Road crashes as a major global public health problem cost 3% of most countries and 5% of low- and middle-income countries gross domestic product (GDP). The World Health Organization has predicted that without sustained action, road traffic crashes will become the seventh leading cause of death by 2030. Objectives: The aim of this study was to analyze the death rate, severity of injuries, and direct medical costs caused by road traffic injuries (RTI) in the city of Tabriz in 2014.Trauma injury admissions due to RTI in Imam Reza Hospital in Tabriz City were investigated in terms of etiology and the direct medical costs during 2014â€“2015. Data were collected using a researcher-made checklist after being confirmed by relevant experts in terms of face validity. All information on direct medical costs are extracted from several sources including hospitals, database of the Ministry of Health and Medical Education, disaster and emergency medical management center, and public and private physiotherapy clinics across the city.Review of the hospital records showed that the mean age of the patients  was 34 ± 17.3 years. In addition, 79.2% of the patients were treated on an outpatient basis, and 20.8% were treated on a hospitalization basis . The mean times of inpatient and outpatient hospitalization for injuries were 3 h and 6.7 ± 5.3 days, respectively. Total direct medical costs were 11.631 dollars, of which 8% was for hospital costs, 9.7% for pre-hospital costs, and 2.3% for physiotherapy costs. Chest and lower part injuries had highest medical costs. From etiological standpoints, the greatest reason of being injured and hospitalization is multiple injuries and bruises, and the prominent cause of death was blow to the head and neck (70%).The results of the present study showed that direct medical costs in Tabriz during 2014-2015 were equal to 0.1% of GDP, which is a considerable amount. High economic and social costs of road accidents and their harmful physical and psychological effects on individuals and community require the attention of professionals and experts in the transportation industry and health-care system to determine appropriate strategies for interventions in reducing accidents burden and injuries.Road traffic injuries, Direct medical costs, Hospital, Trauma, Tabriz, Iran


Arabidopsis thaliana genotypes with Turnip mosaic virus (TuMV) and Cucumber mosaic virus (CMV). We characterized plant life-history trait modifications associated with reduced effects of TuMV and CMV on plant seed production (fecundity tolerance) and life period , both measured as a norm of reaction across viral loads (range tolerance). Also, we analysed resistance-tolerance and tolerance-tolerance trade-offs. Results indicate that tolerance to TuMV is associated with changes in the length of the pre-reproductive and reproductive periods, and tolerance to CMV with resource reallocation from growth to reproduction; and that tolerance to TuMV is traded-off against tolerance to CMV in a virulence-dependent manner. Thus, this work provides novel insights on the mechanisms of plant tolerance and highlights the importance of considering the combined effect of different pathogens to understand how plant defences evolve.Although accumulating evidence indicates that tolerance is a plant defence strategy against pathogens as widespread as resistance, how plants evolve tolerance is poorly understood. Theory predicts that hosts will evolve to maximize tolerance or resistance, but not both. Remarkably, most experimental works failed in finding this trade-off. We tested the hypothesis that the evolution of tolerance to one virus is traded-off against tolerance to others, rather than against resistance and identified the associated mechanisms. To do so, we challenged eighteen  Parasitism is the lifestyle of 50 per cent of all known organisms . This meThe two main host defences against pathogens are resistance, i.e. the host’s ability to limit pathogen multiplication , and tolA body of mathematical work has modelled the conditions in which tolerance evolves. Early models assumed that resources are limited and can be diverted into resistance or tolerance, but not both, and predicted that tolerance or resistance would prevail because they were mutually exclusive . More reA possible explanation for this lack of support of a resistance-tolerance trade-off is that other forces might come into play in shaping the evolution of plant defences. In nature, plant populations are challenged by multiple pathogens , not necTurnip mosaic virus  and Cucumber mosaic virus  and Arabidopsis thaliana . Both viruses are commonly found in wild populations of Arabidopsis at up to 80 per cent prevalence  tolerance to TuMV infection and if such tolerance is related to modifications of plant life-history traits. Specifically, we analysed the association between the effect of infection on plant progeny production (fecundity tolerance) and life period  with resource reallocation from growth to reproduction and with modifications in the length of the growth and reproductive periods. We also analysed resistance-tolerance trade-offs upon infection by TuMV and CMV, and if tolerance to TuMV is traded-off against tolerance to CMV.Raphanus sativus (Brassicaceae) and UK1-TuMV from a plant of Brassica napus (Brassicaceae). LS-CMV was originally obtained from a field-infected plant of Lactuca sativa (Asteraceae). All viruses were derived from biologically active clones , and transcripts were used to infect N.benthamiana plants for virus multiplication. We used a single CMV isolate because previous analyses indicated that, in Arabidopsis, the fraction of the variance in virulence/tolerance explained by the CMV isolate is very low (4%) (Viruses UK1-TuMV (Acc.N. AB194802), JPN1-TuMV (Acc.N. KM094174) and LS-CMV (Acc.N. AF127976) were used. JPN1-TuMV was obtained from a field-infected plant of low (4%) , which ilow (4%) . This al2), with 65–70 per cent relative humidity. In these conditions, plant genotypes conformed two allometric groups , and 0.32 ng of total RNA were added to the Brilliant III Ultra-Fast SYBR Green qRT-PCR Master Mix  according to manufacturer’s recommendations. Specific primers were used to amplify a 70-nt fragment of the TuMV, and a 106-nt fragment of the CMV, coat protein (CP) gene, respectively , inflorescence (IW) and seeds (SW) were obtained. RW was used to estimate plant resources dedicated to growth, and IW and SW were utilized to estimate plant resources dedicated to reproduction . Following IW/RW)i/(IW/RW)m and (SW/RW)i/(SW/RW)m ratios. Values of these ratios greater than one were considered as indicative of such resource reallocation. Seed viability, estimated as per cent germination, did not significantly differ between mock-inoculated (93.0–99.3%) and infected (91.0–99.7%) plants . Also, virus infection did not affect the weight of a single seed  (SW similarly reflects the number of viable seeds in both mock-inoculated and infected plants.Aboveground plant structures were harvested at complete senescence. The weights of the rosette (oduction . The eff≥ 0.110) . Thus, SGP), as days from inoculation to the opening of the first flower; reproductive period (RP), as days from the opening of the first flower to the shattering of the first silique; and plant post-reproductive period (PRP), as days from the shattering of the first silique to plant senescence. In Arabidopsis, the opening of the first flower co-occurs with the end of the rosette growth, and the shattering of the first silique co-occurs with the end of flower production (LP) was quantified as the sum of the three periods. The effect of virus infection on GP, RP and PRP, was quantified as infected to mock-inoculated plants ratios. The (RP/GP)i/(RP/GP)m and (PRP/GP)i/(PRP/GP)m ratios were used to analyse virus-induced alterations of plant development.We recorded growth period i/(IW/RW)m and (SW/RW)i/(SW/RW)m, and (RP/GP)i/(RP/GP)m and (PRP/GP)i/(PRP/GP)m were fitted to a Gamma distribution, and fecundity and mortality tolerance to a logistic distribution according to Akaike’s Information Criteria , where GV is the among-genotypes variance component and EV is the residual variance. Variance components were determined using GzLMMs by the REML method , and the interaction between virus and host genotype was significant . Thus, we analysed accumulation for each virus separately, considering plant genotype and allometric group as factors. For all three viruses, accumulation significantly differed between Arabidopsis genotypes , but not between allometric groups   . Thus, tBroad-sense heritability of virus accumulation ranged from moderate to high depending on the virus: ectively . TherefoSW and LP to virus accumulation regression, respectively) differed depending on the virus . Overall, both tolerance measures were smallest to UK1-TuMV and greatest to LS-CMV, with tolerances to JPN1-TuMV showing intermediate values . Thus, fecundity and mortality tolerances were analysed for each virus independently. From here on, results will be presented firstly for the two viruses at the tolerance extremes (UK1-TuMV and LS-CMV), and lastly for the intermediate state (JPN1-TuMV).Fecundity and mortality tolerances  (iSW/mSW > 1) . The negative slope was stepper for Group 1 genotypes, with none of the infected plants producing seeds, than for Group 2 ones, with only 61.8 per cent of sterilized plants and fertile individuals in 8/11 genotypes (SW to virus accumulation regression for Group 2 genotypes (lower fecundity tolerance) than for Group 1 ones . At odds with UK1-TuMV and LS-CMV-infected plants, in JPN1-TuMV-infected plants the slope of the SW to virus accumulation regression did not differ between allometric groups . However, Group 1 genotypes showed a bimodal response, and were divided into two subgroups according to this response: In Cum-0, Kas-0 and Ll-0 (Subgroup 1a), 50–70 per cent of infected individuals were sterilized and regression slopes were steep. Conversely, infected Cad-0, Cdm-0, Kas-2 and Kyo-1 plants (Subgroup 1b) produced seeds and regression slopes were shallower than for Subgroup 1a    . ExceptiSWm > 1) . Upon UKenotypes . Thus, G × 10−4) . Group 2≤ 0.040) .iLP/mLP < 1) . Mortality tolerance was smaller (steeper negative slope of the LP to virus accumulation regression) for Group 1 than for Group 2 plants  , indicating similar mortality tolerance, with very little effect of virus infection on LP as denoted by iLP/mLP values near one . As for JPN1-TuMV-infected plants, mortality tolerance did not vary between allometric groups . However, again Group 1 plants presented a bimodal response to infection: the slope of the LP to virus accumulation regression was significantly steeper in Subgroup 1a than in Subgroup 1b  and in Group 2 genotypes , which showed similar mortality tolerance    , with mo × 10−4) . In contnear one , and slo= 0.363) . Note th≥ 0.483) .Because fecundity and mortality tolerances are genotype-specific rather than plant-specific variables, by definition heritability for these traits could not be calculated.iRW/mRW, iIW/mIW and iSW/mSW, respectively) , this reduction always depending on the Arabidopsis genotype . For UK1-TuMV-infected plants, all ratios also depended on the allometric group . In Groups 1 and 2, iRW/mRW was greater than iIW/mIW  and iSW/mSW  suggesting no resource reallocation from growth to reproduction. Indeed, (IW/RW)i/(IW/RW)m and (SW/RW)i/(SW/RW)m were always smaller than one  , but not on IW , varied according to the allometric group. For Group 1, the effect of LS-CMV infection on RW was larger than on SW , whereas the opposite was observed in Group 2 . (SW/RW)i/(SW/RW)m differed between allometric groups , values being greater than one only for Group 1  (IW/RW)i/(IW/RW)m  and (SW/RW)i/(SW/RW)m  were also greater for Group 1 than for Group 2 plants, values being similar or greater than one only for Group 1  (SW/RW)i/(SW/RW)m  , indicating that resource reallocation was associated with fecundity tolerance in this subgroup  . In gene × 10−4) . The eff= 0.008) . Similar= 0.008) , for whi Group 1 . These r≤ 0.017) , and the= 0.016) . This rasubgroup .iGP/mGP, iRP/mRP and iPRP/mPRP, respectively) , this ratio being smaller for Group 1 than for Group 2 plants. Interestingly, in Group 2 genotypes the effect of infection on GP was greater than the effect on RP : iGP/mGP was significantly smaller , and iRP/mRP greater , than one. Thus, upon UK1-TuMV infection more tolerant Group 2 genotypes shortened their growth period but elongated the time dedicated to reproduction, as indicated by (RP/GP)i/(RP/GP)m values greater than one in this subgroup  their ratios being always near one in both allometric groups  (RP/GP)i/(RP/GP)m and (PRP/GP)i/(PRP/GP)m were also near one  , and (RP/GP)i/(RP/GP)m and (PRP/GP)i/(PRP/GP)m values smaller , than one. Finally, in JPN1-TuMV-infected plants i/GPmGP, i/RPmRP and i/PRPmPRP did not depend on the allometric group  (RP/GP)i/(RP/GP)m and (PRP/GP)i/(PRP/GP)m did not differ between Groups 1 and 2 and showed values smaller than one  .We also quantified the effect of infection on the plant growth, reproductive and post-reproductive periods (ctively) . Upon UKsubgroup . Note th≥ 0.383) . Accordi≥ 0.116) . Excepti≥ 0.349) . Also, (≥ 0.470) . The effGP, RP and PRP was higher than that of the effect of infection on RW and IW  . Thus, tSW and LP to virus accumulation regression were explored, a significantly negative association indicating a trade-off. No significantly negative association was observed between resistance and the two measures of tolerance for any of the three viruses, neither using the whole set of plant genotypes , nor for each allometric group .To analyse Arabidopsis resistance-tolerance trade-offs to each virus, bivariate relationships between virus accumulation and the slope of the r = −0.62; P = 0.007). No association was found between fecundity tolerance to UK1-TuMV and to JPN1-TuMV , but this was just due to a single outlier value . Finally, no association was detected between fecundity tolerance to JPN1-TuMV and LS-CMV , but again removal of a single outlier resulted in a positive association  . On the other hand, the virus genotype per allometric group interaction was not significant when comparing JPN1-TuMV and LS-CMV      . In both= 0.275) . Given t × 10−4) . The com × 10−4) . Convers × 10−4) . Altogetr = −0.51; P = 0.044), and between tolerance to UK1-TuMV and to JPN1-TuMV when excluding a single outlier . No significant association was observed between mortality tolerance to JPN1-TuMV and to LS-CMV  , whereas no such interaction was detected between JPN1-TuMV and LS-CMV    . Again, = 0.261) . When Gr × 10−4) . These rr ≤ 0.64; P ≥ 0.119). Exception were UK1-TuMV-infected plants when analysed as a whole, in which both tolerances showed a positive association . Thus, when associated, higher mortality tolerance increases fecundity tolerance to a given virus.For each virus, we also analysed potential mortality-fecundity tolerance trade-offs. No significant bivariate associations were found when considering all plant genotypes together, or each allometric group separately in any of the three viruses  interactions. However, the mechanisms by which tolerance is achieved and the forces shaping its evolution are still poorly understood . Using AWe showed that Arabidopsis displays genotype-specific fecundity tolerance to the highly virulent virus UK1-TuMV, with plants of the allometric Group 2 having higher tolerance than Group 1 ones. In Arabidopsis, UK1-TuMV infection often prevents seed production . TheoretIt should be noted that upon UK1-TuMV infection, infected plants of tolerant Arabidopsis genotypes produced on average 30 per cent of the seeds produced by mock individuals. It could be argued that this level of fecundity tolerance is not effective, i.e. seed production of infected plants is far from that of uninfected ones . HoweverFecundity tolerance to UK1-TuMV was associated with genotype-specific modifications of the plant developmental schedule. Particularly, upon UK1-TuMV infection more fecundity-tolerant Group 2 genotypes showed shorter growth, and longer reproductive, periods than mock-inoculated plants. This observation agrees with the prediction of the life-history theory that bringing forward the age at maturity allows infected hosts to reproduce before they experience the full cost of infection, thus compensating (at least partly) the effect on host fitness . These rArabidopsis fecundity tolerance to LS-CMV was higher in Group 1 than in Group 2 genotypes, which was associated with resource reallocation from growth to reproduction, an extensively studied response . NotablyWe failed in finding a negative association between plant resistance and tolerance to the same virus across Arabidopsis genotypes, which indicates the absence of trade-offs between these two defence mechanisms in agreement with previous analyses e.g. . On the A number of experimental works reported that pathogen-driven changes in host life-history traits can be either genetically determined or the consequence of phenotypic plasticity . Thus, iTolerance–tolerance trade-offs may have important implications for understanding the evolution of host defences. To date, most mathematical models on this subject are built on the assumption that tolerance evolves in single host–pathogen interactions . These mveaa019_Supplementary_DataClick here for additional data file.


Unlike HIV-1 Pol (expressed as a Gag–Pol fusion protein), foamy virus (described as an ancient retrovirus) expresses Pol without forming Gag–Pol polyproteins. We placed a “self-cleaving” 2A peptide between HIV-1 Gag and Pol. This construct, designated G2AP, is capable of producing virions with the same density as a wild-type (wt) HIV-1 particle. The 2A peptide allows for Pol to be packaged into virions independently from Gag following co-translationally cleaved from Gag. We found that G2AP exhibited only one-third the virus infectivity of the wt, likely due, at least in part, to defects in Pol packaging. Attenuated protease (PR) activity, or a reduction in Pol expression due to the placement of 2A-mediated Pol in a normal Gag–Pol frameshift context, resulted in significant increases in virus yields and/or titers. This suggests that reduced G2AP virus yields were largely due to increased PR activity associated with overexpressed Pol. Our data suggest that HIV-1 adopts a gag/pol ribosomal frameshifting mechanism to support virus assembly via the efficient modulation of Gag–Pol/Gag expression, as well as to promote viral enzyme packaging. Our results help clarify the molecular basis of HIV-1 gene expression and assembly.The  For ortlication . Within During virus assembly, Gag–Pol recruitment for assembling viral particles facilitates Gag–Pol dimerization, which is believed to trigger embedded PR domain activation . Once acGag–Pol/Gag maintenance at a low expression ratio is critical to virus production, since the artificial overexpression of Gag–Pol or Pol containing an active PR markedly reduces virus yields. Reduced yields are likely due to premature or enhanced Gag cleavage via overexpressed PR activity ,10,11,12It is likely that the regulation of FV PR-mediated virus processing differs from that found in HIV. Alternatively , HIV-1 PR may possess stronger enzymatic activity than its FV counterpart in terms of mediating virus particle processing. One possibility is that an HIV-1 ribosomal frameshift system has evolved to express Pol as a Gag–Pol fusion protein with selectively stronger enzyme activity, despite a marked reduction in expression level. Accordingly, HIV-1 Gag–Pol, even when expressed at a relatively low level, may be capable of efficient packaging, thus providing sufficient enzyme activity for virus replication. The conclusion that HIV-1 Pol can be incorporated into Gag VLPs is largely based on a co-expression system involving the co-transfection of PR-defective Pol and Gag expression vectors ,21. SincGGAAGCGGAGCTACTAACTTCAGCCTGCTTAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTTTTAGGGAAGATCTGGCC-3′ as the forward primer and 5′-GGTACAGTCTCAATAGGGCTAATG-3′ as the reverse primer (HIV-1 nt.2575-52). The forward primer contains a flanking BamHI site, a 2A-like peptide (underlined), and partial N-terminal p6* coding sequences. HIVgpt served as a template. The 2A peptide coding sequence is from porcine teschovirus-1 (Picornaviridae virus family), described as having the highest cleaving efficiency among picornavirus 2A species tested in cell cultures [BamHI and BclI prior to cloning into GP [G2AP was constructed by placing a 2A peptide coding sequence between the Gag C-terminus and Pol N-terminus , using 5cultures . Amplifi into GP . GP  sucrose cushion as described above. Viral pellets were suspended in TSE buffer  and overlaid on a 20–60% (w/v) sucrose gradient consisting of 1 mL layers of 20, 30, 40, 50 and 60% (w/v) sucrose in TSE buffer that had sat for 2 h. Gradients were centrifuged in an SW50.1 rotor at 40,000 rpm  for 16 h at 4 °C; 500 μL fractions were collected from top to bottom. Sucrose density was measured for each fraction. Fraction proteins were precipitated with 10% trichloroacetic acid (TCA) and subjected to Western immunoblotting.Transfected HEK 293T cell supernatant was collected, filtered, and centrifuged through a 2 mL 20% (w/v) sucrose at 274,000× g. Cells were rinsed with ice-cold phosphate-buffered saline (PBS), collected in 1 mL PBS, and pelleted. Viral and cell pellets were suspended in IPB buffer  containing 0.1 mM phenylmethylsulfonylfluoride (PMSF). Supernatant and cell samples were mixed with equal volumes of 2× sample buffer  containing β-mercaptoethanol (5%) and boiled for 5 min. Samples were subjected to 10% SDS-PAGE and electroblotted on nitrocellulose membranes. Membrane-bound Gag and RT detection protocols were performed as previously described [Cells and supernatants from transfected HEK 293T cells were prepared for Western immunoblotting as described previously . Brieflyescribed . Membranescribed . SecondaTo directly observe the impacts of PR-active Pol on virus assembly, processing, and infectivity, a construct designated G2AP was engineered by placing a “self-cleaving” 2A peptide coding sequence between the Gag C-terminus and Pol N-terminus A. A cotrIf 2A is capable of efficiently mediating cleavage involving Gag and Pol, Pol might represent a major virus-associated RT precursor when PR activity is blocked. To test this possibility, we added an HIV-1 protease inhibitor to culture medium following transfection. As shown in Since collected medium was filtered and centrifuged through 20% sucrose cushions, we posited that Gag and RT in culture medium was virus-associated. To confirm the presence of released G2AP Gag as virus particles, pellets derived from G2AP transfectant supernatant were centrifuged with wt viral pellets through the same sucrose density gradient. As shown in A 2A-deficient counterpart designated GP was constructed to confirm that the 2A cleavage function gives G2AP the ability to express Pol independently of the gag reading frame. In addition, 2A mutants defective in cleavage activity  were cloThe G2AP production of virus-associated Gag and RT at levels much lower than those of the wt was in part due to enhanced Gag cleavage by overexpressed Pol containing active PR. Accordingly, virus yields can increase if the Pol expression level decreases to a normal Gag–Pol/Gag ratio. To confirm this assumption, we used an HIV-1 virus-producing vector (designated Dp6*PR) previously described as capable of virus particle assembly and processing in a manner similar to that of a wt . Dp6*PR A band migrating slower than Pr55gag might consist of Gag(MA-CA-NC)-p6*-PR-2A  PR-2A and Pol. We previously reported that MA-CA-NC-p6*-PR is capable of incorporation into viral particles as well as Gag processing mediation, although the latter is less efficient compared to mediation by PR derived from Gag–Pol . MA-CA-NDeficient RT is one possible explanation for the reduced G2AP virus infectivity we observed . In addiFoamy virus (FV) Pol, expressed as a Gag–Pol fusion protein in the absence of spliced Pol, possesses enzymatic activity and the capability to assemble and release as VLPs . HoweverEven though independent HIV-1 Pol expression from the same gag-coding RNA template can generate infectious virions, it is accompanied by a marked reduction in virus titers. Attenuation of PR activity or reduced Pol expression can significantly increase G2AP virus titers and/or virus production. In this regard, HIV-1 may have evolved a Gag–Pol ribosomal frameshifting mechanism that supports efficient Gag and Pol regulation involving the same RNA template, thereby preventing Pol overexpression by expressing Pol as a Gag–Pol fusion protein at about 5–10% the level for Gag. Gag–Pol fusion protein expression rather than Pol expression also ensures the efficient incorporation of viral enzymes into virus particles. We noted a significant reduction in Pol viral incorporation compared to Gag–Pol, supporting the idea that the N-terminal Gag domain is required for efficient Gag–Pol packaging.In conclusion, our results suggest that HIV-1 exploits the Gag–Pol ribosomal frameshift mechanism in support of Pol expression as a Gag–Pol fusion protein at relatively low levels, resulting in the promotion of Gag assembly and Pol incorporation into virus particles. HIV-1 virus assembly and replication apparently benefit from this Gag–Pol ribosomal frameshift mechanism.


Droplet‐based single cell sequencing technologies, such as inDrop, Drop‐seq, and 10X Genomics, are catalyzing a revolution in the understanding of biology. Barcoding beads are key components for these technologies. What is limiting today are barcoding beads that are easy to fabricate, can efficiently deliver primers into drops, and thus achieve high detection efficiency. Here, this work reports an approach to fabricate dissolvable polyacrylamide beads, by crosslinking acrylamide with disulfide bridges that can be cleaved with dithiothreitol. The beads can be rapidly dissolved in drops and release DNA barcode primers. The dissolvable beads are easy to synthesize, and the primer cost for the beads is significantly lower than that for the previous barcoding beads. Furthermore, the dissolvable beads can be loaded into drops with >95% loading efficiency of a single bead per drop and the dissolution of beads does not influence reverse transcription or the polymerase chain reaction (PCR) in drops. Based on this approach, the dissolvable beads are used for single cell RNA and protein analysis. This work reports an approach to fabricate dissolvable polyacrylamide beads, by crosslinking acrylamide with disulfide bridges that can be cleaved with dithiothreitol. The beads can be rapidly dissolved in drops and release DNA barcode primers for droplet DNA barcoding, which demonstrate great potential for high‐throughput single cell sequencing. Droplet microfluidics have proven critical for next‐generation single‐cell analysis.[qv: 1] Coupled with high‐throughput sequencing, droplet‐based single cell sequencing technologies have been developed to analyze RNA, DNA, and even proteins in single cells. To track single cells, barcodes need to be introduced, which is mainly achieved by adding synthetic DNA barcode primers to beads. For these applications, the beads are mostly made of biocompatible polymers, for example, polyacrylamide beads in inDrop,[qv: 2] hydroxylated methacrylic polymer beads used in Drop‐seq,[qv: 3] and polyacrylamide beads used in 10X Genomics.[qv: 8]Each of these chemically different beads has their own advantages and shortcomings. The inDrop polyacrylamide bead can be closely packed in a microfluidic device channel to achieve more than 95% loading of single bead per drop.[qv: 2] However, in the inDrop system, UV light is necessary to release primers from the bead, which somewhat complicates bead fabrication and makes it less cost effective.[qv: 9] Another shortcoming is that UV light may introduce damage to DNA or RNA and skew the results. The Drop‐seq system does not release primers from the bead and the reaction efficiency is low as reactions happen only near the surface of beads.[qv: 3] In the 10X Genomics platform, gel beads can be efficiently delivered into drops, but the comparably high cost and lack of flexibility in designing own primers on the beads are limitations for certain experiments.[qv: 8] What is thus missing today, are barcode beads that are easy to fabricate, can efficiently deliver primers into drops, and thus achieve high detection efficiency.4H10O2S2). As illustrated in FigureN,N′‐Bis(acryloyl)cystamine , a reversible crosslinker,[qv: 14] reacts with acrylamide monomers  to form a porous polyacrylamide hydrogel inside the water drops. The polyacrylamide hydrogel beads serve to i) carry primers by conjugating primers to the hydrogel, ii) deliver primers to individual drops, and iii) release primers by hydrogel dissolution. We used relatively low BAC concentration to make the hydrogel beads easy to dissolve while the beads could still maintain the morphology for carrying and delivering primers. The polyacrylamide bead can be rapidly dissolved in DTT by a thiol–disulfide exchange reaction.[qv: 14] DTT is a common redox reagent that is used to break down protein disulfide bonds and stabilize enzymes, and its addition to dissolve the beads does not influence reverse transcription or PCR. Acrydite‐modified DNA primers can be directly conjugated to the beads during polymerization. The conjugation yield of acrydite‐modified DNA primers to these polyacrylamide hydrogels is more than 50%.[qv: 2] They can be suspended into a homogeneous solution when beads are dissolved. The dissolution products of the beads are polymer fragments and ‐1,2‐dithiane‐4,5‐diol, as shown in Figure −3m DTT, and the primers on the beads can be rapidly suspended into solution. At the same time, we achieve >95% loading efficiency of a single bead per drop. We apply these barcoding beads to single cell RNA and protein analysis. As illustrated in Figure Here, we report a new approach to fabricate reversible polyacrylamide beads, which can be rapidly dissolved in drops and release DNA barcode primers in the presence of dithiothreitol , (C—S), (N—H), (C=O), and (CH2) bond stretching motions, respectively. The peaks at 1456 and 1614 cm−1 are correlated with (CH2) and (NH2) bond deformations, respectively.[qv: 17] These results indicate the successful synthesis of polyacrylamide gel with disulfide bonds.Homogeneous drops that contain acrylamide monomer and BAC are generated using a droplet generation device . The size of the drop can be precisely controlled by the channel size of the device.[qv: 15] We choose conditions to create 70 µm drops to fabricate beads that have an ideal size for barcode addition and that can be encapsulated into drops. Polyacrylamide beads are formed by curing the droplets at 70 °C for overnight . These beads can shrink or expand slightly in buffers with different salt concentrations. Increased ionic strength of the buffer usually causes the polyacrylamide beads to shrink. This is because at low ionic strength, the concentration of bound charges within the gel exceeds the concentration of salt in the external solution, and a large ion‐swelling pressure causes the gel to expand. As the ionic strength increases, the difference between the internal and external ion concentrations decreases and the gel shrinks.[qv: 16] Fourier Transform Raman Spectroscopy was performed to characterize the physicochemical property of the dissolvable polyacrylamide beads, as shown in Figure S3 (Supporting Information). The peaks at 484, 638, 1325, 1664, and 2926 cmFigure−3m DTT, in 3 min with 1 × 10−3m DTT, and in 30 s with 10 × 10−3m DTT. Figure −3m DTT . From 0 to 60 s, partial disulfide bonds are cleaved and the beads expand. From 60 to 120 s, more disulfide bonds are cleaved while breaking beads into fragments. At 180 s, beads can no longer be observed under the microscope.We tested the dissolution time of the beads at different concentrations of DTT Figure2A. The4 Pa s at 37 °C, which is only slightly higher (<3%) than that of the solution without beads, (8.03 ± 0.69) × 104 Pa s. As a control, the solution with intact beads has similar viscosity to the solution without beads . These measurements demonstrate that the dissolved beads have negligible influence on the physical property of the reaction solution.To study whether the dissolution of beads affects the viscosity of reaction solution, we measured the viscosity of solution with dissolved beads and without beads by using tracer particles. The viscosity of the solution with dissolved beads is (8.24 ± 0.72) × 10To investigate its feasibility of primer release, we next conjugated acrydite‐modified DNA primers with 3′ polyT sequence  into the hydrogel beads. A 5′ fluorescence‐labeled polyA primer , complementary to polyT section of AP, was used to light the polyacrylamide hydrogel beads and track the primers on the beads. The primers on the beads are gradually dispersed into the solution along with the bead dissolution within 180 s Figure . This ob−1 for 1 µmol production scale from IDT , while the acrydite photocleavable primers used for the inDrop beads cost 37 $ nmol−1 at the same production scale.[qv: 9] The 12 times lower barcode cost will likely translate into cheaper analysis or the ability to sequence more cells for the same price. More importantly, all bead operation processes can be performed under normal laboratory setting without the need of dark room operation, and the dissolution process is fully compatible with the molecular biology reactions in drops. These properties make dissolvable beads highly appealing for high‐throughput droplet DNA barcoding.Additionally, the acrydite primers used for the dissolvable beads only cost 3 $ nmolFigure9 primers, which is sufficient for capturing the transcriptome of an individual cell. After co‐encapsulation of single cells and the polyT primer conjugated dissolvable beads into drops, the polyT primers are released and bind with mRNA from the cell and start reverse transcription to make cDNA in drop. The cDNAs from the drops with cells could be amplified with targeted primers for GAPDH and TCRA, while the sample without cells did not show any amplification  messenger RNA (mRNA), T‐cell receptor alpha locus (TCRA) mRNA, and total mRNA are measured from single human Jurkat T cells in drops by using the dissolvable beads that carry polyT primers for reverse transcription. PCR with GAPDH primer, TCRA primer, or total mRNA primer is performed to measure the complementary DNA (cDNA) product from the drop. The ability of loading one bead per drop is crucial for the recovery of single cell information. Almost all of the drops (>95%) contained single beads as we were able to closely pack the dissolvable beads in the channel of the microfluidic device to achieve the best possible loading Figure3A. and has been enabled by recent barcoding approaches.[qv: 25] New emerging methods, such as REAP‐seq,[qv: 7] have also been developed to quantify proteins from single cells by combining sequencing with droplet‐based microfluidic devices. Here we test the ability to use the dissolvable beads for single cell protein analysis. Based on earlier work,[qv: 25] we used antibody–DNA conjugates to investigate protein expression Figure . In thisAs a proof of principle, we measured CD3 protein expression in human Jurkat T cells. Anti‐CD3 antibody was conjugated to DNA using trans‐cyclooctene (TCO)‐tetrazine (Tz) click chemistry, and the conjugation was validated using PAGE gel electrophoresis . The antibody–DNA‐labeled cells were then encapsulated into drops containing the dissolvable bead. It is noteworthy that addition of DTT occurs after antibody hybridization, i.e., the DTT does not affect the DNA–antibody barcode prior to cellular binding. The amplicon is designed using the primer on the bead and the DNA conjugated to the antibody. Quantitative polymerase chain reaction (qPCR) amplification curves showed a successful amplification of the product in the presence of antibody–DNA‐labeled cells and no background without Jurkat cells Figure . Agarose−3m DTT, and the primers on the beads can be rapidly and evenly dispersed into the solution when the beads are dissolved. Coupling with droplet‐based microfluidic devices, we successfully demonstrate the ability of using the dissolvable beads as a high‐throughput barcoding cargo for single cell RNA and protein analysis.In this work, we report a new method to fabricate polyacrylamide beads that can be rapidly dissolved in the presence of DTT for high‐throughput droplet‐based DNA barcoding. Imaging experiments show that 70–90 µm beads can be completely dissolved in 3 min with 1 × 10The dissolvable beads deliver primers to each drop with high loading and reaction efficiency for both reverse transcription reaction and polymerase reaction in drops. The dissolvable bead technology overcomes multiple limitations of currently used barcode beads for single cell analysis by achieving both high loading and reaction efficiency in drops. Furthermore, the dissolvable beads can be easily synthesized at low cost. The beads can be dissolved in <3 min at high efficiency, a reaction that primarily depends on the DTT concentration. DTT is fully compatible with almost all molecular biology reactions in drops, which makes the technology easy to be integrated to existing molecular biology assays. These properties will make dissolvable polyacrylamide beads a very attractive tool for both high‐throughput single cell analysis and single molecular analysis. This technology will open up opportunities to better understand and discover heterogeneous and rare molecular signatures that can be a key to diagnostics for various diseases.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file.


Capsid integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses combining azo dye pretreatment with qPCR, has been widely used in recent years; however, variations in pretreatment conditions for various virus types can limit the efficacy of specific protocols. By identifying and critically synthesizing forty-one recent peer-reviewed studies employing capsid integrity qPCR for viruses in the last decade (2009–2019) in the fields of food safety and environmental virology, we aimed to establish recommendations for the detection of infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of attachment or genomic damage, are less well detected using this approach. Although optimizing specific protocols for each virus is recommended, we identify a framework for general assay conditions. These include concentrations of ethidium monoazide, propidium monoazide or its derivates between 10 and 200 μM; incubation on ice or at room temperature (20 - 25 °C) for 5–120 min; and dye activation using LED or high light (500–800 Watts) exposure for periods ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus transmission in routine (water) monitoring settings and during viral outbreaks such as the current COVID-19 pandemic or endemic diseases like dengue fever.  •PMA/PMAxx have higher efficiency removing false negatives from qPCR for DNA/RNA viruses than EMA.•One size fits all pretreatment approaches are possible but lead to lower virus signal reduction.•Capsid integrity qPCR is a valuable tool to adapt existent workflows for improving risk assessment.•Azo dye pretreatment can help refine significance of qPCR during virus outbreaks. Exposure to waterborne and foodborne pathogens through recreational activities, irrigation water, and food consumption, as well as associated occupations, poses a risk to public health in high and low resource environments . To dateWhile still the gold standard, culture-based methods for propagation of infectious human pathogenic viruses in a laboratory environment require specialized facilities, experienced personnel and appropriate cell lines for virus propagation, and test results may only become available after five to ten days . MoleculOne of the most established qPCR modifications to measure infectivity is capsid integrity qPCR, an approach where samples are pre-treated with the intercalating azo dyes propidium monoazide (PMA), ethidium monoazide (EMA) or their derivates PMAxx and PEMAX. First described almost two decades ago by 2Pubmed, Scopus, Ovid, Medline and Web of Knowledge to cover relevant articles in English since the first introduction of the azo dye pretreatment in 2003 , two of3Escherichia coli as their host . As the laboratory safety requirements, as well as workload for culture-based detection, of those phages are significantly lower than for enteric viruses, the necessity to establish alternative methods to determine phage infectivity is not as urgent as for enteric viruses  for significant capsid damage; lower temperatures (85 °C) and proteolysis were not effective . These azo dyes became available for research purposes in the early 2000s, but a number of studies reported that the more charged EMA tended to enter bacterial cells with intact membranes, thus potentially resulting in false-negative qPCR results and cytotoxic effects . SeventyAzo dye concentrations are quite evenly distributed, ranging from less than 10 μM to greater than 200 μM. More than half of the articles under investigation reported either 50 μM or 100 μM , which i6Approximately two thirds of the studies included in this review applied an incubation time with the azo dye of 5 or 10 min . A quart7Early publications utilizing azo dyes for the removal of non-infectious virus particles exclusively applied 500- to 800-Watt halogen light sources used in stage lighting . Besides84) and Cis-dichlorodiammineplatinum (CPPD) are long known to chelate in mammalian cells by nucleic acid ligands (E. coli and Cronobacter skazakii (Addition of non-ionic surfactants like Triton X-100  and sodi ligands , and havskazakii .9Various methods to inactivate viruses were used in the studies included in this review. The intention was either to evaluate disinfection efficiency as it is currently used in food safety and water treatment  or to ac10The utilization of azo dye pretreatment can be recommended for applications where culture assays take too long to inform necessary remedial actions, and under low resource conditions either in developing countries or in laboratories with only basic analytical capabilities and biosafety levels. The recent outbreak of SARS-CoV-2 associated COVID-19 represents an example where knowing the ratio of infectious to non-infectious virions would help in determining whether symptomatic or asymptomatic carriers need to be isolated  and coulTo determine the validity of azo dye pretreatment of environmental samples, a robust set of controls for environmental and foodstuff related samples should be included. Such controls could involve viral targets like pepper mild mottle virus  and crAs11•PMA and the derivate PMAxx show a higher efficiency in removing false positive signals from qPCR for both DNA and RNA viruses than EMA and PEMAX.•Incubation duration and temperature, reagent concentration as well as light source and exposure time need to be optimized and validated for the virus under investigation.•“One-size-fits-all” pretreatment approaches are possible but might lead to lower signal reduction rates of individual viruses.•Capsid integrity qPCR can be a valuable tool to adapt existent workflows and qPCR protocols to reflect the ability of viruses to infect humans, thus improving risk assessment and consumer safety derived from these measurements.•Capsid integrity is a strong indicator of virus infectivity, which allows for the establishment of robust assays to assess the infectivity of novel viruses in the event of outbreaks like the 2014 Ebola virus epidemic and the current COVID-19 pandemic.An evaluation of methods on the application of azo dye pretreatment to determine virus infectivity by qPCR revealed a great diversity of viruses that have been tested under a range of treatment conditions. The systematic literature comparison led to the following conclusions:M.L. and K. S. conceived and planned the literature review. M. L., E. S. and D. C. S. performed the title and abstract as well as full-text screening. M. L. took lead in writing the manuscript, C. D. and M. L. conceptualized and generated figures. S. W., S. M. and K. S. provided valuable feedback and helped shape the discussion, analysis and narrative. All authors contributed to editing and proofreading of the manuscript.10.13039/501100001381Singapore National Research Foundation and 10.13039/100009950Ministry of Education under the Research Centre of Excellence Programme.This research was supported by the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Herpez zoster ophthalmicus is a severe variant of shingles (herpes zoster), which occurs when the immune system is weakened and the virus responsible for chickenpox reactivates.Herpes zoster (shingles) is an infection caused by re-activation of the varicella zoster virus, which causes chickenpox. Primary infection usually happens in childhood. After the primary infection, the patient is immune to the virus, which then lies inactive in the dorsal root ganglia or cranial ganglia. Eventually, as the patient's natural immunity reduces as a result of ageing or disease, the virus can reactivate and spread to the skin, via the afferent nerves, to cause herpes zoster. By the time they are 40 years of age, the vast majority (99.6%) of individuals worldwide have antibodies to varicella zoster virus and are therefore at risk of herpes zoster.About one-third of individuals will get herpes zoster in their lifetime. Herpes zoster can appear at any age, although there is a strong tendency for it to develop after 60 years of age due to natural weakening of the immune system. There has been a worldwide increase in the prevalence of herpes zoster. The reason for this is not known; however, with an ageing population, the incidence is likely to increase.“About one-third of individuals will get herpes zoster in their lifetime.”The majority of individuals who get herpes zoster are otherwise healthy. However, it is more common in immunocompromised individuals  or with some diseases . In some regions, herpes zoster may signal underlying immunosuppression from HIV/AIDS, especially in young adults; these patients are also at risk of severe complications and systemic involvement. Recurrent attacks of herpes zoster are uncommon, but they are a feature of HIV/AIDS.Herpes zoster most frequently involves the chest, abdomen, face or genitals. The distribution corresponds to the distribution of the nerve (dermatome) that has been affected. The disease does not cross the midline, but two adjacent dermatomes on the same side can be involved. Herpes zoster ophthalmicus (HZO) is a particularly severe variant of herpes zoster and accounts for about 20% of all cases. The eye is not always involved, but there is a particularly high risk (50%) of ocular involvement if the first division of the fifth cranial  nerve is affected, with vesicles extending to the tip of the nose, known as Hutchinson's sign .1,2The clinical signs of HZO are usually preceded by tingling over the affected area, and patients generally feel unwell and have a headache. After 2–3 days, a painful rash develops, consisting of blisters that ooze fluid for 3–5 days. The blisters then dry out and form scabs that heal after four weeks. The skin remains painful until the rash has gone.Post-herpetic neuralgia sometimes develops. It involves pain, itching, and burning over the area of the initial rash that is still present after three months. This develops in about 13% of all cases, and half of individuals over 70 years will still have pain from it after a year. The incidence of post-herpetic neuralgia increases with age of onset of HZO, with eye involvement, and with the severity of the initial attack. It can be debilitating, preventing sleep and daily activities, and severely affect quality of life.HZO can affect all parts of the eye, with an onset of disease 2–4 weeks after the first appearance of the rash. Anterior segment complications can be severe. They include dendritic keratitis, secondary corneal anaesthesia, persistent epithelial defect, secondary infection, stromal neovascularisation, and corneal opacity .Patients with HIV/AIDS, in particular, can develop disfiguring scarring and pigmentation of the face and lids. There may also be chronic granulomatous uveitis, iris atrophy, scleritis, and acute retinal necrosis. Vasculitis is less common but can cause ischaemic optic neuropathy, extraocular muscle palsies and a contralateral stroke.Varicella zoster virus is the most common cause of acute retinal necrosis. However, acute retinal necrosis is not restricted to patients who have had HZO, which suggests that blood, rather than nerves, are responsible for the spread of the virus to the eye.The appearance of HZO is very characteristic, and this gives the diagnosis in almost all cases. However, tingling or pain exacerbated by light touch may develop several days before the rash or, rarely, a rash may never develop (zoster sine herpete). In these patients, identifying the cause of the neuralgia can be difficult. Herpes virus DNA can sometimes be detected in the tear film by polymerase chain reaction (PCR), although this is an expensive test and not widely available. As the majority of adults have had chickenpox, they will have antibodies against varicella zoster virus and serology is rarely helpful. It is not necessary to perform extensive investigation for underlying diseases, unless HIV/AIDS is suspected.Herpes simplex infection of the lid and cornea can mimic HZO, especially if there is bacterial superinfection of severe atopic dermatitis (eczema herpeticum). Contact dermatitis from plants, or a reaction to locally applied medications, can mimic HZO. Consider herpes simplex if patients have had multiple recurrences of ‘shingles’. A bilateral rash is unlikely to be HZO.Medical staff should wear gloves if touching skin with blisters; however, if they have had chickenpox in the past, they are not at risk of infection. Wash the affected skin with soap and water and pat dry, cover with a loose dressing, and use a dry cold compress if available. Don't use dressings that can stick to the skin. Antibiotics are not necessary unless secondary skin infection is suspected, and don't use topical antivirals on the skin.There is no agreed consensus for the medical management of HZO. If a patient is seen within 72 hours of the onset of the blisters, they should be given a course of oral aciclovir tablets: 800 mg five times daily for seven days . Provide this treatment at the start of any new ocular involvement. Intravenous aciclovir should be considered for individuals with HIV/AIDS to reduce the risks of disseminating varicella zoster infection. Antiviral treatment reduces the risk of chronic ocular complications by 20% to 30%. It does not prevent post-herpectic neuralgia but reduces the duration of the pain by about 50%.Dendritic keratitis and uveitis may indicate chronic HZV activity. Keratitis should be treated with topical aciclovir 3% ointment or ganciclovir 0.15%, five times daily for at least 5 days, reducing to twice daily until the dendrites have resolved. Add a topical steroid if there is stromal disease or uveitis. Slow tapering of topical steroid may be required, and some individuals need long-term drops once a day to control inflammation. Long-term prophylactic antiviral treatment  is often used to treat these complications and prevent reactivation of the varicella zoster virus in the eye, but this is not yet evidence based.Patients can develop complete corneal anaesthesia after HZO, known as neurotrophic keratitis. This can lead to sight loss, as the anaesthetic cornea is at risk of developing an epithelial defect, secondary infection, vascularisation and scarring. Frequent lubricating drops are helpful to prevent epithelial breakdown. A chronic epithelial defect may heal with a temporary tarsorrhaphy, bandage contact lens or amniotic membrane graft. Unfortunately, a proportion of patients eventually require a permanent tarsorrhaphy or conjunctival flap to protect the eye. Severe post-herpetic neuralgia may be helped by giving oral analgesics , anticonvulsants  or tricyclic anti-depressants. Acute retinal necrosis requires specialist management and patients should be referred to a tertiary centre if possible. Treatment includes intravenous aciclovir 10–15 mg/kg for 2 weeks, followed by oral valaciclovir 1 g three times daily for a further 6 weeks.Corneal anaesthesia is a common feature of HZO, and progressive corneal disease may pass unnoticed. Review patients periodically or ask them to monitor their vision themselves and seek help if it deteriorates. Patients receiving topical steroid treatment should be monitored for complications.A vaccine against chickenpox is available in many countries. This uses a live, attenuated virus, and is also used at a higher dose to prevent (not treat) herpes zoster. Because it is a live virus, do not give this vaccine to people who have impaired immunity . A recombinant virus vaccine is also available to treat herpes zoster and, as it does not contain a live virus, it can be used in immunocompromised patients. Both vaccines boost cell-mediated immunity against varicella zoster virus, which then reduces the risk of reactivation. Regional guidelines vary, but vaccination against herpes zoster is usually only recommended for individuals over 60 years of age. Both vaccines are safe to use in patients who have had chickenpox and individuals do not need to be tested for immunity against the virus before they are vaccinated. A large randomised-controlled clinical trial found that, after three years, the live attenuated vaccine had reduced the incidence of herpes zoster amongst adults over 60 years of age by 51% and the burden of illness by 66.5%.Unfortunately, the effect of vaccination only lasts for about eight years, and the vaccine is less effective in patients older than 70 years. The recombinant vaccine is more effective than the live attenuated vaccine, and it reduces the incidence of herpes zoster and post-herpetic neuralgia by 90%; it is now the preferred option in some countries.Cost is a consideration in the widespread introduction of vaccination against herpes zoster. Prophylactic low-dose oral aciclovir (400 mg twice daily) is a less effective alternative to vaccination for patients at high risk of herpes zoster.A patient with HZO can spread chickenpox in non-immune individuals, but HZO cannot be transmitted directly to cause HZO in another person. Transmission of the virus typically requires direct contact with oozing skin. The risk of transmission stops when the rash crusts and stops leaking. Patients who are immunocompromised  are more contagious because they shed more virus particles. A person who has HZO blisters should avoid direct contact with babies less than 1 month of age, pregnant women who have not had chickenpox, and individuals with a suppressed immune system.


Background: The number of people living with HIV (PLHIV) in need of treatment monitoring in low-and-middle-income countries is rapidly expanding, straining existing laboratory capacity. Point-of-care viral load (POC VL) testing can alleviate the burden on centralized laboratories and enable faster delivery of results, improving clinical outcomes. However, implementation costs are uncertain and will depend on clinic testing volume. We sought to estimate the costs of decentralized POC VL testing compared to centralized laboratory testing for adults and children receiving HIV care in Kenya. Methods: We conducted microcosting to estimate the per-patient costs of POC VL testing compared to known costs of centralized laboratory testing. We completed time-and-motion observations and stakeholder interviews to assess personnel structures, staff time, equipment costs, and laboratory processes associated with POC VL administration. Capital costs were estimated using a 5 year lifespan and a 3% annual discount rate. Results: We estimated that POC VL testing cost USD $24.25 per test, assuming a clinic is conducting 100 VL tests per month. Test cartridge and laboratory equipment costs accounted for most of the cost . Costs varied by number of VL tests conducted at the clinic, ranging from $54.93 to $18.12 per test assuming 20 to 500 VL tests per month, respectively. A VL test processed at a centralized laboratory was estimated to cost USD $25.65. Conclusion: POC VL testing for HIV treatment monitoring can be feasibly implemented in clinics within Kenya and costs declined with higher testing volumes. Our cost estimates are useful to policymakers in planning resource allocation and can inform cost-effectiveness analyses evaluating POC VL testing. In 2019, an estimated 38 million people were living with HIV (PLHIV) worldwide, with the vast majority residing in resource-limited settings . In addiPoint-of-care (POC) VL testing is a promising strategy recommended by the WHO that can increase VL testing coverage and provide rapid delivery of results ,7. POC cKenya is one of the countries hardest hit by the HIV epidemic, with an adult HIV prevalence of 4.9% . There aClinicalTrials.gov Identifier: NCT03820323) and the parallel cohort study (Opt4Mamas), which are evaluating the impact of POC VL testing for children aged 1–14 years and pregnant women, respectively. The Opt4Kids study enrolled participants from March to December 2019, and the Opt4Mamas study from February 2019 to June 2020. Study participants are receiving HIV care at five Ministry of Health facilities in western Kenya. Study procedures for Opt4Kids have been previously described and are similar in Opt4Mamas [The present costing study was conducted alongside the ongoing randomized controlled trial . It has been validated and found feasible and reliable in rural African communities [® equipment, herein referred to as “POC equipment” can be purchased to accommodate different numbers of simultaneous tests (ranging from 4 to 80 samples processed at the same time), greatly impacting the cost of the machine. For this costing analysis, we assumed POC equipment would run 4 samples simultaneously. The 4-module machine was used by three of the four on-site laboratories participating in this study and is the most commonly utilized GeneXpert® equipment in Kenyan clinics. Further, the 4-module machine was identified as the most cost-effective size for the average clinic testing volume based on calculations by the Kenyan Ministry of Health National Laboratory at the time that national roll-out began .POC VL testing in the studies is conducted via the Xpertmunities . This asmunities . DiffereWe conducted detailed microcosting from the provider perspective to compare the per-test cost of administering an HIV POC VL test to a centralized VL test from the payer perspective ). Costs (2020 USD) were collected from expense reports, staff and expert interviews, literature, and were divided into personnel, clinical supplies, equipment, office supplies, and laboratory space. All blood samples for POC VL and centralized laboratory tests were obtained through venous blood draws. We assumed that the following would remain the same across POC and centralized VL testing: personnel time to draw blood from client, supplies needed to collect blood samples, and office supplies. We assumed clinics would purchase a 4-module processing GeneXpert IV machine, which would be housed in the clinic laboratory. We assumed a 5-year instrument life and a 3% annual discount rate for POC equipment costs. Costs were collected in Kenyan Shillings and converted to USD using the exchange rate of 107.77 Kenyan Shillings to USD $1 reported at the midpoint of the data collection time period (August 2020). We did not include societal costs such as those incurred by patients to attend a clinic visit, including transport and opportunity costs of missed work.We developed an initial list of main activities by reviewing the parent study protocols  and discWe assumed that POC test material and supplies reflect preferential pricing provided by manufactures for low and middle-income countries (LMICs). As the equipment component of the cost per test depends on the number of tests conducted, we calculated POC VL test cost per patient at varying VL testing volumes. The lower bound of the testing volume range was estimated via communications with clinic staff, and the upper bound was estimated as the maximum capacity of POC VL tests that the machine could run in a month, assuming that machines were only used for POC VL testing. Assuming one 4-cartridge GeneXpert machine, the maximum capacity would be 24 tests per 7-h workday, 480 per month, and 5760 tests per year.Based on conversations with in-country experts, we assumed clinics would not have a stable power supply (works approximately 60% of the time), and therefore included costs of a small back-up generator and battery for the GeneXpert equipment. Although most back-up generators can serve multiple machines (including larger POC machines) and refrigerators in a laboratory, hence sharing the costs across these units, we included the full cost as part of the start-up. Without a back-up generator, we estimate that sample wastage occurs 15% of the time compared to 1% of the time with back-up generator. We estimated electricity costs using published data on the average energy consumption by a GeneXpert instrument in low and middle-income countries . The avePOC testing platforms already exist at several facilities across Kenya and are primarily used for diagnostic testing for tuberculosis (TB) and other diseases. The national prevalence of TB in Kenya is estimated to be approximately 558 per 100,000 adults , howeverThe fee-for-service cost for conducting a centralized laboratory VL test in the Kenya public health sector (USD $24.63) was obtained from USAID data;  this cosWhen viral load tests repeatedly indicate poor management of HIV treatment, and poor adherence is ruled out, regardless of the viral load testing method , a drug resistance test (DRT) can be requested. The per-test cost to run a DRT at the National HIV Reference Laboratory in Nairobi, Kenya, is USD $67.64 . This feWe conducted time and motion observations over six weeks in November 2019–August 2020 in study clinics to estimate staff time needed to implement POC VL testing. When a study participant arrived in the clinic room, a stopwatch was started and the amount of time taken for each activity was recorded using pen and paper . Time and motion observations were conducted until we reached information saturation, and data were extracted into Microsoft Excel  to calculate total and unit costs associated with the intervention. Saturation of information was defined as occurring when additional observations did not change the estimates regarding how long an activity took. Resources and time spent on research activities  were removed from programmatic costs.We estimated that POC VL testing cost USD $24.25 per test, assuming a clinic volume of 100 VL tests conducted per month . Costs wIn this microcosting study, we estimated the per-test POC VL costs at varying clinic volumes compared to centralized laboratory testing in Kenya. Overall, we found that the POC VL testing’s per-test cost is higher than centralized laboratory testing, particularly for lower volume clinics. However, POC test costs declined rapidly with higher VL testing volumes since the POC instrument’s cost is spread over a larger number of tests. At clinics conducting 500 VL tests per month or with cartridge costs reducing to $12.00, POC testing had a lower cost than centralized laboratory testing. Overall, we find that POC HIV VL testing can be implemented into routine care at reasonable costs in clinics with moderate to large VL testing loads. Since POC testing costs were highly sensitive to the number of tests performed on each instrument, clinic volumes will be important as countries consider how to scale up POC VL technology to maximize resources within a limited budget. Based on consultation with in-country staff, we assumed a typical large Kenyan clinic conducts 100 viral load tests per month. Our study is among the first to estimate results of counseling costs by viral suppression among children and adults; we find that adherence counseling costs are highest for virally unsuppressed children and lowest for virally suppressed adults.Our findings are similar to previous costing studies, which estimated the cost per POC VL test to be between $25 and $30 ,13. One ® cartridge ($14.90/test) makes up the majority of the POC VL costs (72–91%), which is consistent with other costing studies [® platforms across Kenya that are currently being used to test for TB and other sexually transmitted infections [® equipment and recurring costs could be shared with other diseases’ diagnostics to improve cost efficiency. Patients attending low-volume clinics in rural settings may benefit from POC testing since poor quality roads can increase transport time to centralized laboratories leading to delayed results, degrading of samples, and increased loss to follow up [Identifying cost drivers of POC VL testing can inform strategies to reduce costs. We find that the cost of a GeneXpert studies ,25. Negofections . We findollow up . AlthougLayering on spoke-and-hub models for low volume or rural facilities may further decrease POC VL costs, which Kenya has already implemented for other disease testing platforms using GeneXpert. A study in Zimbabwe found that among women on Option B+, those who attended antenatal care at smaller volume clinics were 36% less likely to undergo VL testing than women at higher volume hospitals . While lThe demand for VL testing in 130 predominantly LMICs is projected to increase dramatically from 14.7 million tests in 2017 to over 28.5 million in 2022 ,29.Our study has several limitations. We conducted microcosting for one region of Kenya, which may not generalize nationally to other areas. Furthermore, costs were collected alongside a research trial, which may not be representative of routine clinical practice. However, our time and motion observations included data on routine clinic visits and we excluded research-related time costs. We used published cost estimates for centralized laboratory costs; therefore, we were not able to disaggregate unit costs for comparison to POC VL test costs. Our study did not account for the overhead, administrative and supervisory costs needed to implement POC nationally; future studies are needed to assess POC VL test implementation costs beyond the clinic level. We obtained instrument costs from the Cepheid distributor in Kenya rather than from the Kenyan Ministry of Health, so it is possible that costs estimates may vary depending on the source. However, the costs obtained from the Cepheid distributor reflect the country-negotiated costs and are consistent with the published literature ,13. FurtThe strengths of our analysis include detailed time and motion data based on observations of clinic care, laboratory processes, and adherence counseling. Data collection was carried out at five study sites with varying resource levels, patient volumes, and staff, contributing to the generalizability of our findings. We estimated costs of POC VL implementation for both adults and children living with HIV and we provide estimates of adherence counseling costs separately by viral suppression category.We estimated POC VL test costs at varying testing volumes and assess the impact of cost-sharing with other diagnostic testing, providing nuanced costs to inform policymakers as POC is rolled out nationally in sub-Saharan Africa. In summary, we find that POC VL technology can be implemented at reasonable costs for clinics with moderate to large patient loads. Future studies are needed to evaluate these aspects of POC testing in hard-to-reach, non-research facilities across various clinic volumes in order for this promising intervention to improve patient health outcomes for people living with HIV.


We describe two novel fluorescent peptides (compounds 1 and 2) targeting oxytocin with a boron-dipyrromethenyl group as the fluorophore bound to an artificial peptide based on the oxytocin receptor, and their application for the analysis of oxytocin levels in human serum using nanometer-sized magnetic beads modified by fluorescent peptides (FMB-1 and FMB-2). Under the optimized experimental protocols, FMB-1 and FMB-2 emitted low levels of fluorescence but emitted much higher levels of fluorescence when associated with oxytocin. The detection limit of oxytocin by FMB-2 was 0.4 pM, which is approximately 37.5 times higher than that of conventional methods, such as ELISA. Using these fluorescent sensors, oxytocin was specifically detected over a wide linear range with high sensitivity, good reusability, stability, precision, and reproducibility. This fluorescent sensor-based detection system thus enabled the measurement of oxytocin levels in human serum, which has widespread applications for oxytocin assays across varied research fields. Oxytocin is a pituitary peptide produced in the paraventricular nucleus of the hypothalamus and stored in the posterior pituitary ,2,3,4. OTo date, various biosensing methods have been developed for the detection of oxytocin, mostly by ELISA and LC–MS ,14,15,16Currently, sensors based on nanomaterials have been developed for the detection of various chemical substances by combining them with optical analytical methods such as fluorescence spectroscopy ,18,19,20To design fluorescent probes for use in an oxytocin assay, we assumed a weak-to-strong performance enhancement of the fluorescence signal upon binding to oxytocin. In addition, we assumed that the introduction of a selective recognition site to the probe would not lead to losses in optical properties and stability. This allowed for the development of probes with a reduced background, high sensitivity, high selectivity, and reduced interference from other substances.We previously developed a highly sensitive fluorescent reagent for oxytocin detection . HoweverTherefore, in this study, we aimed to develop an improved method for oxytocin detection that makes use of the properties of fluorescent molecular probes. The probes were constructed using the boron-dipyrromethenyl group as a fluorescent unit because of its interesting spectral properties, such as high quantum yields, large extinction coefficients ,30, highAll the chemicals used in this study were of analytical grade and were purchased from Tokyo Chemical Industry , Wako Pure Chemical Industries, Ltd. , and GE Healthcare . Magnetic beads were purchased from Tamagawa Seiki Co., Ltd. . Fluorescence spectra were recorded at 25 °C using a JASCO FP-6500 fluorophotometer and FP-8300 fluorophotometer.Control serum was purchased from Wako Pure Chemical Industries, Ltd. . This reagent contained IgG (1005 ± 100 mg/dL), IgA (193 ± 19 mg/dL), IgM (77 ± 7 mg/dL), C3 (110 ± 11 mg/dL), C4 (19.4 ± 1.9 mg/dL), CRP (0.79 ± 0.07 mg/dL), RF (23 ± 3 IU/mL), and ASO (90 ± 13 IU/mL).N--2-hydroxybenzyl)-N-methylglycine from 4-hydroxybenzaldehyde was performed according to our previously published method + = 6077.81 .Compound 2: Yield 85%; HPLC purity 95.7%; MALDI-TOF MS (+): [M + H]N-Hydroxysuccinimide. The reaction was then monitored by varying the reaction time . The optical change in the fluorescence emitted by the fluorescent magnetic beads (FMB-1 and FMB-2) was investigated by the addition of 0.5 nM oxytocin. We determined the optimal experimental conditions based on the combination of reaction time and peptide concentration that gave the highest fluorescence reading in the presence of oxytocin.Immobilization of the fluorescent peptides onto magnetic beads, detection of oxytocin using fluorescent peptides, and the detection of oxytocin using fluorescent peptides immobilized on magnetic beads have been described previously . The optFMB-1 or FMB-2 were added to HEPES buffer solution (pH 7.0) to obtain a final concentration of 80 μg/mL. After the addition of 100 μL oxytocin (0–1000 pM) to 100 μL of FMB-1 or FMB-2, the reaction mixture was incubated for 50 min. After concentrating FMB-1 or FMB-2 using a magnet, FMB-1 or FMB-2 were washed briefly, and the fluorescence spectra were recorded.Selectivity was measured by comparing the response of 100 μL of FMB-1 or FMB-2 after the addition of 100 μL of oxytocin or other substances, including albumin, globulin, sodium, potassium, calcium, magnesium, glucose, lactic acid, creatine, creatinine to control serum. The reaction mixture was incubated for 50 min. After concentrating FMB-1 or FMB-2 using a magnet, the beads were washed briefly, and fluorescence spectra were recorded.The reusability of the beads was tested by performing multiple cycles of the reaction between FMB-1 (or FMB-2) and oxytocin and brief washing with aqueous NaOH to remove oxytocin from the magnetic beads. After completing the assay, the fluorescent magnetic beads were reused to measure the residual activity.The stability of FMB-1 and FMB-2 was analyzed by incubating the beads at 4 °C and calculating their residual activity by measuring the fluorescence intensity over an extended time period.Artificial fluorescent peptides, designated as compound 1 and compound 2 , were imOxytocin concentrations ranging between 0 and 1000 pM were added to a suspension of fluorescent magnetic beads and incubated for 50 min. After magnet-driven concentration and a brief wash, the response of the fluorescent magnetic beads before and after the addition of oxytocin was monitored. Typical absorption spectra and the relationships between the concentration of FMB-1 or FMB-2 and the measured absorbance are shown in The magnetic beads exhibited a very weak fluorescence emission in the absence of oxytocin; however, a strong fluorescent signal was observed after the addition of oxytocin . The fluThe emission intensities of FMB-2 at 525 nm were plotted as a function of oxytocin concentration. The detection limit and detectable range of FMB-2 were 0.4 pM  and 0.4~1000 pM for oxytocin, whereas those of FMB-1 were 2.1 pM  and 2.0~500 pM. The fluorescent peptides (compound 1 and compound 2) immobilized on these magnetic beads have amino acid sequences that are based on the oxytocin receptor , and theThe response of FMB-2 to various contaminants, such as inorganic salts, proteins, and other foreign substances which may be present in biological samples, was tested to investigate the specificity of the fluorescent magnetic-bead based oxytocin assay. The fluorescence ratios of FMB-2 at 525 nm were monitored before and after the addition of the interfering substances . A largeTo investigate the pH dependence of the reaction time between oxytocin and FMB-2, the fluorescence intensities of FMB-2 mixed with 0.5 nM oxytocin were measured at pH values ranging from 3 to 11. Our fluorescent magnetic beads exhibited inhibition at high pH (10–11) and low pH (3–4). However, the fluorescence intensities were nearly unchanged when the pH was in the range of 5 to 9, which corresponds to the physiological pH of the human body b. FluoreWe next examined the reusability of the fluorescent magnetic beads for the detection of oxytocin. Any clinical assay would greatly benefit from the reusability of consumables, which decreases costs as well as reducing the environmental impact of producing and disposing these consumables. Reusability was measured after cycles of the reaction were performed using the magnetic beads and oxytocin, with brief washes between each cycle to remove unreacted oxytocin. After the release of oxytocin from the surface of the magnetic beads by the addition of NaOH(aq) and washing, we monitored the initial fluorescence intensity, as shown in The stability of FMB-2 is an important factor for the long-term measurement of oxytocin. The fluorescent magnetic beads were stored in 1.5-mL tubes at 4 °C, and the fluorescence ratio was monitored at intervals of 10 days. FMB-2 preserved over 98% of its initial fluorescence intensity for up to 5 months d. HoweveFinally, the fluorescent magnetic beads were applied to measure the levels of oxytocin in human serum that had been spiked with oxytocin representative of physiological oxytocin levels. FMB-2 exhibited a rapid increase in fluorescence intensity with the addition of human serum samples. We quantified the oxytocin concentrations in the human serum with CV values of 2.7% and recovery rates of approximately 100.9%. The data are summarized in We compared the characteristics of our novel magnetic bead-based assay for oxytocin with those of existing methods . The enzIn the present study, we developed a novel fluorescent peptide for the detection of oxytocin, and nanometer-sized magnetic beads containing these peptides were synthesized using a conventional synthetic method. Changes in fluorescence indicate the successful binding of the target oxytocin with the fluorescent peptide. We observed large changes in fluorescence in the presence of oxytocin, indicating a high sensitivity and selectivity, as well as demonstrating good reusability and durability of these magnetic beads. These parameters contribute to the detection of oxytocin in human serum samples with high precision. This sensing system is easy to use and offers rapid and highly sensitive detection based on the unique properties of fluorescence enhancement. We are currently investigating other possible molecular probes for this system, which could be used to detect a range of biological substances that may be useful in research and medical fields.


Emerging studies increasingly demonstrate the importance of the throat and salivary glands as sites of virus replication and transmission in early COVID-19 disease. SARS-CoV-2 is an enveloped virus, characterized by an outer lipid membrane derived from the host cell from which it buds. While it is highly sensitive to agents that disrupt lipid biomembranes, there has been no discussion about the potential role of oral rinsing in preventing transmission. Here, we review known mechanisms of viral lipid membrane disruption by widely available dental mouthwash components that include ethanol, chlorhexidine, cetylpyridinium chloride, hydrogen peroxide, and povidone-iodine. We also assess existing formulations for their potential ability to disrupt the SARS-CoV-2 lipid envelope, based on their concentrations of these agents, and conclude that several deserve clinical evaluation. We highlight that already published research on other enveloped viruses, including coronaviruses, directly supports the idea that oral rinsing should be considered as a potential way to reduce transmission of SARS-CoV-2. Research to test this could include evaluating existing or specifically tailored new formulations in well-designed viral inactivation assays, then in clinical trials. Population-based interventions could be undertaken with available mouthwashes, with active monitoring of outcome to determine efficacy. This is an under-researched area of major clinical need. Viral e,It is widely known that interfering with the lipid envelope represents a virucidal strategy to target many coronaviruses, with a large body of work evidencing the impact of many agents.https://apps.who.int/iris/bitstream/handle/10665/331138/WHO-WPE-GIH-2020.1-eng.pdf; https://www.epa.gov/pesticide-registration/list-n-disinfectants-use-against-sars-cov-2; https://www.who.int/gpsc/tools/GPSC-HandRub-Wash.pdf) . At the same time, there has been no discussion of oral antiviral strategies, apart from a recent response to an article in the British Medical Journal calling for protection for healthcare workers against infection (https://www.bmj.com/content/369/bmj.m1324/rr-5). Properly designed clinical trials that address this issue are currently lacking in the literature. Current WHO interim guidance on clinical management of SARS-CoV-2 in the home is focused on the use of personal protection, including face masks, along with hand, clothing, and surface sanitation, to reduce risk of airborne and direct spread of the virus, but does not mention oral hygiene (https://apps.who.int/iris/handle/10665/331133). Thus, its utility in the setting of SARS-CoV-2 has not been considered systematically, and there is a lack of either positive or negative robust clinical evidence.We are now widely encouraged to use soap or 60%–70% alcohol-based gels to inactivate SARS-CoV-2, based on the view that these agents damage the lipid envelope, including in recent World Health Organisation (WHO) and Environmental Protection Agency recommendations  in the United Kingdom, Europe, and the United States. Prompted by this, we reviewed the available scientific literature to establish whether oral treatment using ethanol-based or other types of mouthwashes could present a strategy to either dampen or reduce viral load, to potentially restrict virus transmission in the current pandemic situation, particularly for vulnerable individuals or healthcare workers. We found that there is a paucity of data systematically testing the impact of lower (less toxic) ethanol concentrations on enveloped virus inactivation, with most simply reiterating the use of the higher concentrations described above.,It is becoming increasingly recognized that the throat is a major site of replication and shedding of virus in COVID-19 illness, and that viral load is important.The route of SARS-CoV-2 infection is currently considered to be via respiratory droplets, similar to SARS-CoV,,At this time, there is incomplete information on how SARS-CoV-2 moves from the throat and nose to the lungs, and this could include (1) viral shedding, (2) the aspiration of necrotic cell debris, or (3) direct infection of neighboring cells. Assuming viral shedding is involved, the oral rinses that target the viral lipid envelope represent a potential method to remove/rinse or inactivate infective particles generated in the throat. The specific intracellular replication cycle for SARS-CoV-2 in humans is not yet known. Based on nonsynchronized replication cycles that take <24 h, virus is likely to be secreted almost constantly.When considering lower  ethanol concentrations, the literature on mammalian cells (from where the lipid envelope originates) provides a close comparator. We also reviewed studies on model membrane vesicles comprising phospholipids such as phosphatidylcholine; however, as these are protein-free, the impact of nonlipid components on ethanol toxicity is not accounted for. Bacterial pathogens contain very different membranes in lipid and protein composition, including lipopolysaccharides and peptidoglycans, so they are not considered here. Below we summarize the literature on impact of ethanol on cell/model membranes .Interdigitation refers to the process whereby the presence of short-chain alcohols enables the methyl group of the fatty acyl chains to move beyond the midplane of the bilayer, penetrating the opposite monolayer, and appears to be an event that precedes and promotes vesicle fusion and leakage.,interdigitated phospholipid sheets from small unilamellar vesicles (SUVs), which then annealed to form larger interdigitation-fusion vesicles (IFVs).,,,,,,,Biophysical studies in the 1980s and 1990s compared various alcohols  for their ability to perturb model phospholipid membranes. Many were optimizing generation of lipid vesicles for drug delivery; however, the toxicity of most short-chain alcohols prohibits oral use. Here, we reviewed reports on the properties of ethanol on model membranes. Few studies directly investigated lysis, instead focusing on membrane fluidity, permeability, interdigitation, thickness, and other parameters. In one study, ethanol >3.4 M (20% v/v) resulted in membranes not being considered “stable”.We also reviewed the impact of ethanol on mammalian cells in vitro. Of direct relevance to coronaviruses, a study on corneal epithelial cells showed that a 30-s incubation with 20% ethanol led to around 40% loss of viability, which increased to 70% loss at 40% ethanol. There was significant leakage of intracellular contents following 20% ethanol for 30 s.https://www.astm.org/Standards/E2197.htm), or in the United Kingdom, the equivalent British Standard (BS) Norme Européenne (EN) standard , https://shop.bsigroup.com/ProductDetail?pid=000000000030401479). Also, it was conducted at 22°C, rather than the more relevant 36.8°C oral temperature, where the impact of denaturation agents would be greater.In 2007, Roberts and Lloyd found that 20% ethanol completely inactivated three enveloped viruses: Sindbis, herpes simplex-1, and vaccinia, in vitro, while having no effect on the nonenveloped poliovirus-1.In 2017, Siddharta et al. tested WHO recommended formulations against enveloped viruses, including coronavirus. Focusing on WHO formulation I, which contains 85% (v/v) ethanol, 0.725% (v/v) glycerol, and 0.125% (v/v) hydrogen peroxide, they measured in vitro infectivity in the presence of a soil load . A 30-s exposure of a dilution containing 34% (v/v) ethanol (40% of neat) completely prevented subsequent viral replication.So far, we found only studies that tested the impact of reduced ethanol amounts on enveloped viruses. Both were conducted in vitro, and show positive outcomes in relation to virus denaturation.These studies indicate that relatively dilute ethanol will be highly effective against enveloped viruses. However, there is an urgent need to determine how coronaviruses are impacted by dilute alcohol under biologically relevant conditions , and whether in combination with nontoxic, membrane disrupting agents, oral inactivation of SARS-CoV-2 could be achieved. A minimum amount of ethanol, for example, 10%–30% (v/v) would be effective, and contact time will also be an important parameter that may reduce ethanol exposure required. Ethanol impacts membrane properties of artificial lipid membranes, causing leakage of contents even in the absence of complete lysis. The ability of the virus to infect host cells could also be modified by inducing biophysical changes to the virus membrane which impact on protein function. The spike glycoprotein which is required for SARS-CoV-2 infectivity contains a transmembrane domain that is inserted into the viral envelope,The concept that perturbing the membrane could inactivate viruses has recently been tested in relation to membrane-disrupting agents, and an in vitro screen of 1000 compounds identified a series of lipidomimetics that can alter the membrane and dampen infectivity of HIV-1.We investigated the potential for commercially available mouthwashes to disrupt viral lipid envelopes, either due to ethanol , or othe10 lower than baseline, demonstrating a modest impact on viral titer. Longer contact times  were not tested.The virucidal actions of 21% (v/v) ethanol with essential oils toward an enveloped virus were reported in humans in vivo in 2005. A 30-s rinse reduced infectious virions of herpes simplex types I and II to effectively zero.A study in 1995 tested 26.9% ethanol with essential oils against herpes, influenza, rotavirus, and adenovirus in vitro. Here, an impact on the viral lipid envelope was speculated since herpes and influenza were significantly impacted, while adenovirus and rotavirus (nonenveloped) were not.https://iadr.abstractarchives.com/abstract/2010dc-131191/evaluation-of-h1n1-antiviral-properties-of-an-essential-oil-containing-mouthrinse).A follow-up unpublished study in 2010 determined that a 30-s in vitro exposure to 21.6% ethanol with essential oils led to >99.99% reduction of infectivity of H1N1 Influenza A pandemic strain . Notably, there is published evidence that eucalyptus oil and thymol have significant antiviral properties toward herpes simplex virus at these concentrations, hypothesized to relate to disruption of the viral lipid envelope.These studies provide proof-of-concept that mouthwashes containing essential oils with 21%–27% ethanol can inactivate enveloped viruses, both in the lab and in humans, with the likely mechanism being damage to the lipid envelope. Here, ethanol in combination with essential oils may provide a more effective formulation. Thus, these types of mouthwash may be effective against SARS-CoV-2, although studies have not been conducted. While other commercially available ethanol mouthwashes generally contain lower levels without essential oils, an impact on membrane biology may remain theoretically possible, and studies are required.,Chlorhexidine is widely used for oral health in the United Kingdom, being especially effective against Gram-positive bacteria, but to a lesser extent Gram-negative bacteria and fungi.Chlorhexidine mouthwashes have been a critical clinical tool for over 40 years to reduce oral bacterial flora and prevent infection and mucositis in cancer patients receiving chemotherapy and radiotherapy.,P < 0.05) was found.https://www.who.int/medicines/publications/essentialmedicines/en/). It should be noted that rare allergic reactions have been reported for PVP-I.Povidone-iodine (PVP-I) mouthwash has been widely studied in relation to broad-spectrum antimicrobial and virucidal actions. At 0.23%, which is routinely used in Japan, this rapidly inactivates SARS-CoV, MERS-CoV, influenza virus A (H1N1), and rotavirus in vitro.Studies from Japan surprisingly found that gargling with chlorinated tap water reduced respiratory infections, and in one, was even better than PVP-I. In one, three groups of around 130 age- and gender-matched human subjects were studied .https://www.cdc.gov/parasites/naegleria/sinus-rinsing.html).Separate to oral rinsing it is worth noting that nasal rinsing with saline is a popular method promoted to clear nasal passages for sufferers of colds and allergies. Given that virus is recovered in the nasopharynx, a similar consideration of how this might be used as preventative measure could be made. As for mouthwash, clinical studies have not systematically examined how effective nasal rinsing is for preventing respiratory infections. Notably, rare reports of serious illness when not properly cleaned, due to the presence of parasitic amoebae in unboiled tap water, has led to recommendations on careful disinfection of rinsing syringes being made by CDC . Here, the impact of not only dose/composition but also the critical issue of contact time with agents, which is a known modifiable parameter of virucidal activity can be easily tested. The virucidal mechanisms can be determined, conducting lipidomics analysis of the envelope along with assays that determine spike protein conformation and activity. While a particular ethanol concentration may achieve full inactivation, lower amounts could either help to remove virus or lead to membrane damage (permeability/leakage) that may impact throat cell virus infectivity, for example, through potential modification of the ability of the spike glycoprotein to interact with receptors on host cells. In this regard, for HIV-1, the lipid membrane stabilizes membrane glycoproteins, regulating their sensitivity to antibody neutralization.The impact of temperature and soil load needs to be considered for in vitro studies, applying the ASTM or EU standard protocols (https://www.nature.com/articles/d41586-020-00698-x).Most virucidal research uses in vitro models, where the response to the agent will be different, and also does not take into account the impact of host immunity. There is an absence of animal model studies on coronavirus respiratory illness, although macaques and mice transgenic for humanized ACE2 are beginning to be studied with early indications being that both develop mild illnesses in response to SARS-CoV-2 virus , chemokines , and matrix metalloproteases (MMP9) were all upregulated at the mRNA levels 1–3 days after a 30-s exposure to 20% (v/v) ethanol.Available research approaches include:We highlight a list of excellent review articles that were consulted as part of preparing this review and were a source of primary research cited herein:Budding of viral lipid membranes: Simons and GaroffImpact of virus on lipid metabolism: Sanchez and LagunoffReview of inactivation of coronaviruses on surfaces: Wolff et al.Emergence of SARS-CoV in 2003: Peiris et al.,Two papers on the impact of ethanol on interdigitation of membranes: Slatter and HuangControl of infection using Povidone-Iodine: EggersSummary of the composition of lipid membranes in cells: van MeerSince the review covers many areas from basic biochemistry, virology, and microbicidal research, as well as clinical information both in medicine and dentistry, multiple sources were consulted. Most references were identified from PubMed, ResearchGate, or Google, using search terms including “virus,” “coronavirus,” “lipid envelope,” “alcohol,” “membrane,” “chlorhexidine,” and others, alone and in combination. Many references that were first identified, were then investigated further to find additional source material and the original primary research which was then included. The idea for drafting the review was initiated by V.B.O. on March 21, 2020, through reaching out in person to various international experts, to get their views and input directly, via phone calls and then follow-up emails. Boots UK was approached initially to discuss the ideas and for information on formulation of available mouthwashes. Boots researchers and scientists contacted Johnson & Johnson, who provided proprietary information. Boots researchers  then conducted further searches of PubMed, Google, and ResearchGate, using the same terms. Academic and clinical expertise was consulted for virology , clinical/ICU (Fegan and Wise), dental practice (Thomas), immunology (Jones), lipid biochemistry , and microbicides (Sattar and Maillard). Phone/email correspondence with experts generated input, opinions, and identified additional references. No timeline for references was used and no languages were excluded.