 289   and UniP(NifBAv) , K. oxyt(NifBKo) , Clostri(NifBCp) , M. infe(NifBMi) , Methano(NifBMa) , Methano(NifBMt) , and C.  Table 2 . The fre3, an online tool that uses the full NCBI taxonomy to generate phylogenetic trees .Twenty-eight NifB proteins representing all phylogenetic groups known to contain diazotrophs  Supplem were sel4 was used to generate protein alignments and neighbor joining (NJ) phylogenetic trees  and a 64 contiguous amino acid sequence used to generate the NifX-like domain trees . Phylogenetic trees shown in Figures E were resolved using the Interactive Tree of Life online tool6   and UW140 (ΔnifB) (K. oxytoca strains UC9 (ΔnifB) and UC16  (The strains and plasmids used in this work are listed in Supplementary Table  (ΔnifB)  have bee-nifBKo)  have beeM. infernus  and C. tepidum  nifB sequences were codon-optimized and synthesized by GenScript  for expression in E. coli. Plasmids pRHB557 and pRHB558 contained the CtnifB and MinifB genes, respectively, cloned into the NdeI and EcoRI sites of pRHB258 for the expression of His9-tagged proteins under the control of the nifH promoter (A. vinelandii UW140 (ΔnifB) by homologous recombination at the D-sequence, a 1.1-kb DNA fragment from the chromosomal region downstream of Avin02530 . Transformants were selected in agar plates of NH4+-free Burk’s modified medium . The exponential growth rate constant corresponds to ln2/td, where td represents the doubling time.For diazotrophic growth rate A. vinelandii in vivo nitrogenase activity determinations strains were grown at 30°C on NH4+ supplemented Burk’s medium and then collected, washed and derepressed for nitrogenase as previously described (For escribed . Acetyleescribed .tac::Kogst-nifB) is a derivative of pGEX-4T-3  that contains the entire KonifB gene  fused to a gst-encoding gene , was cloned into the NdeI and XhoI sites of pGEX-4T-3 to generate plasmid pRHB554. K. oxytoca UC9 (ΔnifB) strain was transformed with pRHB554 to generate strain UC28 . Positive transformants were selected in LC agar plates containing ampicillin (150 μg/ml) and carbenicillin (800 μg/ml).Expression plasmid pRHB233 , and the in vivo nitrogenase activity was determined by ethylene production at 30°C for 30 min in 1-ml culture samples at a normalized OD600 value of 1, as previously described (For diazotrophic growth rate and  acetate . Cells wescribed . The groAzotobacter vinelandii UW422 cells overexpressing NifBCt under the control of a nifH promoter were grown in 32-l batches in a 300-l fermenter (Bioprocess Technology). Nitrogenase derepression and cell collection were carried out as described in  supplemented with protease inhibitors (200 μM PMSF and 1 μg/ml leupeptin) and 5 μg/ml DNAse inside a Coy Labs glovebox for 30 min. Cells were pelleted at 14,000 × g for 10 min at 4°C and then transferred back inside the glovebox. Pellets were lysed by osmotic shock in 450 ml buffer B . A cell-free extract was obtained by collecting the supernatant after centrifugation at 70,000 × g for 1 h at 4°C. The cell-free extract was supplemented with NaCl to a final concentration of 180 μM and loaded onto a 25-ml IMAC column  previously charged with Co2+ and equilibrated in buffer C  at 4°C. Column was washed with 3 column volumes of buffer C, followed by 7 column volumes of buffer C supplemented with 50 μM imidazole. NifBCt was eluted using buffer C supplemented with 300 μM imidazole. Eluted fractions were analyzed by SDS-PAGE and Coomassie staining. Fractions containing pure NifBCt were pooled and desalted using a HiPrep 26/10 desalting column  previously equilibrated with buffer C. Purified NifBCt was stored in liquid N2 as pellets.Purification of His-NifBCt Native Molecular Weight was determined by size-exclusion chromatography using a HiLoad 16/600 Superdex 200 column attached to an AKTA FPLC . The column was equilibrated with 50 μM Na2HPO4, pH 7.6, 180 μM NaCl, 10% glycerol, 5 μM 2-mercaptoethanol and 2 μM Na2S2O4 and the chromatography was run with the same buffer at a flow rate of 1 ml/min. The column was calibrated for molecular mass determination by using the molecular weight standard proteins aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), and carbonic anhydrase (29 kDa) .NifBCt samples were diluted in 50 μM Tris-HCl (pH 8) buffer containing 200 mM KCl and 10% glycerol to a final concentration of 10 μM NifBCt. Samples were then incubated during 2 h at 37°C with a 12-fold molar excess of Fe2+ [(NH4)2Fe(SO4)2] and S2- (Na2S), in the presence of 10 μM DTT. The Fe and S excess was removed from reconstituted preparations by filtration in a HiPrep 26/10 desalting column  equilibrated in dilution buffer. After desalting, Fe content of reconstituted NifBCt samples was quantified as described by As isolated NifBAzotobacter vinelandii UW140 (ΔnifB) cell-free extracts were obtained as described above and used for biochemical complementation assays. Purified NifBCt (0.16 μM) was added to reaction mixtures containing 0.2 ml of UW140 cell-free extract (4.4 μg protein/ml) in 22 μM Tris-HCl (pH 7.4), 17.5 μM Na2MoO4, 175 μM R-homocitrate, 400 μM (NH4)2FeSO4, 400 μM Na2S, 880 μM SAM, 1.32 μM ATP, 18 μM phosphocreatine, 2.2 μM MgCl2, 3 μM Na2S2O4, 3.5% glycerol, 40 μg creatine phosphokinase, and 2 μM NifH at a final volume of 400 ml. Control reactions contained 1.4 μM pure NifB-co instead of NifBCt. Reactions were incubated at 30°C for 45 min inside an MBraun glovebox (O2 < 0.1 ppm) to allow for FeMo-co synthesis and insertion into apo-NifDK present in the UW140 extract. Acetylene reduction activity of reconstituted NifDK protein was quantified after addition of 0.1 μg NifH, changing the vial gas phase to 100% argon, and finally injecting 0.5 ml acetylene. Reaction mixtures were incubated in a water bath at 30°C for 15 min and 600 rpm shaking and then stopped by addition of 0.1 ml of 8 M NaOH. Ethylene formation was measured in a Shimadzu GC-2014 gas chromatograph equipped with a Porapak N80/100 column.CE-E, SA, EJ-V, and AS carried out experimental work; CE-E, SA, EJ-V, SB, and LR carried out experimental design and data analysis; CE-E, SB, and LR wrote the paper.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.


The Spondweni serogroup includes Zika and Spondweni viruses. Both viruses have been historically misidentified and their diseases have been misdiagnosed due to their serological cross-reactivity and similar clinical presentations. Early case reports indicate a subset of patients present with clinical manifestations suggestive of serious illness.Flaviviruses have a high serological cross-reactivity. Before the advent of genetic sequencing, serological assays such as virus neutralization and hemaglutination-inhibition were used to differentiate virus species. Much of the early work differentiating flaviviruses into various serogroups was later confirmed by sequencing and phylogenetic analyses. Historically, serological assays  were also used to determine evidence of prior infection and geographic distribution. Both viruses in the Spondweni serogroup exhibit serological cross-reactivity and cause non-specific febrile illness in humans, making diagnosis challenging in regions where both viruses circulate.––Aedes aegypti mosquitoes,Zika virus was first isolated in Uganda in 1947 (strain MR-766),Both Zika and Spondweni viruses are primarily transmitted to humans through the bite of an infective mosquito, and the majority of infections are asymptomatic. In symptomatic Zika and Spondweni virus cases, signs and symptoms appear as early as three days post-infection.,,,,The six well documented cases of Spondweni virus infections report signs and symptoms of fever (100%), headache (83.3%), nausea (83.3%), myalgia (66.6%), arthralgia (50.0%), vertigo (33.3%), conjunctivitis (16.7%), maculopapular and pruritic rash (16.7%), epistaxis (16.7%), photophobia (16.7%), vomiting (16.7%) and disorientation (16.7%).,,,–While most symptomatic Zika and Spondweni virus infections present with mild to moderate febrile illness, a subset of cases present with short duration clinical manifestations suggestive of more serious illness. Before 2014, conjunctivitis, maculopapular and/or pruritic rash, hematuria, hematospermia, aphthous ulcer and epistaxis — indicating vascular leakage — as well as reports of photophobia, vomiting, vertigo, disorientation, meningismus and bilateral transient ocular paresis — indicating neurological involvement — had been reported.in utero may be a result of undetected endemicity: girls exposed before puberty would be immune during their reproductive years.While Zika virus has a wide geographic distribution,As infectious disease surveillance activities shift to focus on the current Zika virus epidemic, it is critical to remember that numerous other mosquito-transmitted viruses can cause infections resulting in non-specific febrile illness. These infections may clinically resemble symptomatic Zika or Spondweni virus infections and require appropriate laboratory diagnostics to identify.


Opportunity costs of bed‐days are fundamental to understanding the value of healthcare systems. They greatly influence burden of disease estimations and economic evaluations involving stays in healthcare facilities. However, different estimation techniques employ assumptions that differ crucially in whether to consider the value of the second‐best alternative use forgone, of any available alternative use, or the value of the actually chosen alternative. Informed by economic theory, this paper provides a taxonomic framework of methodologies for estimating the opportunity costs of resources. This taxonomy is then applied to bed‐days by classifying existing approaches accordingly. We highlight differences in valuation between approaches and the perspective adopted, and we use our framework to appraise the assumptions and biases underlying the standard approaches that have been widely adopted mostly unquestioned in the past, such as the conventional use of reference costs and administrative accounting data. Drawing on these findings, we present a novel approach for estimating the opportunity costs of bed‐days in terms of health forgone for the second‐best patient, but expressed monetarily. This alternative approach effectively re‐connects to the concept of choice and explicitly considers net benefits. It is broadly applicable across settings and for other resources besides bed‐days. Hence, choosing to treat or care for one patient means a lost opportunity to treat or care for another patient in the presence of an unmet demand, as exemplified by waiting lists. It is such a trade‐off that loosely embodies the economic notion of “opportunity costs”.Most economic textbooks define opportunity costs similar to “the value of the next‐best alternative” forgone, and others consider “ what is given up”  economic textbooks  and EconLit (Ovid). Additionally, the reference lists of all articles screened in full‐text were subsequently checked for relevance. The search was last updated on 02 December 2016, using the following search terms: “bed‐day” AND ; “opportunity costs” AND  for details of the syntax see Table Third, we compared and appraised the different approaches to explore their impact on bed‐day values. Drawing on these findings, we developed a novel approach for valuing the opportunity costs of bed‐days in line with economic theory and from the perspective of a decision maker aiming to maximise health with limited resources.33.1Early economic theorists initially interpreted opportunity costs in terms of units of a displaced alternative product: “If among a nation of hunters […] it usually costs twice the labour [time] to kill a beaver which it costs to kill a deer, one beaver should naturally exchange for or be worth two deer” Smith, . In thisDeparting from natural units was seen as necessary by economists to account for the monetary value used in almost all kinds of exchange  when providing/treating one more unit of option i. Also, “next‐best” here means “second‐best” when referring to Methodologies A and B, but it means the alternative with the greatest value when referring to Methodology D. The four methodologies then read:ju denotes the number of units of the next‐best alternative forgone, the net benefit is calculated by subtracting the marginal expenditure of the next‐best alternative, Cj, from its marginal gross benefit, jB, and iC is the marginal expenditure of the alternative chosen. Generally, expenditures are a sacrifice, typically of money, and benefits a gain, typically valued as the minimum willingness to trade‐off, that is, pay or sell depending on the perspective, also known as the marginal rate of substitution  3.2.1Two approaches used the first methodology by expressing units of the second‐best alternative as patient‐equivalents (Approach 1) or treatment‐equivalents (Approach 2).Patient‐equivalents were calculated in terms of the number of alternative patients that could have been treated using the same resources, for example, bed‐days, differently. Frequently, the alternative patient was (implicitly) approximated by the average patient population likely to occupy that bed. One article additionally adjusted for an occupancy rate of 0.75  occupancy rate of bed‐days, iC is the expenditure incurred, and jC is the expenditure of the forgone alternative use . For didactic reasons, we have separated fractions into two terms to demonstrate how to value the units consumed by patient i.Based on Equation 3.2.2The second methodology was used by four approaches that valued the units measured either monetarily (Approach 3) or in terms of the health benefit, usually QALYs, using patient‐equivalents (Approach 4) and/or local cost‐effectiveness thresholds (Approaches 5 and 6). Not all articles using patient‐equivalents reported them separately.Monetary values took on providers' forgone gross expenditures, payment losses (mostly diagnosis‐related groups), or net revenue losses. One article adjusted the revenue in sensitivity analyses by 0–25% to account for an occupancy rate of 0.75–1.00  or present them separately (Approach 8).The  economic convention of valuing the actually chosen alternative was widely followed by taking either market prices, national tariffs and payment sources, average expenditures from budgets and accounts, or reference costs. Stated‐preference techniques such as willingness‐to‐pay surveys were also used to elicit values.Given that the opportunity costs of fixed resources such as bed‐days or clinic slots are not always adequately reflected by the monetary value of prices and payments, especially in the short run, they may be separated from the variable costs related to other consumables. In the short term, variable costs better indicate cost changes according to changes in resource consumption as no cash savings will be realised for the fixed costs proportion . Opportunity costs were represented as the  economic costs for providers, even though most articles identified opportunity costs as only the forgone net revenues; for instance: “The sum of opportunity cost and total cost defines the true cost of a surgical device” ; in our example patient P2 and not P3. Note that the highest valued patient and the second‐best patient are unlikely to be each other's second‐best alternative; this is only true for the special case of identical marginal opportunity costs.Table i  according to the nine approaches is shown in Table The value of the opportunity costs for the 10 bed‐days consumed by treating one patient 3.4The approach used to value bed‐days, and consequently the units in which their costs are expressed, must be chosen to match the decision‐making agent's objective(s). In our illustration based on the existing approaches, different agents such as providers, payers, or society may seek to maximise natural units, expenditures, revenues, net revenues, or health outcomes.i less favourable, which likely distorts the bed‐days' true value.Approaches 1 and 2 lend themselves for analyses of natural units, for example, to maximise throughput. Expressing the relative costs of production as the exchange rate between natural units (Approach 1) reflects the true value of competing resource consumptions more accurately than the exchange rate between the expenditures associated with the natural units (Approach 2) given the independence of monetary inflation. In fact, if the expected exchange value differs from the expected cost value, a change in the optimal choice may occur  has been criticised for the missing link to actually displaced or unfunded services , and to capture any changes in health occurring without the treatment; cf. discussion. In healthcare settings operating near full capacity and in the presence of an unmet demand from otherwise treated patients awaiting admission, the term OCR is to be omitted.Expressing the health benefit in monetary terms is exactly what is captured with the net monetary benefit, NMB Claxton, , which ii equal £14,000 in terms of net benefits forgone for the second‐best patients j. By considering health maximisation as objective and the net monetary value of the forgone QALYs, this differs from the values calculated with the existing approaches of Methodology B, that is, providers' expenditures of £10,000, revenue of £12,000, net revenue of £2,000, gross monetary benefit of £24,000, and 1.2 gross QALYs forgone. Moreover, if the chosen alternative had not been optimal, Methodology D would need to be used.Applied to the data in Table 1 was the optimal choice as indicated in Table 1 were the sum of the expenditure and the highest net benefit forgone smaller than the benefit incurred. Thus, the true value of the opportunity costs for the 10 bed‐days is here equal to the forgone second‐best net benefit of £14,000.However, P4This paper explored different estimation techniques for valuing the opportunity costs of resources. Although seen as fundamental in defining economics  as the bulk of treatment costs can be expected to occur towards the beginning of a stay . To enhance generalisability, a pragmatic compromise may be to use patient groups from the regular patient population awaiting admission to a particular ward. Potential heterogeneity of patients should be taken into account by, for example, considering patient sub‐groups. From a payer perspective, specific reimbursement payments may be taken as expenditure for the different patient groups, possibly adjusted for price distortions such as subsidies  such as infectious disease epidemics . Additionally, the opportunity costs of bed‐days lost due to isolation or closure of wards can be calculated on the same scale as those directly consumed by patients. By identifying bed‐days as resources, this paper concentrated on the last two steps of costing of identification, measurement, and valuation  revenue maximisation but not equity concerns.Although our approach values opportunity costs with the NMB, it is not used incrementally here to evaluate two or more different healthcare interventions  be aware of the underlying assumptions and resulting biases when applying these approaches, which frequently remain unmentioned and unquestioned; (b) carefully consider the adequate costing approach dependent on an agent's objective and perspective; and (c) explicitly state any potential implications in a manner that is comprehensible to decision makers. For decision makers aiming to maximise health, we propose a novel alternative for bed‐days that effectively re‐connects to the concept of choice and explicitly considers net benefits.The authors have no conflict of interest.Table A1: Search syntaxes used for the scoping literature review of existing applications suitable to estimate the opportunity costs of bed‐days (last search on 02 December 2016).Figure A1. Flowchart of the scoping literature review of existing applications suitable to estimate the opportunity costs of bed‐days.Table A2: Overview of relevant papers detailing existing applications suitable to estimate the opportunity costs of bed‐days.Table A3. Input data to illustrate the approaches to value the opportunity costs: gastroenteritis casesTable A4. Overview of approaches to value the opportunity costs of bed‐days used for patients with acute gastroenteritisTable A5. Opportunity cost results of the 80 bed‐days used for gastroenteritis cases using Approach New5.Table A6. Input data to illustrate the value of the opportunity costs: TLH in terms of TAH.Table A7. Overview of approaches to value the opportunity costs of bed‐days for TLH surgeryTable A8. Opportunity cost results of the 3,760 bed‐days used for TLH procedures using Approach New1.Click here for additional data file.


Ixodes ricinus tick from a natural focus where human neurological disease is rare. The strain shows unique characteristics in neuroinvasiveness and neurovirulence.We report here the complete genome sequence (GenBank accession no. KX268728) of tick-borne encephalitis strain HB171/11, isolated from an  Flavivirus in the family Flaviviridae, is the most important tick-borne virus in Europe and Asia. The virus genome consists of a single-stranded 11-kb (+ [positve sense]) RNA coding for one large polyprotein, yielding three structural proteins  and seven nonstructural proteins . TBEV strains differ in their neuropathogenicity. We present the whole genome of a unique low-pathogenic TBEV strain, adding more data on the neuropathogenicity of TBEV strains.Tick-borne encephalitis virus(TBEV), a member of the genus The clinical symptoms of the infection by TBEV strains may range from subclinical to severe and fatal forms. The basis of this variability of clinical symptoms is unknown. However, there is some evidence that beside individual patient characteristics , viral characteristics also may determine the outcome of the clinical form of infection. For the pathogenicity of a neurotropic virus, its ability to invade the central nervous system (CNS) from the peripheral route of infection (neuroinvasiveness) and its ability to cause pathological changes in the CNS (neurovirulence) in laboratory mice can be distinguished . In natuIxodes ricinus ticks from a natural focus in southeastern Germany. In this natural focus, several human cases with mild, mainly gastrointestinal, and constitutional symptoms, but no neurological symptoms, occurred in the nearby village. Ticks were crushed and nucleic acid extracted and tested as described  (KX268728.The whole-genome sequence of MucAR HB171/11 has been submitted in GenBank under the accession number


Corticoviridae is a family of icosahedral, internal-membrane-containing viruses with double-stranded circular DNA genomes of approximately 10 kb. Only one species, Pseudoalteromonas virus PM2, has been recognized. Pseudoalteromonas virus PM2 infects Gram-negative bacteria and was isolated from seawater in 1968. Pseudoalteromonas virus PM2 is the first bacterial virus in which the presence of lipids in the virion has been demonstrated. Viral lipids are acquired selectively during virion assembly from the host cytoplasmic membrane. The outer protein capsid is an icosahedron with a pseudo T=21 symmetry. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Corticoviridae, which is available at www.ictv.global/report/corticoviridae.The  T=21 lattice [Icosahedral virions consist of an internal membrane and an outer protein capsid that has a diameter of 57 nm between facets , Fig. 1. lattice . Lipids The genome is a highly supercoiled, circular, double-stranded DNA of 10 079 bp . The DNAXII, shares significant sequence similarity with the superfamily I group of replication initiation proteins [Replication of the Pseudoalteromonas virus PM2 genome, most probably by a rolling circle mechanism, takes place in proximity to the host cytoplasmic membrane. The largest virus protein P12, encoded by gene proteins . The genCorticoviridae family consists of only the genus Corticovirus, containing one recognized species – Pseudoalteromonas virus PM2  Report:


Eudromia elegans) in Germany. The full-length genome of this virus shares <76% sequence identity with other avihepadnaviruses. The virus may therefore be considered a new extant avian hepadnavirus.In 2015, we identified an avian hepatitis B virus associated with hepatitis in a group of captive elegant-crested tinamous ( Hepadnaviridae, members of which constitute 2 major extant genera: Orthohepadnavirus, which infect mammals, and Avihepadnavirus, which infect birds  belongs to the family Eudromia elegans), a member of the ancient group of birds the Palaeognathae, which includes emus (Dromaius novaehollandiae) and ostriches (Struthio spp.).Hepadnaviruses generally are characterized by their narrow host range and strong hepatotropism  underwent necropsy at the University of Veterinary Medicine Hannover, Foundation . Initial histologic examination revealed moderate, necrotizing hepatitis and inclusion body–like structures within the hepatocytes. To identify a putative causative agent, we isolated nucleic acids from the liver and prepared them for sequencing on an Illumina MiSeq system  . We compAvihepadnavirus, forming a new clade (The newly identified ETHBV shared <76% nt sequence identity with other avian HBVs  Table 1.ew clade , panel Aew clade  Figure 1The identification of ETHBV prompted us to retrospectively screen the flock of 7 elegant-crested tinamous at Wuppertal Zoo and the 6 that had died within the past 4 years and had undergone necropsy at the University of Veterinary Medicine Hannover, Foundation  Table 4.To further characterize ETHBV, we confirmed infection in the liver using an in situ hybridization protocol , panel BETHBV can be considered a new extant hepadnavirus associated with hepatitis in the elegant-crested tinamou. Whether ETHBV can infect other species within the Palaeognathae or whether it is host restricted within other tinamou species remains to be elucidated. The discovery of ETHBV suggests that other avian species may harbor as-yet undiscovered HBVs. The pathogenesis of avian hepadnavirus infections and the mechanisms of virus transmission in captive tinamou flocks warrant further investigation.Additional materials and methods for analysis of probable new avian hepadnavirus in an elegant-crested tinamou, Germany.


Chikungunya virus is a mosquito-borne alphavirus which causes an acute febrile illness associated with polyarthralgia. Beginning in August 2013, clinicians from the Yap State Department of Health in the Federated States of Micronesia (FSM) identified an unusual cluster of illness which was subsequently confirmed to be chikungunya virus disease. Chikungunya virus disease previously had not been recognized in FSM.Information from patients presenting to healthcare facilities was collected and analyzed. During August 11, 2013, to August 10, 2014, a total of 1,761 clinical cases were reported for an attack rate of 155 clinical cases per 1,000 population. Among residents of Yap Main Island, 3% were hospitalized. There were no deaths. The outbreak began on Yap Main Island and rapidly spread throughout Yap Main Island and to three neighboring islands.Chikungunya virus can cause explosive outbreaks with substantial morbidity. Given the increasing globalization of chikungunya virus, strong surveillance systems and access to laboratory testing are essential to detect outbreaks. Aedes species of mosquitos and immunologically-naïve populations. In August 2013, chikungunya virus disease was identified for the first time in the Federated States of Micronesia. The explosive outbreak that followed is described in this report. Given the increasing globalization of chikungunya virus, strong surveillance systems and access to laboratory testing are essential to detect outbreaks. When outbreaks occur, a swift vector control response and implementation of prevention measures are important to limit disease spread.Chikungunya virus can cause large outbreaks with substantial morbidity, especially in places with certain  Chikungunya virus is a mosquito-borne alphavirus capable of causing large outbreaks of acute febrile illness with severe polyarthralgia ,2. FirstDuring August to October 2013, clinicians from the Yap State Department of Health in FSM identified an unusual cluster of illness characterized by acute fever and arthralgia. Among 51 patients with samples initially sent to the U.S. Centers for Disease Control and Prevention (CDC), 38 (75%) had evidence of recent chikungunya virus infection by detection of ribonucleic acid (RNA) or anti-chikungunya virus immunoglobulin (Ig) M and neutralizing antibodies. Testing for other arboviruses, including dengue, Zika, Ross River, and O’nyong-nyong viruses by reverse transcription-polymerase chain reaction (RT-PCR) or IgM tests was negative. We describe the epidemiologic features of FSM’s first known chikungunya virus disease outbreak.Yap State is a collection of islands in the western part of FSM in the western Pacific Ocean. It consists of Yap Main Island, which is divided into 10 municipalities, and 10 inhabited neighboring islands and atolls . Yap StaWe defined a clinical case of chikungunya virus disease as a Yap State resident with acute onset of fever and new onset of arthralgia or arthritis on or after August 11, 2013 (the onset date for the first laboratory-confirmed case). A laboratory-confirmed case was a clinical case with chikungunya virus RNA or IgM antibodies detected in serum. Clinical cases who resided on an island with no laboratory-confirmed cases were excluded from the analysis. A clinical case without detectable IgM antibodies in serum collected ≥8 days after illness onset was considered not to have chikungunya virus infection.Cases were identified among patients presenting to any health care facility in Yap State, including Yap Memorial Hospital on Yap Main Island or any outpatient clinic located throughout Yap State. All patients presenting with fever or new onset of arthralgia or arthritis were evaluated by a healthcare provider. For patients who met the clinical case definition, health care providers on Yap Main Island completed a case report form and on neighboring islands maintained a line list with patient demographic and symptom information.After identification of chikungunya virus as the causative agent of the outbreak, only a subset of subsequent clinical cases had serum samples tested for chikungunya virus infection. Testing of all samples was not possible for logistical reasons. Clinical cases from new geographic areas, who were hospitalized, and those aged <5 years were prioritized. In most weeks a convenience sample of clinical cases from Yap Main Island also had samples tested.Samples collected from patients beginning January 1, 2013, as part of routine dengue fever surveillance on Yap Main Island also were available. There samples were retrospectively tested to establish the onset date of the chikungunya virus disease outbreak as accurately as possible.Serum samples were tested for chikungunya virus RNA byRT-PCR and/or anti-chikungunya virus IgM antibodies by enzyme-linked immunosorbent assay (ELISA). The test used depended on the timing of sample collection, with RT-PCR typically being used for samples collected ≤5 days after illness onset and IgM ELISA for samples collected ≥5 days after illness onset . Nearly Data were entered into a Microsoft Access database and analyzed using Microsoft Excel 2010 and Epi Info 7.1.4. For persons who presented on two or more occasions, only data from the first visit were included. Chikungunya virus disease attack rates per 1,000 population were calculated using 2010 census data . HospitaThis assessment was judged to be non-research public health practice, and therefore it was not subject to institutional review board review requirements. For epidemiological analysis, the data were anonymized.From August 11, 2013, to August 10, 2014, a total of 1,761 clinical cases were reported to the Yap State Department of Health. This represents an attack rate of 155 clinical cases per 1,000 persons i.e., 15% of the population sought health care for a clinically-compatible illness. Overall, 904 (51%) clinical cases were female, and the median age was 30 years (range: 3 weeks–92 years). All age groups had attack rates greater than 110 per 1,000 population, with the highest attack rates in persons aged ≥30 years . Among tThe outbreak began on Yap Main Island in the northeastern municipality of Tomil . The firOverall, 171 (10%) clinical cases across Yap State had serum samples tested and 119 (70%) had laboratory-confirmed chikungunya virus infection. Four (2%) had equivocal or indeterminate results. Forty-four (26%) had negative chikungunya virus laboratory results, but their serum samples were collected too early (<8 days) after illness onset to exclude infection. Only four (2%) clinical cases had no detectable anti-chikungunya IgM antibodies in serum collected ≥8 days after illness onset and were thus considered not to have recent chikungunya virus infection.Two-hundred nine (12%) clinical cases had dengue testing performed by NS1 antigen or IgM antibody testing. One patient with illness onset in September had dengue virus IgM antibody detected; this patient also had chikungunya virus infection confirmed by IgM antibody testing.Sixty-nine patients on Yap Main Island with dengue-like illness during January 1 through August 10, 2013, had samples retrospectively tested for evidence of recent chikungunya virus infection. None were positive for chikungunya virus RNA or IgM antibodies.Beginning in August 2013, Yap State in FSM experienced an explosive chikungunya virus disease outbreak. Initially limited to Yap Main Island, the outbreak spread to two neighboring islands within 3 months, and to a third within 6 months. By August 2014, 15% of Yap State’s population had sought healthcare for symptoms compatible with chikungunya virus disease.We believe this was FSM’s first chikungunya virus disease outbreak. Previous dengue and Zika virus disease outbreaks in FSM were investigated without finding evidence of chikungunya virus infections in either humans or mosquitoes –8. FurthAedes hensilli and Ae. aegypti mosquitoes in Yap State serving as disease vectors [It is unknown how chikungunya virus was introduced into Yap State, but it was likely imported by an infected person, as has occurred in previous chikungunya virus disease outbreaks ,10. Gene vectors .Previous studies have suggested considerable morbidity among patients affected by chikungunya virus disease. During an outbreak on Grand Comore Island, 52% 79/152) of ill persons missed work or school for a mean of 7 days . Further52 of illThere were two main limitations to our analysis. First, cases were limited to patients who presented to medical care and were reported to the Yap State Department of Health by a healthcare provider. Because we were unable to identify sick persons who did not present for medical care, the reported attack rate is likely an underestimate of the true rate. Second, some clinical cases might not actually have had chikungunya virus infection as not all clinical cases were tested. However, there was minimal circulation of dengue virus and no circulation of other arboviruses that cause similar clinical syndromes, and our clinical case definition was similar to one shown to have 84% sensitivity and 89% specificity during a chikungunya outbreak in Mayotte . TherefoAedes species of mosquitos and immunologically-naïve populations. Given the increasing globalization of chikungunya virus, strong surveillance systems and access to laboratory testing are essential to detect outbreaks [Chikungunya virus is capable of causing explosive outbreaks with substantial morbidity, especially in places with certain utbreaks . When ou


Grapevine rupestris vein feathering virus and Grapevine yellow speckle viroid 1 were recorded in the Czech Republic for the first time.Comprehensive next generation sequencing virus detection was used to detect the whole spectrum of viruses and viroids in selected grapevines from the Czech Republic. The novel NGS approach was based on sequencing libraries of small RNA isolated from grapevine vascular tissues. Eight previously partially-characterized grapevines of diverse varieties were selected and subjected to analysis: Chardonnay, Laurot, Guzal Kara, and rootstock Kober 125AA from the Moravia wine-producing region; plus Müller-Thurgau and Pinot Noir from the Bohemia wine-producing region, both in the Czech Republic. Using next generation sequencing of small RNA, the presence of 8 viruses and 2 viroids were detected in a set of eight grapevines; therefore, confirming the high effectiveness of the technique in plant virology and producing results supporting previous data on multiple infected grapevines in Czech vineyards. Among the pathogens detected, the  The infection of plant tissues by viral pathogens can cause significant economic losses to agriculture crops . Many diThe availability of such a broad-spectrum detection technique provides powerful support to certification processes, which are supposed to guarantee the clean health status of propagation materials .In addition, the use of the metagenomic approach in vegetatively propagated species would help in preventing the spread of viruses  as well https://www.ncbi.nlm.nih.gov/guide/all/). Genomic resources specific to viruses can be accessed at https://www.ncbi.nlm.nih.gov/genome/viruses/.The NGS method generates a very large amount of sequencing data from each sample, and thus the high demand for proper data processing and analysis arises. UNIX-based open-source platforms for the entire process exist, as well as commercial ones such as CLC Genomic Workbench, Geneious, or Lasergene. The need for reference sequences is primarily provided by the NCBI, with its large and freely available databases  present in plant tissues , 8. TherTechniques for the molecular detection of grapevine viruses are based on the analysis of RNA, isolated either from vascular tissues or from softer tissues such as leaves and petioles. NGS techniques more frequently use RNA isolated from leaves and petioles as the starting materials , 9.The present work was aimed at demonstrating the NGS technique's utility for the detection of grapevine viruses using phloematic tissues as the starting material, and in obtaining a picture of the virome of different Czech grapevine cultivars.All plants analyzed in the work belong to the public collection of the Crop Research Institute, Prague, Czech Republic. They are officially recognized as genetic resources under “The National Program on the conservation and utilization of genetic resources of microorganisms important for food and agriculture”. Their free use for research purpose is guaranteed by the Czech national legislation, namely Act no. 148/2003 Coll., on the conservation and utilization of genetic resources of plants and microorganisms important for food and agriculture.Grapevine cuttings were collected in November 2013 from grapevines maintained under controlled conditions in a screenhouse of the Crop Research Institute, Prague, Czech Republic, ensuring freedom from infection of some additional pathogens.All the grapevines had already been partially characterized, and the present work was focused on those plants known to contain larger numbers of viruses, as well as those showing symptoms of an unclear etiology. Further criteria for plant selection were their having been different cultivars and having different origins from divergent parts of the Czech Republic. A descriptive list of the samples is provided in Vitis berlandieri × Vitis riparia), originating from Modřice, South Moravia, coordinates 49.1252N, 16.6031E. It was tested for the first time in 2005 during the ELISA (enzyme-linked immunosorbnet assay)-based grapevine virus survey [Grapevine leafroll-associated virus-1 (GLRaV-1), Grapevine virus A (GVA), Grapevine virus B (GVB), and Grapevine fleck virus (GFkV). Testing by RT-PCR also revealed infection by the Grapevine rupestris stem pitting-associated virus (GRSPaV) [Plant G1 is grapevine rootstock Kober 125 AA (origin s survey . During (GRSPaV) .Cuttings from the plant were taken in 2005, from which a new plant was grown and cultivated at the Crop Research Institute Prague (CRI) in a screenhouse, where it was labeled as TI23. The plant did not show any symptoms of virus infection.Plant G2, labeled as TI21 in the screenhouse of CRI , of grapevine variety Pinot Noir, had recently been bought in a market. It was selected as being representative of certified propagated material. The origin of the plant was Mělník, Central Bohemia, Czech Republic. The plant showed mild mosaic symptoms, and was positive in RT-PCR, using generic primers for tymoviruses .Grapevine Pinot gris virus (GPGV) [e.g., stunting, mottling and leaf deformations) [Plant G3, labeled as TI25 in the CRI screenhouse, is a interspecific grapevine hybrid of variety Laurot, having originated from a vineyard of genetic resources held in the Mendel University in Brno, Faculty of Horticulture, Lednice, South Moravia. The cultivar is a product of crossing Merlan (Merlot × Seibel 13 666) × Fratava (Frankovka × Svatovavřinecké); while the Seibel 13666 is a complex interspecific variety, used as a donor of resistance against fungal diseases. The G3 plant is the first grapevine in the Czech Republic found to be infected by the newly emerged mations) .Plant G4, labeled as TI15 in the screenhouse of the CRI, is grapevine variety Müller-Thurgau, having originated in the Karlštejn Research Station of Viticulture, Central Bohemia, coordinates 49.9350611N, 14.1652414E. This Research Station is a unit of the Crop Research Institute, Prague, Czech Republic. The plant was previously found to be infected with GLRaV-1, GVA, and an unspecified tymovirus. The plant was showing symptoms of a mild mosaic, and downrolling of the leaf blades.Plant G5, labeled as TI30 in the screenhouse of the CRI, grapevine variety Guzal Kara, having originated from Modřice, South Moravia, coordinates 49.1252N, 16.6043E, was showing symptoms of irregular and dichotomous growth, typical for nepovirus infection, although no nepovirus had thus far been detected. In 2006, the plant was also replanted into the screenhouse of the Crop Research Institute, Prague.Plants G6-G8 were originally selected from production vineyards in Perná, South Moravia for containing different viruses based on previous commercial tests. These plants showed symptoms such as leaf mottling, mosaic, and leaf deformations; but not symptoms typical for GPGV . The graTotal RNAs were extracted from 1 g of scraped phloem according to . The lowhttp://www.ncbi.nlm.nih.gov/) with an e-value threshold of 10−6 in both. Contigs mapping to the Vitis vinifera L. genome, as well as those mapping to bacterial and fungal genomic fragments or their viruses were omitted from further work. A list of the potential viruses and viroids present in the analyzed samples was created, and reference sequences were selected for mapping of NGS reads. As the first choice, sequences from the RefSeq database (NCBI) were used, because they contain full-length sequences of particular viruses and viroids. For viruses with a lower homology with RefSeq-originated data, sequences with a higher homology were used for mapping, despite the fact they were not of full length. This was especially the case for GRSPaV, GVA, and GVB viruses. A list of reference sequences is presented in The sequence quality was checked by use of fastQC-0.10.1 , and a FAfter mapping the unique reads on the reference sequences, the presence of the studied viruses in grapevine vascular tissues were then determined. The data carried with BLAST and mapping analyses adjudged that viruses whose genome coverage were at least 20%, and with a sequencing depth greater than 5, were expected to be present in the sample. Further confirmation of the presence of a virus or viroid was done by RT-PCR as a second method, and a positive reaction in RT-PCR was the definitive criterium for estimating the presence of a virus or viroid.A genome coverage threshold of 20% was used according to the authors' experience, based especially on the analysis of the presence of real viruses in examined grapevines. A sequencing depth of 5 was used according to published works –23.™ Plant Total RNA Kit . Reverse transcription was performed with a Transcriptor First Strand cDNA Synthesis Kit , using random hexamers for reaction priming according to the manufacturer's suggestions. FastStart Taq DNA Polymerase  was used for PCR. The conditions and primers used for the detection of individual viruses and viroids are listed in The isolation of RNA from vascular tissues was performed using a SpectrumIn the case of unclear results, and a first record of the pathogens, the products of RT-PCR, were cloned into pGEM-T Easy vector and sequenced. At least five clones from every sample were commercially sequenced. The resulting sequences were compared, the genetic divergence was estimated, and a phylogenetic tree was constructed using the MEGA7 program  to ensur6, G2 = 1.5×106, G3 = 2.7×106, G4 = 2.1×106, G5 = 1.9×106, G6 = 1.3×106, G7 = 8.9×105, G8 = 8.2×105 unique reads, respectively , Closteroviridae (Grapevine leafroll-associated virus 1), and Betaflexiviridae ; whereas, the two viroids (Hop stunt viroid and Grapevine yellow speckle viroid 1) belong to the family Pospiviroidae.Plants of various varieties and geographical origins were included in the analyzed set; primarily from Moravia , but also from wine regions in Bohemia . Libraries representative of the sRNA populations extracted from grapevines G1-G8, and sequenced by Illumina technology, contained G1 = 1.6×10ectively . Furtherapevines . The eigThe sequences obtained were submitted to GenBank NCBI under the following accession numbers: KT000353-KT000360 (HSVd), KP693444-KP693448 (GPGV), KT000346-KT000352 (GYSVd-1), KT000361-KT000362 (GFkV), KT000363-KT000365 (GLRaV-1), and KT000366-KT000371 (GRSPaV). Sequences of GVA, GVB, GRVFV, and GSyV-1 were not completed because of the low coverage of the full-length reference sequences.The results of sRNA sequencing were checked by RT-PCR detection; thus meaning that all detected pathogens (viruses and viroids) had their presence proven in the tested materials by at least two methods see . All mapTo compare the read mapping efficiency of two frequently used programs , belonging to the genus Foveavirus, family Betaflexiviridae, was confirmed to be present in all eight plants. A high coverage of mapped reads occurred in the majority of vines (6 out of 8) ranging from 97% to 100%; with low coverage in plants G1 and G4, reaching only 46% and 25%, respectively. The greatest amount, corresponding to sRNA GRSPaV, was detected in the G3  and G5 plants , with 100% coverage of the genome. Regarding the high level of GRSPaV genome variability, we used as a reference sequence KX274277.1 for mapping on sample G4 with 25% resulting genome coverage; the mapping on reference NC_001948.1 provided genome coverage that was too weak (only 16%); however, the other results suggested the presence of GRSPaV.Grapevine Pinot gris virus (GPGV), genus Trichovirus, family Betaflexiviridae, is a newly-emerged virus, recently identified in Italy [in Italy , and subin Italy , 34, 35,in Italy , 36. GPGGrapevine virus A (GVA), genus Vitivirus, family Betaflexiviridae, was detected in plants G1 and G4. GVA is a common grapevine virus in the Czech Republic [Republic . It had Grapevine virus B (GVB), genus Vitivirus, family Betaflexiviridae, was detected in plant G1. The virus is very rare in the Czech Republic [Republic . In factGrapevine leafroll associated virus 1 (GLRaV-1), genus Ampelovirus, family Closteroviridae, was detected in plants G1, G4, and G6. The virus had the greatest number of reads among all of the viruses. This is caused by the fact that it has the largest genome (more than 18 kb) compared to e.g., the 6–8 kb in fovea-, tricho-, and tymoviruses. GLRaV-1 occurrence is common in the Czech Republic [Republic .Grapevine fleck virus GFkV, genus Maculavirus, family Tymoviridae, was detected in plants G2 and G7. GFkV is a common grapevine virus in the Czech Republic according to the previous surveys [ surveys .Grapevine rupestris vein feathering virus (GRVFV), genus Marafivirus, family Tymoviridae, was detected in plants G1, G4, G7, and G8. RT-PCR with primers designed on the contigs and sequencing of the cloned PCR product confirmed the presence and proper taxonomic identity of the virus in all of the plants. The sequence of the PCR product was submitted into GenBank under reference number KX465108.This is the first confirmation of the presence of GRVFV in the Czech Republic.Grapevine Syrah virus 1 (GSyV-1), genus Marafivirus, family Tymoviridae, was detected in plants G1 and G3. The presence of this virus on the examined plants was also confirmed by [irmed by .Grapevine yellow speckle viroid 1 (GYSVd-1), genus Apscaviroid, family Pospiviroidae, was found to be present in 7 out of the 8 plants. To our knowledge, this is the first described occurrence of Grapevine yellow speckle viroid 1 in the Czech Republic. The viroid is reported to occur worldwide; e.g., in Italy [in Italy , USA [37in Italy , and Newin Italy .Hop stunt viroid (HSVd), genus Hostuviroid, family Pospiviroidae, was detected in all eight grapevine plants. HSVd was earlier detected in Czech grapevines by [The next viroid, vines by . HoweverVitis sp.Plant G1, which is grapevine rootstock Kober 125AA, does not show symptoms of virus disease, although it is infected with at least 7 viruses and viroids. As this genotype is an indicator for rugose wood complex viral disease, it should show some symptoms, as the plant is infected with several viruses responsible for expression of the disease mentioned. However, after grafting inoculum from this plant into the sensitive LN33 indicator, symptoms will appear . It is tPlant G2, showing symptoms of a mild mosaic, was found to be infected with several pathogens including GPGV . Mosaic Plant G3, showing symptoms of mosaic and vein banding, was known to contain GPGV. The presence of GPGV was confirmed together with other viruses and viroids. The typical symptoms of GPGV were not observed.Plant G4, showing symptoms of downrolling of its leaf blades, was found to contain GLRaV-1, GVA, GRSPaV, and viroids. GLRaV-1 is the cause of the downrolling of the leaf blades.Plant G5, showing dichotomous growth, was expected to contain some nepovirus. However, no nepovirus was detected by NGS, only GRSPaV and viroids. The symptoms are probably due to another endogenous factor causing a phytohormone imbalance .Plants G6 to G8 were showing symptoms of leaf mottling and deformations, but not those typical for GPGV. All three had a confirmed infection by NGS of GPGV. Since all three come from the cultivar Chardonnay, we can conclude that GPGV can cause symptoms of leaf mottling and deformations in this cultivar under Czech conditions.Based on the results of sRNA NGS detection, RT-PCR with specific primers to the corresponding pathogens were applied to RNA isolated from vascular tissues to confirm virus and viroid presence in the analyzed plants. The results are given in Using the RT-PCR method, GYSVd-1 in plant G6 was not detected, while the mapping of reads covered 57% of the reference sequence. Additionally, not one contig was identified to be of GYSVd-1 origin. The other seven plants were positive for the viroid in RT-PCR, with coverage ranging from 91–100%. We can conclude that the viroid is not present in plant G6.GPGV was detected by RT-PCR, even in plant G4; although the coverage was only 18%, which is under the 20% threshold. The numbers of mapped reads are shown in Tymoviridae, Grapevine rupestris vein feathering virus (GRVFV) was detected in plant G1. Its presence was confirmed by RT-PCR and sequencing of the PCR product. As mentioned in the introduction about plants tested in the present work, the G1 plant was originally positive in ELISA for GFkV. Since no GFKV is present in plant G1 (but GRVFV is), the antibodies were also probably accidentally raised against another virus other than GFkV. GRVFV was also detected in plants G4, G7, and G8.GFkV was detected by NGS in plants G2 and G7, and also confirmed by RT-PCR. In its first occurrence in the Czech Republic, another virus from the family For all of the reasons delineated, and due to the end-point nature of RT-PCR, as well as the massively parallel nature of sRNA NGS, sensitivity comparisons of the two methods can be very difficult. This subject should continue to be studied diligently. Further, it needs to be ascertained what kind of coverage and sequencing depth is sufficient to determine a positive sample. A comprehensive database of virus sequences should be used for NGS contig identification, and the one with the best matching score should then be used for genome mapping. For many grapevine viruses, full-length genome sequences from the Central European region are still missing.The diagnostics of RNA pathogens (viruses and viroids) was carried out using sRNA sequencing. This method seems very promising, using the metagenomic approach –4, consiThe plants used in this study were all tested and previously found positive for the presence of some RNA viruses. However, the spectrum range of RNA pathogens detected was still unexpected. In every one from the evaluated set of 8 plants, at least 3 RNA pathogens were detected. This study also provides an unique comparison of various varieties, grown in different wine regions of the Czech Republic. The relatively high occurrence of GLRaV-1 and GFkV in the Czech Republic is a well-known fact , 41. HowDistribution of foveavirus GRSPaV in the Czech Republic has not yet been studied; however, thus far, our experience with grapevine virus detection shows that its occurrence in Czech grapevines will be very high , 42.This study is also the first work to prepare libraries for sRNA sequencing from grapevine vascular tissues.  used wooThe virus detection percentage rates obtained in this study among the set of 8 plants from 5 localities are: GRSPaV—100%, GPGV—75%, GRVFV—50%, GLRaV-1–38%, GFkV—25%, GVA—25%, GSyV-1–25%, and GVB—13%. Viroid HSVd was detected in 100% of the plants, GYSVd-1 in 88% of the plants. This result is especially interesting due to the different geographical origins of the plants tested. According to the requirements of Czech legislation , plant propagation materials have to be tested for GFLV, ArMV, GLRaV-1, and GLRaV-3 (as well as for GFkV in the case of rootstock mother plants), which are all widespread in the Czech Republic [Use of the coverage value set at 20% proved to be a good threshold for estimating virus presence, taking into consideration use of suitable reference sequences for mapping, mostly the one giving the best identity during the BLAST search.The fact that all of the tested plants turned out to be positive for the presence of viroids is another interesting observation of this study.As a general conclusion, the results presented in this study demonstrate the great importance of correct and proper processing of NGS data, as well as the need for localized reference data on viruses and viroids.S1 File(PPTX)Click here for additional data file.S2 File(PPTX)Click here for additional data file.S1 Table(PPTX)Click here for additional data file.


Ticks are efficient vectors of arboviruses, although less than 10% of tick species are known to be virus vectors. Most tick-borne viruses (TBV) are RNA viruses some of which cause serious diseases in humans and animals world-wide. Several TBV impacting human or domesticated animal health have been found to emerge or re-emerge recently. In order to survive in nature, TBV must infect and replicate in both vertebrate and tick cells, representing very different physiological environments. Information on molecular mechanisms that allow TBV to switch between infecting and replicating in tick and vertebrate cells is scarce. In general, ticks succeed in completing their blood meal thanks to a plethora of biologically active molecules in their saliva that counteract and modulate different arms of the host defense responses . The transmission of TBV occurs primarily during tick feeding and is a complex process, known to be promoted by tick saliva constituents. However, the underlying molecular mechanisms of TBV transmission are poorly understood. Immunomodulatory properties of tick saliva helping overcome the first line of defense to injury and early interactions at the tick-host skin interface appear to be essential in successful TBV transmission and infection of susceptible vertebrate hosts. The local host skin site of tick attachment, modulated by tick saliva, is an important focus of virus replication. Immunomodulation of the tick attachment site also promotes co-feeding transmission of viruses from infected to non-infected ticks in the absence of host viraemia (non-viraemic transmission). Future research should be aimed at identification of the key tick salivary molecules promoting virus transmission, and a molecular description of tick-host-virus interactions and of tick-mediated skin immunomodulation. Such insights will enable the rationale design of anti-tick vaccines that protect against disease caused by tick-borne viruses. Dermacentor andersoni in 1907, ticks have been identified as vectors of a huge range of viral, bacterial, and protozoan agents of diseases, and have become a major focus of medical and veterinary research  or “tiboviruses” , and the families Nairoviridae, Phenuiviridae, and Peribunyaviridae in the new order, Bunyavirales  (mosquito-borne), and Nairobi sheep disease virus (1910) and louping ill virus (LIV) (1929) (tick-borne), opened the floodgates for the discovery of over 500 arboviruses during the ensuing years , was reported in Kansas in 2014  serocomplex, have emerged or re-emerged, and/or spread, posing an increasing threat to human and animal health. For example, a rise in the incidence of human infections caused by Powassan virus (POWV) in the USA , the genus Orthonairovirus of the Nairoviridae comprises 11 other species, 9 of which are TBVs (Table Phlebovirus genus (Phenuiviridae) first reported in China , transmitted by soft ticks, other DNA viruses may be transmitted by ticks. Lumpy skin disease virus , the cause of skin disease in cattle, is transmitted by blood-feeding insects such as mosquitoes and stable flies  , or detection of the presence of viral RNA or DNA in a tick, do not necessarily prove that a particular tick species is a competent vector of the virus Nuttall, . To deteOrnithodoros, Carios, and Argas, and hard ticks of the genera Ixodes, Haemaphysalis, Hyalomma, Amblyomma, Dermacentor, and Rhipicephalus  are vectors of a few TBV species, whereas others can transmit several different TBV species   and its principal vectors, Ixodes persulcatus and I. ricinus. Several experimental studies have been carried out to explain the TBEV—vector—host interactions. For example, tick infestation of viraemic laboratory animals indicated that most of the tested hard tick species  acquired TBEV from the infected blood meal and maintained the virus transstadially  is unclear in tick antiviral defense  of partially fed R. appendiculatus or A. variegatum females in comparison to ticks feeding on guinea pigs inoculated only with the virus  that had undetectable or very low levels of viraemia. In contrast, co-feeding transmission was about four-times lower to ticks feeding on tick-resistant bank voles (Clethrionomys glareolus) that produced significantly higher viraemia and virus loads in lymph nodes and spleen than Apodemus mice    are not able to maintain the virus, LIV can persist through NVT in mountain hares (Lepus timidus) populations. Mountain hares that did not develop detectable viraemia were shown to support NVT of LIV between co-feeding infected and non-infected I. ricinus ticks; the efficiency of NVT was significantly reduced when ticks co-fed on virus-immune hares , but not for I. ricinus or soft ticks (non-competent vectors) , but also in Metastriata species  cells, dendritic cells (DCs), T cell subpopulations , B cells, neutrophils, mast cells, basophils, endothelial cells, cytokines, chemokines, complement, and extracellular matrix -like helicases (RLHs) detecting nucleic acids within the cytosol  systems such as Toll-like receptors (TLRs) located at the cell surface and within endosomes, or the retinoic acid-inducible gene I -alpha is a powerful cytokine secreted by several cell types after viral infection. Together with IL1, TNF-alpha is known to promote DC migration from the skin into regional lymph nodes. TBEV infection was found to induce the release of TNF-alpha and IL6 by DCs, but undetectable levels of IL10 and IL12p70 were measured in DC cultures infected with the virulent Hypr strain in contrast to DCs infected with the less virulent Neudoerfl strain , a TLR-4 ligand, by inhibition of the activation of the ERK 1/2 and p38 MAP kinases, leading to increased production of IL10 and reduced synthesis of IL12p70 and TNF-alpha , to CCL4  (via CCR1), and to CCL5 (RANTES)   is also able to bind to human CCL3 and mouse CCL3 , and the IFN-gamma receptor subunit, IFNGR1 -induced production of IFN-alpha, IFN-beta, and IFN-gamma .Immune IFN, known as type II IFN or IFN-gamma, is secreted mostly by activated NK cells and macrophages during the early stages of infection  gene expression in a similar manner as the expression of type I IFN genes that is induced by transcriptional mechanisms involving IRF's and NF-κB , cytokines (Il1b) and anti-microbial molecules, and downregulation of genes related to DNA repair, transcription, chromatin remodeling, transcription factor binding, RNA splicing, and mRNA metabolism was demonstrated  that may be related to TNF-alpha, AP-1, and growth factor responses at the tick bite site, were upregulated while genes encoding cytoskeletal elements , signaling molecules, growth factor receptor (Pdgfrb), or growth factor (Tgfb3) were downregulated . After 3 h of POWV-infected tick feeding, cutaneous gene expression analysis revealed a complex proinflammatory environment, which included significant upregulation of proinflammatory cytokines related to neutrophil and phagocyte recruitment, migration, and accumulation . The most distinct histopathological difference between the POWV-infected vs. uninfected tick feeding sites was observed at 3 hpi, when higher levels of cellular infiltrates (mostly neutrophils and some mononuclear cells) were detected at the POWV-infected tick feeding sites compared to the uninfected feeding sites  microplus. They work by eliciting antibodies in immunized animals. When taken up in the bloodmeal of feeding ticks, the antibodies bind to the antigen resulting in damage to the midgut. The consequent impact on feeding success and reproductive output causes a gradual reduction in tick numbers and tick-borne infections  microplus] although other species  are being investigated. Most strategies favor the “hidden” or “concealed” antigen approach, as illustrated by the Bm86/Bm95-derived anti-tick vaccines. These are antigens not normally exposed to the host immune system during blood feeding. The most promising candidates include subolesin, ferritins, and aquaporins. Tick subolesin is functionally related to mammalian akirin-2, a downstream effector of the Toll-like receptor required in the innate immune response. A combination of subolesin and Bm86 has been patented as a more effective formulation for controlling cattle tick infestations  antigen that cross-reacts with a midgut  antigen  DNA levels were reduced ;Does not induce adverse host responses Cost-effective and practicalThe prospects of developing a single anti-virus vaccine against all TBVs are unrealistic at this point in time as no common target has yet been identified against which vaccines can be developed. However, the idea of a single anti-tick vaccine that provides universal protection against TBV infections is not quite so far-fetched given that TBVs have a common target: they are reliant on a tick vector to survive. Thus, if a tick antigen is found that is common to tick vector species, and immunization with the antigen induces a host response that interferes with virus transmission, the possibility of developing a universal TBV vaccine becomes real. The “wish list” for an ideal universal TBV vaccine looks something like this:Theileria parva, the protozoan tick-borne agent of East Coast fever in cattle, it was ineffective although 64TRP was effective against TBEV , we should move closer to specifying the ingredients required to generate an anti-tick vaccine that controls TBVs. Defining an environment at the tick-host interface that is “hostile” to TBVs opens up the possibility of creating a universal vaccine.Ticks succeeded in their role as blood-feeders and vectors of TBV thanks to their complex life history and feeding biology. Components in tick saliva have been found to play a crucial role in tick feeding and mediating transmission of TBV. Nowadays, an increasing body of evidence exists on (i) manipulation of host defenses by ticks to enhance feeding and promote pathogen transmission and (ii) strategies used by tick-borne pathogens to evade host immunity and ensure survival in different biological systems. However, much of this knowledge comes from tick and tick-borne bacteria interaction studies. Information on tick and TBV interaction is still limited and so far no tick molecules enhancing virus transmission have been identified. The systems biology approach employing transcriptomics and proteomics has started to reveal molecular mechanisms constituting the survival strategy and persistence of TBV in their vectors and vertebrate hosts as well as the interactions at the tick-virus-host interface determining virus transmission. Identification of SAT factors enhancing TBV transmission during the early phases of tick attachment in the host skin, but mainly the understanding of the complexity of the relationships between ticks, TBV, and their vertebrate hosts, will enable novel strategies for controlling ticks and viral tick-borne diseases.MK, PB, IS, ST, PN, MH, and VH conducted the literature search and wrote the paper. PB, PN, and MK prepared the figures and tables. All authors critically read and revised the manuscript.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.


With 71% of Malawians living on < $1.90 a day, high household costs associated with severe malaria are likely a major economic burden for low income families and may constitute an important barrier to care seeking. Nevertheless, few efforts have been made to examine these costs. This paper describes household costs associated with seeking and receiving inpatient care for malaria in health facilities in Malawi.A cross-sectional survey was conducted in a representative nationwide sample of 36 health facilities providing inpatient treatment for malaria from June–August, 2012. Patients admitted at least 12 h before study team visits who had been prescribed an antimalarial after admission were eligible to provide cost information for their malaria episode, including care seeking at previous health facilities. An ingredients-based approach was used to estimate direct costs. Indirect costs were estimated using a human capital approach. Key drivers of total household costs for illness episodes resulting in malaria admission were assessed by fitting a generalized linear model, accounting for clustering at the health facility level.Out of 100 patients who met the eligibility criteria, 80 (80%) provided cost information for their entire illness episode to date and were included: 39% of patients were under 5 years old and 75% had sought care for the malaria episode at other facilities prior to coming to the current facility. Total household costs averaged $17.48 per patient; direct and indirect household costs averaged $7.59 and $9.90, respectively. Facility management type, household distance from the health facility, patient age, high household wealth, and duration of hospital stay were all significant drivers of overall costs.Although malaria treatment is supposed to be free in public health facilities, households in Malawi still incur high direct and indirect costs for malaria illness episodes that result in hospital admission. Finding ways to minimize the economic burden of inpatient malaria care is crucial to protect households from potentially catastrophic health expenditures. Plasmodium falciparum malaria with artemisinin-based combination therapy (ACT), and management of severe malaria with parenteral artesunate or quinine contributes to reducing malaria mortality [Despite significant reductions in malaria burden in recent years, malaria remains a major cause of morbidity and mortality in Malawi. With an estimated 3.3 million malaria cases and 7200 deaths in 2015, malaria accounts for 34% of all outpatient consultations and remains the leading cause of death among children under 5 years old [The perceived and actual financial costs of care for malaria are important barriers to accessing prompt and effective malaria treatment and these costs can be economically catastrophic for many households –8. In a Few studies have examined the household costs of uncomplicated malaria and febrile illness treatment in Malawi and there are virtually no studies that have evaluated the household costs of inpatient malaria treatment. One study in 2004 found that mean reported direct costs per febrile episode amount to $1.05 (or $1.28 in constant US 2012 dollars) in urban areas compared to $0.21 ($0.26 in constant 2012 dollars) in rural areas . These cIn order to ensure equitable access to malaria treatment, especially among the most vulnerable populations, it is essential to understand the household costs of inpatient management of malaria. This study examines the direct and indirect household costs associated with malaria illness episodes resulting in inpatient treatment in Malawi, as well as the most important drivers of those costs. This analysis was conducted within a larger study of inpatient malaria case management at public and private nonprofit mission health facilities in Malawi. The study assessed different aspects of facility readiness to manage malaria cases, including availability of antimalarial and other supplies, availability of malaria diagnostic services, health worker training and adherence to national guidelines for the diagnosis and treatment of severe malaria. Details of this study are published elsewhere .Malaria is endemic throughout Malawi, where the entire population of 17.2 million is considered at risk . The mosHealth care is delivered free of charge at Ministry of Health (MoH) facilities, whereas Christian Health Association of Malawi (CHAM) facilities receive some government subsidies but charge a nominal fee . An esseA nation-wide, cross-sectional survey was conducted in 36 health facilities from June through August of 2012. The sampling strategy is described in more detail elsewhere [Patients were eligible to participate in the inpatient interview if they had been admitted with any malaria diagnosis, or admitted with another diagnosis but started on an anti-malarial treatment, at least 12 h prior to the initial arrival of the study team. Patients who were to be discharged on the day of the interview or had sought care at another facility before hospital admission were eligible to provide information on household costs associated with their present malaria episode. Caregivers were asked to respond for patients aged < 12 years or those unable to speak for themselves.Trained interviewers used Dell Axim X51 personal digital assistants (PDAs) to collect demographic and cost information from eligible patients or their caregivers. The questionnaires were programmed in visual CE . Questionnaires elicited demographic information for patients and their caregivers  and information about the patient’s current malaria episode and treatment seeking . Information on household assets and economic characteristics was also elicited. Patients were then asked to provide cost information for their visit to the current facility as well as any previous visits to health facilities for the current malaria episode. Total costs associated with malaria episodes resulting in inpatient admission was considered the primary outcome, while direct and indirect costs were secondary outcomes.Direct medical and non-medical costs were estimated using an ingredients-based approach in which the patient or caregiver was asked to recollect the costs incurred for individual items throughout their illness episode , 24. DirIndirect costs were estimated using a human capital approach , 26. ForAll statistical analyses were performed using SAS version 9.4  and STATA IC/14 . Frequencies and cross tabulations on patient demographics and mean and median direct and indirect costs were calculated using survey procedures with a cluster statement to account for clustering of individual observations at the health facility level. Household wealth index was calculated from household characteristic and asset data using principal component analysis . HousehoStandard diagnostics showed that the primary outcome (household costs) was highly skewed to the right and heteroskedastic Fig. a, b, prei is a vector of independent factors known or presumed to influence the household costs as outlined above. Two models were run; in the full model, all independent factors were included. In the restricted model, a backward stepwise regression was used, beginning with the full model, and retaining only independent factors with corresponding p values < 0.05. Robust standard errors clustered at the health facility level were generated. The exponential of coefficients (βi) in GLMs with a log link are interpreted as the ratio of arithmetic means between groups [The empirical GLM took the form:n groups .Ethical approval from both the US centers for disease control and prevention institutional review board  and the university of Malawi college of medicine research and ethics committee  was granted prior to study implementation. Written informed consent was obtained from patients 18 years and older, or caregivers for incapacitated patients or patients less than 18 years prior to data collection. Patients aged 7–18 years and not incapacitated were asked to provide written assent as well.p = 0.01), but otherwise did not differ based on demographics, illness, and treatment seeking characteristics. Among patients who did provide cost information, the mean age was 13 years ; 31 (39%) of the patients were under 5 years old, 46 (57%) were 5 years or older, and 3 (4%) were of unknown age. Approximately half (54%) were female, and the majority (94%) lived in rural areas.Out of 100 patients who were eligible to provide cost information, 80 (80%) provided cost information about their current malaria episode and were included in this analysis  prior to seeking care at the current facility, and spent a mean of 2.5 days admitted to the health facility . A total of 60 (75%) patients had sought care at another facility prior to admission at the study facility, and 26 (32.5%) patients were discharged on the day of the study. Patients lived a mean distance of 10.2 kilometres  away from the health facility, and more than half (55%) lived 5 or more kilometres away. Patients travelled for an average of 0.2 days  to and from the facility in order to access care.p = 0.06). Children under 5 years incurred a mean total cost of $9.74  compared to $22.86  for those 5 years and older (p < 0.01). Patients from the least poor wealth tertile had higher mean costs at $28.06  than those in the middle tertile  and poorest tertile  (p < 0.01). Household wealth was not associated with the likelihood of patients attending a CHAM vs. MoH facility (p = 0.14).The mean household cost of malaria episodes resulting in inpatient admission, including direct and indirect costs, was $17.48 . In bivariate analysis (Table p < 0.01). Patients who had sought prior care for their present malaria episode had significantly higher mean costs at $19.00  compared to $12.94  for patients who had not sought prior care (p = 0.02). Mean household costs were $11.56 per night admitted . Mean costs for patients who were discharged the day of the interview were $13.89 , compared to $16.05  for patients who were not discharged the day of the interview (p = 0.62).Patients from rural residences incurred lower costs at a mean of $15.03  compared to $50.68  for patients from urban or small town residences (p < 0.01). Household costs for children under 5 years were 40% lower than for patients 5 years and older (p < 0.01). Household costs were 40% lower for patients that lived < 5 km away from the health facility compared to those who lived 5 km or greater away (p < 0.01). Each day in the hospital was associated with a 2% increase in overall household costs (p = 0.03). Costs for households in the wealthiest tertile were 82% higher than costs for households in the middle and poorest tertiles (p < 0.01).Multivariate GLM regression was used to determine patient and facility characteristics associated with total household costs per malaria episode Table . In the p < 0.05) (Table p = 0.06), and no other predictors were found to be significant. Direct medical costs averaged $2.39 , with medications accounting for almost all direct medical costs . These direct medical costs differed significantly by facility ownership, averaging $1.06  at MoH health facilities compared to $4.19  at CHAM facilities (p = 0.01). Direct non-medical costs averaged $5.20 ; these averaged $4.54  for MoH facilities and $6.09  at CHAM facilities (p = 0.42). Meals and travel accounted for the largest proportion of direct non-medical costs and were the two most substantial contributors to overall direct costs, but were not significantly different by CHAM vs. MoH management type.The mean direct cost incurred by patients admitted for malaria episodes was $7.59  Table . Costs dOverall, each malaria episode resulted in a mean loss of 8.5 productive days , including lost productive days for both patients and their caregivers. This was associated with a mean lost productivity cost of $9.90 per episode  Table . While pp = 0.26). Households in the least poor wealth index tertile lost an average of 16.5 productive days , compared to 7.0 days  for households in the middle wealth tertile and 9.0 days  for households in the poorest tertile (p = 0.07).When associated caregiver costs are excluded, patients 14 years and older lost an average of 10.4 productive days , which was associated with an average lost productivity cost of $15.89 . Likewise, excluding patient costs, caregivers alone lost a mean of 9.2 days  caring for and accompanying sick patients, with an associated lost productivity cost of $10.65 . Caregivers accompanying patients under 5 years lost 7.9 productive days , compared to 9.9 productive days lost  for those accompanying patients 5 years and older (Despite policies aimed at providing free healthcare through the formal healthcare system , househoManagement of malaria episodes that result in admission poses a much greater economic strain on Malawian households than uncomplicated, outpatient malaria episodes. Studies in Malawi have reported direct household costs for uncomplicated malaria ranging from $1.28 to $5.52 in constant 2012 dollars , 17, 19.The high direct costs for management of malaria episodes resulting in hospitalization in Malawi are similar to those observed in Mozambique, Ghana, and Uganda for inpatient management of malaria episodes , 10, 33,Patients who visited CHAM-owned facilities incurred higher costs than those who visited MoH facilities. Although CHAM facilities receive some government subsidies for their operations , they chThe apparently higher cost of medication at CHAM facilities represents an important avenue for policy intervention; additional governmental subsidies for anti-malarial medications at CHAM facilities and increased attention to supply chain issues could reduce direct medical costs for patients visiting CHAM facilities. This is especially important given that family wealth status was not a predictor of which type of facility patients visited, so poorer families who visit CHAM facilities could benefit substantially from such policies. Additionally, continued medication subsidies should ensure that medications remain affordable at both CHAM and MoH facilities.Despite government efforts to expand healthcare access in rural areas of Malawi , increasThis study has several limitations. Because it was conducted as part of a larger study, it was not explicitly powered to examine cost outcomes. The retrospective study design introduced the possibility of recall bias, as patients may not have accurately remembered prices incurred for malaria treatment at previous health facilities, although cost information collected was limited to the current malaria episode in order to minimize recall bias. Additionally, the lost costs associated with school fees for absent days of school were not included in the indirect cost estimates, as data on school fees were not collected. Thus, the costs presented here may underestimate the true household costs of inpatient malaria episodes.Complete malaria episode information was only available for patients who were discharged the day of the survey. Thus, the cost estimates presented here may underestimate the true overall costs. However, total costs for patients who were discharged the day of the interview compared to those who were not yet discharged were not significantly different. Furthermore, patients were not followed longitudinally. Thus, even for patients discharged the day of the survey, information on after-care, including productive time lost by patients and their caregivers during recovery, and any recrudescence and related costs were not collected. Again, this likely resulted in underestimation of overall household costs associated with malaria episodes. Finally, the findings may not be generalizable to other settings where malaria care subsidies and cost drivers vary substantially.In settings such as Malawi where health care is heavily subsidized but direct costs remain relatively high, policy efforts to address both medical and non-medical drivers of these costs are crucial. Direct non-medical household costs may be mitigated by subsidizing meals and travel for families under a certain income level through the use of cash transfers or establishing health equity funds. Community health insurance schemes may also address the cost of care-seeking for acute febrile illness episodes. Meanwhile government subsidies should be continued and strengthened to ensure medications, consultations, and other direct medical costs remain affordable at both CHAM and MoH facilities. This is especially important given that the costs of inpatient malaria treatment might otherwise be catastrophic for the poorest families, and out of pocket costs are a major barrier to malaria care seeking among poorer households . With 71Indirect costs may be addressed through continued malaria prevention and education efforts. With more than 3 million malaria cases every year in Malawi, the economic costs due to productive days lost for patients and their caregivers are staggering, making prevention campaigns a good investment both for public health and economic growth , 38. ExpThe findings of this study contribute to the evidence highlighting the substantial economic burden of malaria episodes resulting in hospitalization on households. While direct expenditures are an important component of household costs, indirect costs due to lost productivity magnify the economic burden of inpatient malaria management on families, and costs were notably higher for patients from hard-to-reach households and for those who visited CHAM compared to MoH facilities. As perceived and actual costs of care are important dimensions of access, minimizing these costs is crucial to protect households from potentially catastrophic health expenditures, and ensure that all patients receive prompt and effective care.