Salmonella spp. This work designed an approach for phage therapy using encapsulation by ionotropic gelation of the lytic bacteriophage S1 for Salmonella enterica in 2% w/v alginate beads using 2% w/v calcium chloride as crosslinking agent. This formulation resulted in beads with an average size of 3.73 ± 0.04 mm and an encapsulation efficiency of 70%. In vitro, the beads protected the bacteriophages from pH 3 and released them at higher pH. To confirm that this would protect the bacteriophages from gastrointestinal pH changes, we tested the phage infectivity in vivo assay. Using a model chicken  infected with Salmonella Enteritidis, we confirmed that after 3 h of the beads delivery, infective phages were present in the chicken’s duodenal and caecal sections. This study demonstrates that our phage formulation is an effective system for release and delivery of bacteriophage S1 against Salmonella Enteritidis with potential use in the poultry sector.Modern bacteriophage encapsulation methods based on polymers such as alginate have been developed recently for their use in phage therapy for veterinary purposes. In birds, it has been proven that using this delivery system allows the release of the bacteriophage in the small intestine, the site of infection by  Salmonella can infect humans and animals. Two of the main pathogens of these genera infecting animals, and specifically poultry, are Salmonella enterica serovar Pollurum (S. Pullorum) and Salmonella enterica serovar Gallinarum  [Salmonella enterica serovar Enteritidis (S. Enteritidis), which can infect humans through consumption of contaminated meat and eggs. At the same time, poultry infected with S. Enteritidis infect their healthy neighbors contributing to further economic loss. S. Enteritidis is one of the main foodborne pathogens responsible for spreading Salmonella in humans, representing a public health issue [Salmonella. This can include applications intended to limit the spread of the bacterium or directly for the treatment of salmonellosis in poultry, an approach known as phage therapy [Bacteria of the genus linarum) . Their elinarum) ,2. Anothth issue ,4. The i therapy ,10,11,12Salmonella that colonize the digestive tract [Salmonella Enteritidis in cecal tonsils of day-old chickens. Five days after treatment, Salmonella Enteritidis loads decreased by 3.5 orders of magnitude. Andreatti-Filho et al. [Salmonella Enteritidis in cecal tonsils during the first 24 h. However, the treatment failed to achieve a significant reduction after 48 h. Despite these reported successes of the solutions and cocktails, they have limited stability due to loss of the phage titer during processing and storage, which in a few months could represent losses as high as 99% of the initial titer.Currently, a wide variety of studies on phage therapy in the poultry sector demonstrated that its use is effective in reducing bacterial loads of ve tract ,14,15. Ive tract  demonstro et al.  demonstrSalmonella spp. phage Felix O1 in a chitosan-alginate- calcium chloride system, demonstrating that the ALG allowed Felix O1 to be infective in the gastrointestinal tract (GIT), promoting the phage delivery in their intestines.In fact, one of the main challenges of phage delivery forms has been their stability, which is why different authors have been inclined to look for solid pharmaceutical options such as nanosomes, liposomes or microspheres . CurrentSalmonella enterica in vitro [Salmonella enterica to humans.We previously isolated and partially characterized a phage, “S1”, capable of infecting in vitro . BrieflyIn order to use S1 in phage therapy, a thorough genetic characterization of the phage is required to ensure it is virulent (strictly lytic) and carries no genes that would be a barrier to its use; furthermore, as the gastrointestinal pH of chickens varies from 1 to 4 in the gizzards, while ranging from 5 to 7 in the duodenum, the phage would experience a wide-range of pHs which could impair its efficacy ,23. For Salmonella enterica serovar Enteritidis phage type 13A (SE PT13A), was obtained from the USDA National Veterinary Services Laboratory  and donated by Dr. Billy Hargis and Dr. Guillermo Tellez of the Department of Poultry Science, University of Arkansas. SE PT13A was grown on tryptic soy agar (TSA)  or tryptic soy broth (TSB)  according to the assay needs. Strains Salmonella enterica serovar Pullorum (MDR-MC862-A) and Salmonella enterica serovar Gallinarum (MDR-MC862-B), were isolated from clinical trials in poultry and donated by the Faculty of Veterinary, National Autonomous University of Mexico. The strain Salmonella enterica serovar Cholerasuis (S. Cholerasuis), Salmonella enterica serovar Typhi (S. Typhi), Salmonella enterica serovar Typhimurium (S. Typhimurium) and Citrobacter freundii (C. freundii), Enterobacter cloacae (E. cloacae) and Escherichia coli (E. coli) were isolated from clinical trials in humans . The supernatant was filtered by passage through 0.45-µm cellulose acetate membrane filter  and stored at 4 °C.Phage S1 was isolated from wastewater from the city of Cuernavaca Morelos, Mexico . Propagaw/v  to a tube containing 3 mL of bacteriological agar at 0.7% w/v and was poured in triplicate into TSA plates that were incubated at 37 °C for 24 h.The enumeration of phage S1 was performed using the double-layer agar method described by Adams , with so11 PFU/mL) of phage S1, a phage DNA extraction was performed with Invitrogen PureLink Viral RNA/DNA Mini Kit  following the kit protocol. Library preparation of the resultant DNA was performed by tagmentation using Illumina Nextera protocol. The final library was prepared for sequencing with the reagent MiSeq kit v2  and sequenced using Illumina MiSeq System. The fragments obtained were paired-end reads with lengths of 150 bp. Host reads and adapters were trimmed using Trimmomatic v0.36 [Salmonella phage ZCSE2 (NC_048179) and S144 (MT663719) was used to identify protein hits and their e-values. For each protein comparisons the e-value from the full sequence was taken into consideration. Additional protein searching was made with the open reading frames (ORF’s) using HHpred toolkit [Loughboroughvirus genus  were uploaded into the web server to generate the phylogenetic tree. The completeness of the final assembly was confirmed by mapping the sequences from the last and first portions of S1 genome and compared with the linear assembly. The obtained tandem repeat was visualized by Tablet v.1.17.08.17 [After obtaining a high titer . Additionally, the EOP was tested in other gram-negative species; C. freundii, E. cloacae and E. coli  of phage S1 was determined in five serovars of  E. coli . The EOPThe EOP value obtained was classified as high (EOP ≥ 0.5), medium (0.5 ≤ EOP > 0.01), and low (EOP ≤ 0.1) .w/v and 1.19 × 1011 PFU of phage S1 was added, the mixture was extruded using a 5 mm internal diameter needle to a sterile calcium chloride solution 2% w/v . To remove excess calcium chloride solution from the beads, three washes were made with 150 mL of sterile deionized water for 3 min and kept at room temperature for two hours to remove excess water. Finally, 100 beads were measured with a digital calibrator UltraTech®  and stored at 4 °C.Bacteriophage S1 was encapsulated using the ionic gelation technique by extrusion ,19,38. Aw/v. The determination of the total PFU was made using the double layer agar method by Adams [The encapsulation efficiency (%EE) was determined according to what was reported by Colom et al.  and Boggby Adams . The %EEby Adams (2)%EEenw/v. An aliquot of 500 µL of each dilution per triplicate was seeded in TSA plates by spatulation technique and incubated at 37 °C for 24 h. The bacterial titer (CFU/mL) was calculated according to equation 3 as follows:SE PT13A cultures were grown to the middle of the exponential growth phase, then inoculated with 1 g of alginate beads with and without phage S1. The cultures were maintained at 37 °C with constant agitation at 120 rpm. Samples were taken at 0, 12, 24, 36 and 48 h after inoculation and ten-fold dilutions were performed with sterile saline solution 0.85% 8 PFU were mixed on 10 mL of sterile saline solution 0.85% w/v, the final pH was adjusted to 3, 5, 7 and 8.5 with HCl 0.2 M and NaOH 0.2 M , accordingly. The mixture was incubated at 40 °C and in agitation for 100 rpm, aiming to mimic the average temperature of the chicken GIT, and at either pH 3 for 45 min, pH 5 for 15 min, pH 7 for 30 min, and pH 8.5 for 20 min, aiming to mimic the pH and transit time of the proventriculus + gizzard, duodenum, jejunum + ileum or cecum, respectively [Salmonella grows in an optimum pH of 6.5 to 7.5 and its growth is inhibited at pH < 3.8 [To determine whether the alginate beads protect bacteriophage S1, we designed the following experiment. Free phages at a fixed concentration of 9.56 × 10ectively ,23,40. TpH < 3.8 .On the other hand, 1 g of ALG beads containing the phage were incubated under the same conditions as the free phages described above. After incubation, the beads on pH 3 and 5 were adjusted at pH 7 with 0.2 M NaOH for 5 min and rinsed with sterile distilled water three times and placed in 20 mL of the broken microsphere solution at pH 7.5 and stirred at 120 rpm until macroscopic and visible disintegration for the release of the phage . The PFUSalmonella, upon arrival, samples were taken from the cloaca with sterile swabs that were sown in TSB and incubated at 37 °C for 24 h. The animals were kept in the conditioning phase for seven days by supplying them with commercial growth food FlagasaTM  and water ad libitum. Thirty chickens were distributed in five groups for elimination test and eight were used for the identification of the degradation of the ALG beads and phage S1 release test. Finally, the animals were euthanized by cervical dislocation in accordance with the 4th edition of the Guide for the Care and Use of Agricultural Animals in Research and Teaching [A sample size of 38 one-month-old chickens was calculated taking into consideration 5% of chicken loss during the transportation. We obtained all animals from a commercial supplier . The Institutional Project Review Board “Ad hoc Committee for admission to Doctorate in Pharmacy” assigned by Faculty of Pharmacy, UAEM approved the ID# 2019/003 research protocol in 17 June 2019. Chickens were transferred and handled in accordance with the Official Mexican Standard NOM-051-ZOO-1995, Humanitarian treatment in the mobilization of animals . After oTeaching  Chapter Teaching .9 PFU phage S1. Two randomly chosen chickens were sacrificed at 1, 3, 5 and 18 h after administration and macroscopic search for ALG beads was performed in crop, gizzard, cecum and duodenum cavities of the GIT. In addition, the presence of phage S1 was determined as follows: the contents of each of the mentioned cavities was homogenized in 20 mL of sterile saline solution 0.5% w/v and centrifuged at 1300× g for 15 min, the supernatant was filtered using a 0.45-µm filter. An aliquot of 50 µL from that filtrate was inoculated on TSA plates which contained a lawn of SE PT13A (spot test technique). Plates were incubated at 37 °C for 24 h. The presence of lytic zones was considered as a positive for phage activity in each of the analyzed chicken organs.To observe the degradation process of the ALG beads during their passage through the gastrointestinal tract, a total of eight chickens were administered 1 g of alginate beads containing 4.9 × 10Salmonella spp. infects. The chickens’ infection with SE PT13A and administration of the corresponding treatments were performed as indicated in g for 15 min. The supernatant was analyzed by spot test as described previously, to determine if the encapsulated phage S1 had passed through the GIT, been released, and remained infectious.Knowing that phages are biological agents that replicate in the host cell, we designed an assay that would corroborate their presence in organs that We obtained a complete and novel assembled phage genome of 53, 394 base pairs in length, with an average coverage of 7000× and genomic guanine and cytosine (G + C) content of 45.8%. The genome annotation of phage S1 resulted in 75 ORF’s, while the prediction of the protein functions assigned only 29 ORF’s  and phage terminase proteins. We did not detect tRNAs, transposases, integrases or phage repressors. A schematic gene organization is shown in To predict its lifecycle, we used a predictive analysis tool (PhageAI), which anticipated phage S1 as 100% virulent. We confirmed the completeness of the phage S1 after mapping their reads from the end and beginning of its linear sequence.The coverage of these tandem repeats resulted in an average 50×. However, we couldn’t determine the phage termini due the tagmentation protocol used in the library preparation.Salmonella phages; S144 (97.64%), ZCSE2 (97.58%) and SE4 (87.05%), the accession numbers are NC_048179, MT663719 and NC_048764 respectively. These three phages belong to the family Myoviridae and genus Loughboroughvirus. We carried out a phylogenetic analysis using a full viral protein alignment to classify S1 through the database from ViPTree. Phage sequences from ZCSE2 and S144 are considered relatively new. The release of these sequences to the NCBI database happened during 2020. For that reason, we added the new genomes into the webserver to identify the phylogeny of S1. The proteomic tree showed that S1 had the closest relationship with phage ZCSE2, suggesting that S1 comes from the same linage, the genus Loughboroughvirus. Also, we denoted a second cluster showed a close relationship between ZCSE2 and S144 and a third cluster showing the relation of SE4 with the rest of phages from the same genus  up to 24 h [Phage ZCSE2 it is a mere 571 bp shorter than ZCSE2 and 97.58% identical over the entire length of its genome arising two gaps at the beginning and end of their sequences. S1 encodes 75 proteins, all but 73 of which can be found in S1 as well (85% amino acid similarity cutoff). We noticed that the ORFs 50 and 56 in phage S1 are unique proteins that are not present in any other phage from the same genus (at more than 85% amino acid similarity cutoff). These are small proteins with 50 and 58 amino acids, respectively , with th to 24 h . These fSalmonella enterica, indicating that SE4 also has a broad spectrum of bacterial killing [Salmonella spp.A phage less closely related to S1 but from still the same genus is SE4, which is 100 bp longer than S1 and 87.05% identical over the entire length of its genome. From the 75 proteins in S1, only 57 were found in S1 and in the others with 85% amino acid similarity cutoff . The maj killing  and highThe conserved proteins across their phylogenetic neighbors is shown in S. Pullorum MDR-MC862-A was 0.61 while with S. Gallinarum MDR-MC862-B was 0.58 . This encapsulation percentage was slightly less than reported in other studies where the beads disintegrated under the same conditions, such as that by Moghtader et al. [w/v alginate beads coated with chitosan and polyethyleneimine, as well as the study by Yongsheng et al. [5 up to 1011 [The phage S1 encapsulated by ionic gelation in ALG beads using calcium chloride as a crosslinking agent resulted in an average size of 3.73 mm with a range of 3.69 to 3.77 mm. The size of the beads formed by the ionic gelation technique by extrusion depends mainly on the diameter of the nozzle used, which is the determining factor for obtaining the desired size . Consider et al. , who repg et al. , who obt to 1011 ,61. For We first set out to ensure the alginate beads would not interfere with our ability to detect phage activity in vitro. We compared the bacterial growth exposing the ALG beads and ALG beads + phage. When SE PT13A was grown in the presence of ALG beads, the viable bacterial counts increased X-fold over the first 24 h, with a drop of less than 2-fold over the following 24 h , consistSalmonella lives, from duodenum to the cloacal zone, where the pH ranges from 6 to 8 on average [The alginate bonds are strengthened when they are in the presence of acidic pH values. For this reason, the pH is relevant to preserve ALG beads made by ionic gelation. As more basic pH, the beads depolymerize, resulting in the delivery of phages ,63. In c average . This isw/v of ALG beads protects the phage when it is exposed at acidic pH, keeping it infective in the duodenum and cecum, cavities that are infected by the Salmonella genus.The passage of the beads in the GIT of the chicken was visualized macroscopically when dissecting two animals at random at 1, 3, 5, and 17 h after administration. It was only possible to observe the alginate beads in the crop of chickens sacrificed 1 h after administration. These results are in agreement with the literature, indicating that there exists a decrease in particle size of chicken feed caused by the process of crushing and maceration by the gizzard ,67. BesiSalmonella in the experimental animals and after the conditioning phase through cloacal swabs, the corresponding infection was performed in each group. Salmonella colonizes the GIT of birds, mainly the cecum and cloaca. The incubation period of Salmonella infection in poultry varies from four to seven days, so the cloacal samplings obtained at three, five, and seven days after infection with SE PT13A showed that at days three and five, in all groups, the presence of lactose-positive, nonproducing hydrogen sulfide colonies, not consistent with the biochemical characteristics of SE in the TSA, were observed [Gallus gallus domesticus model. Group C had a visibly decreased bacterial growth compared with group D. The results obtained are consistent with those reported by Andreatti Filho et al. [S. Typhimurium by a cocktail of phages encapsulated in liposomes and detected a significant reduction in the cecum of 3.8, 3.9, and 1.5 logarithmic units of S. Typhimurium on days eight, 10 and 15 posttreatment, respectively. Likewise, Bardina et al. [S. Typhimurium twice a day on days four and five after infection, which reduced the bacterium by one logarithmic unit on days five and six after treatment. This reduction in bacterial loads and shedding of phages could also have further benefits, as it could actively lower Salmonella loads in the environment, reducing the potential for feedback contamination [By ensuring the absence of the genus observed . Finallyo et al.  who evalo et al.  measureda et al.  administmination ,71.Salmonella enterica tested. Ionic gelation was an efficient encapsulation process, conferred protection of the phage from low pHs, and enabled the release of infectious particles capable of preventing Salmonella growth both in vitro and in vivo. Further work quantifying in vivo release and efficacy would be warranted to enable regular therapeutic or prophylactic use of encapsulated phage S1 in this context.Bacteriophage S1 is a virulent phage lacking any genes that would prevent its use in therapy, and is capable of lysing three of five strains of


N = 7), Senegal (N = 11), and Mali (N = 5). We modelled costs for programme transition (2021) and early scale-up (2022–2023). Between July 2019 and September 2020, a total of 51,028, 14,472, and 34,353 HIVST kits were distributed in Côte d'Ivoire, Senegal, and Mali, respectively. Across countries, 64–80% of HIVST kits were distributed to FSW, 20–31% to MSM, and 5–8% to PWUD. Average costs per HIVST kit distributed were $15 for FSW , $23 for MSM , and $80 for PWUD , driven by personnel costs , and HIVST kits costs (2–20%). Average costs at scale-up were $11 for FSW , $16 for MSM , and $32 for PWUD . Cost reductions were mainly explained by the spreading of IPO costs over higher HIVST distribution volumes and progressive IPO withdrawal at scale-up. In all countries, CSO-led HIVST kit provision to KP showed relatively high costs during the study period related to the progressive integration of the programme to CSO activities and contextual challenges . In transition to scale-up and integration of the HIVST programme into CSO activities, this model shows large potential for substantial economies of scale. Further research will assess the overall cost-effectiveness of this model.Despite significant progress on the proportion of individuals who know their HIV status in 2020, Côte d'Ivoire (76%), Senegal (78%), and Mali (48%) remain far below, and key populations (KP) including female sex workers (FSW), men who have sex with men (MSM), and people who use drugs (PWUD) are the most vulnerable groups with a HIV prevalence at 5–30%. HIV self-testing (HIVST), a process where a person collects his/her own specimen, performs a test, and interprets the result, was introduced in 2019 as a new testing modality through the ATLAS project coordinated by the international partner organisation Solthis (IPO). We estimate the costs of implementing HIVST through 23 civil society organisations (CSO)-led models for KP in Côte d'Ivoire ( In Western and Central Africa, 5 million people are living with HIV, representing a prevalence of 1.4% in 2019 , and 95% of those on ART to have their viral load suppressed by 2030 .Conventional facility-based HIV testing services (HTS) does not adequately reach those KP due to stigma, discrimination, and health services not responding to needs specific to each group. Local civil society organisations (CSO) providing mostly community-based HIV testing services using peer educators have proven successful in reaching the core members of these populations, linking, and retaining them into care , 8.Auto Test VIH, Libre d'Accéder à la connaissance de son Statut) (HIV self-testing (HIVST) is defined as a process where a person collects his/her own specimen , performs an HIV test and interprets the result, often in private . Followi Statut) . The pro Statut) , 18.OraQuick® HIV self-tests have been subsidised by the Bill and Melinda Gates Foundation, then proposed by Orasure Inc. at US$2 per kit in 50 low- and middle-income countries for public sector distribution . HoweverIn this study, we estimate the costs of implementing HIVST through CSO for KP in Côte d'Ivoire, Senegal, and Mali. We also assess the costs of scaling up this model to guide project national scale-up, propose costed operational plans, and inform on the sustainability of this distribution model.N = 7), Senegal (N = 11), and Mali (N = 5) from July 2019 to September 2020. Implementing partners' key characteristics are presented in development phase (June 2018–March 2019): all activities that identify sustainable distribution models for each country, to fully integrate HIVST into existing programmes; (2) start-up phase : adaptation of self-testing information materials to the local context, development of training manuals, training of HIVST providers, sensitisation of key actors and building partnerships with local partners (regardless of when the costs were incurred), and other start-up costs; and 3) early implementation phase (up to September 2020): demand creation, HIVST kits distribution, and project supervision  to capture the impact of not discounting or using a higher local central bank discount rate such as in Mali . We evalWe also modelled costs at scale-up when HIVST kit distribution volumes would increase following each country's National Strategic Plan for HIV testing to predict the variation of average cost between the implementation and scale-up phases. The production function, developed by Cobb and Douglas, describes the relationship between outputs and factors of productions (inputs) . AccountWhere:C: Total costj: inputs differentiating intervention levels—international, national, district, and communityFCj: Fixed cost (independent of Sj) for fixed input j VCj: Variable cost for input j UCj: Unit cost per variable inputs j for one output (the type of unit depends of each category): new staff to train, HIVST kits to distribute, etc.Sj: Scale variable for input j to reach desired number of outputs: number of new providers required for scale-up, total number of providers at scale-up, number of HIVST kits to distribute.scenario 1). IPO's goal to progressively disengage to promote local project ownership overtime was considered. Note that we still account for 15% of international costs in 2023 because we assume another coordination component will still exist (and incur costs) within the local health system at central level. Year 2023 would then represent what it costs for the country to support HIVST post-ATLAS (scenario 2). We also assessed the impact of optimising delivery channels by simplifying the model of partners/sub-partners and decreased CSO headquarter costs by 20%, which is reasonable to assume when evaluating interventions transitioning from pilot (ATLAS) to routine implementation phase (scenario 3) (scenario 4). Finally, we combined all scenarios above to assess the global impact on average costs at scale per KP and scale-up year.In anticipation of planned project scale-up by respective country ministries of health and post-ATLAS transition, we conducted a series of scenario analyses varying some of the key model parameters by country and by scale-up year, considering 2021 as a transition year, 2022 partial scale-up, and 2023 as full scale-up. Four potential scenarios are presented in nario 3) . FinallyThis study was approved by the London School of Hygiene and Tropical Medicine  WHO Ethic Research Committee , and by three national ethic committees: Comité National d'Ethique des Sciences de la vie et de la Santé de Côte d'Ivoire , Comité National d'Ethique pour la Recherche en santé du Sénégal , and Comité d'Ethique de la Faculté de Médecine de Pharmacie et d'Odonto-Stomatologie de l'Université des Sciences et des Techniques de Bamako au Mali .N = 33,647) were distributed to FSW, 26%  to MSM, and 8%  to PWUD. In Senegal, 64% of kits  were distributed to FSW, 31%  to MSM, and 5% (N = 662) to PWUD. In Mali, 80% of kits  were distributed to FSW, and 20%  to MSM.During the costing period, 51,028, 14,472, and 34,353 HIVST kits were distributed in Côte d'Ivoire, Senegal, and Mali through a total of 161, 48, and 191 peer educators, respectively. These volumes corresponded to 68% (Côte d'Ivoire), 103% , and 42%  of planned targets. The average number of HIVST kits distributed was 7,290  across 7 CSO in Côte d'Ivoire, 3,618  across the main four models composed of 11 CSO in Senegal , and 6,871  across 5 CSO in Mali. In Côte d'Ivoire, 66% of kits  compared to the one in Senegal and Mali (3 months), start-up costs as percentage of total costs were higher in Côte d'Ivoire.Wide variations of average costs per HIVST kit distributed were found between CSO . In CôteThe major driver of these cost differences both between and within key groups for all countries was the number of kits distributed per dispensing agent, except in Côte d'Ivoire where the average number of kits distributed per dispensing agent was comparable between groups. Another important driver of cost variation between and within groups for all countries was the total number of HIVST kits distributed by a CSO. An increase of any of these two drivers would lead to a reduction in average costs.1a), Senegal (1b), and Mali (1c). Our unit costs per HIVST kit distributed remained robust when key cost parameters were varied. In Côte d'Ivoire, varying life of start-up sensitisation and training between 1 and 3 years had the strongest effect on costs ranging between $12–$17, $14–$19, and $14–$20 for FSW, MSM and PWUD, respectively. The life year of development and start-up phases, allocation factor swapping (for FSW and MSM) had a moderate effect with less than a dollar variation. The variation of discount rate almost had no effect on costs. In Senegal, the discount rate applied had the strongest effect with average costs varying between $17–$19, $26–$30, and $141–$163 for FSW, MSM, and PWUD, respectively due to higher proportion of capital costs compared to Côte d'Ivoire. Allocation factor swapping from trained distributors had an effect on average costs for PWUD (reduction to $127), while swapping from time-motion study results had no effect. In Mali, swapping of allocation factors has the strongest effect, but overall, average costs only varied by <2 dollars suggesting our average costs were quite robust.Reaching HIVST distribution targets greatly reduced costs , and $14 (PWUD) in Côte d'Ivoire; $13 (FSW), $23 (MSM) and $50 (PWUD) in Senegal; and $10 (FSW), and $17 (MSM) in Mali. We note the significant reduction of average costs at scale-up vs. observed average costs for FSW and MSM in Côte d'Ivoire, PWUD in Senegal, and all groups in Mali. Across countries, years, and key groups, the trend is an overall increase in total costs as expected. Although we estimate variation between countries and key groups, in transition and scale-up, overall cost drivers are fixed costs such as sensitisation activities, and headquarter-based personnel costs at national and sub-national level, and variable costs such as training and HIVST kits costs (varying with HIVST distribution targets). In Senegal, we estimate higher personnel costs at CSO level (headquarter- and field-based).As the scale-up model does not account for other contextual factors related to the transition post-ATLAS, analyses of plausible scale-up scenario are presented in For all countries and key groups, we find that HIVST volumes are the major determinants of costs per HIVST kit distributed , followed by IPO withdrawal starting in 2022, reduction of implementers' central costs, and the estimated reduction of HIVST kit price. Accounting for all these factors together would increase estimated scale-up average costs between $9 (FSW−2023) and $18 (PWUD−2021) in Côte d'Ivoire, from $12 (FSW−2023) to $65 (PWUD−2021) in Senegal, and from $9 (FSW−2023) to $21 (MSM−2021) in Mali.Coup d'Etat in August 2020, and ongoing armed conflict with intermittent suspension of fieldwork activities. Indeed, estimated average costs per kit would be as low as $7 (FSW) and $8 (MSM) assuming targets were reached in Mali. Finally, the COVID-19 pandemic also led to reduced  or suspended  activities during 2–3 months, leading to high observed costs, although self-testing was shown to be a timely alternative to provider-delivered HIV testing during periods of lockdown and reduced social interactions , and CSO had to suspend their field activities for security reasons, contributing to an unstable, and therefore costly, delivery system of kits for this group. In Mali, there were safety concerns due to the country's ractions .Important average costs variations between CSO were observed. High number of kits distributed per dispensing agent led to a reduction in average costs and depended on the type of HIVST distribution activity with high distribution in bars and brothels, and low distribution in small gatherings at KP's house. CSO-specific policy with monthly maximum targets of kits distribution per agent could potentially lead to higher average costs. Small number of HIVST kits distributed per CSO was also driving average costs high and was explained by the type of population reached , and the CSO size. To a lesser extent in Mali, numerous HIVST delivery models per CSO (some not presented here such as Index and STI services) could lead to higher spreading of central costs across models, and therefore, a reduction of average costs.HIV Self-Testing AfRica) project  for FSW and MSM at $16 and $21, respectively , 63% (MSM), and 10% (PWUD) of average cost reduction in Côte d'Ivoire, 19% (FSW), 12% (MSM), and 66% (PWUD) in Senegal, and 35% (FSW), 41% (MSM) in Mali. Beyond scale economies, other contextual factors were considered, such as accounting for progressive integration of the ATLAS project to existing CSO and withdrawal of the IPO. The scenario analysis suggests that, overall, even if target were not reached, costs at scale would decrease in Côte d'Ivoire (except PWUD) and Mali. However, results are more nuanced for Senegal with constant (FSW) or increasing average costs  due to high fixed costs at sub-national level.Our study has several limitations. First, our outcome metric “per HIVST kit distributed” does not fully capture the HIVST cascade. For example, there remain uncertainties related to the true percentage of kits use, the actual final users of the kit , and among those with a reactive HIVST the linkage rate to confirmatory testing. However, there is now large evidence on high acceptability of HIVST kits in the general population and among KP , 44–47. In three countries of West Africa, HIVST kit provision to KP through CSO had higher initial costs during the study period, related to the progressive integration of HIVST to CSO activities, and a challenging implementing environment . The analysis of costs at scale suggests that, in transition to scale-up and further integration of the ATLAS project, this model shows large potential for substantial economies of scale as programmes scale-up and mature.Recent modelling studies in Cameroon, Senegal, Côte d'Ivoire, and South Africa show that key populations and their sexual partners, particularly FSW and their clients, can play an important role in HIV transmission in both low and high HIV prevalence settings due to prevention gaps , 4, 48. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the London School of Hygiene and Tropical Medicine Research Committee  WHO Ethic Research Committee , and by three national ethic committees: Comité National d'Ethique des Sciences de la vie et de la Santé de Côte d'Ivoire , Comité National d'Ethique pour la Recherche en santé du Sénégal , and Comité d'Ethique de la Faculté de Médecine de Pharmacie et d'Odonto-Stomatologie de l'Université des Sciences et des Techniques de Bamako au Mali . The patients/participants provided their written informed consent to participate in this study.Amani Elvis Georges ; Badiane Kéba ; Bayac Céline ; Bekelynck Anne ; Boily Marie-Claude ; Boye Sokhna ; Breton Guillaume ; d'Elbée Marc ; Desclaux Alice , Montpellier, France/CRCF, Dakar, Sénégal); Desgrées du Loû Annabel ; Diop Papa Moussa ; Ehui Eboi ; Jean Kévin ; Keita Abdelaye ; Kouassi Kra Arsène ; Ky-Zerbo Odette ; Larmarange Joseph ; Maheu-Giroux Mathieu ; Moh Raoul ; Mosso Rosine ; Ndour Cheikh Tidiane ; Paltiel David ; Pourette Dolorès ; Rouveau Nicolas ; Silhol Romain ; Simo Fotso Arlette ; Terris-Prestholt Fern ; Traore Métogara Mohamed .Diallo Sanata ; Doumenc ; Geoffroy Olivier ; Kabemba Odé Kanku ; Vautier Anthony .Abokon Armand ; Anoma Camille ; Diokouri Annie ; Kouame Blaise ; Kouakou Venance ; Koffi Odette ; Kpolo Alain ; Tety Josiane ; Traore Yacouba .Bagendabanga Jules ; Berthé Djelika ; Diakite Daouda ; Diakité Mahamadou ; Diallo Youssouf (CSLS/MSHP); Daouda Minta (Comité scientifique VIH); Hessou Septime ; Kanambaye Saidou ; Kanoute Abdul Karim ; Keita Dembele Bintou ; Koné Dramane ; Koné Mariam ; Maiga Almoustapha ; Saran Keita Aminata ; Sidibé Fadiala ; Tall Madani ; Yattassaye Camara Adam ; Sanogo Abdoulaye .Bâ Idrissa ; Diallo Papa Amadou Niang ; Fall Fatou ; Guèye NDèye Fatou NGom ; Ndiaye Sidy Mokhtar ; Niang Alassane Moussa ; Samba Oumar ; Thiam Safiatou ; Turpin Nguissali M.E. .Bouaré Seydou ; Camara Cheick Sidi ; Kouadio Brou Alexis ; Sarrassat Sophie ; Sow Souleyman .Md'E and FT-P designed the study. Md'E coordinated, conducted data analysis, and wrote the paper. MT and KB conducted data collection/analysis. AV, AF, OK, NR, PG-F, MM-G, M-CB, GM, and JL provided logistical support and intellectual inputs. All authors revised the manuscript and agreed for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Perhabdovirus perca. However, it was distinct enough from these viruses to form a separate genetic lineage. Multiple substitutions along the genome caused non-detection using a range of conventional PCRs previously shown to target four known genogroups of perhabdoviruses. However, various generic PCRs efficiently detected the isolated virus. The origin of this virus remains to be elucidated. It may have been introduced into the farm via wild genitors. This finding provides new evidence of the high genetic diversity of percid perhabdoviruses and the potential of new genotypes to emerge as threats for fish farming. Efforts to improve the existing diagnostic methods and control this large group of viruses are still needed.A perhabdovirus was isolated from a mortality episode affecting a fish farm in 2019 in Western Europe. This virus was produced in cell culture and was readily detected by a species-specific real-time PCR assay. The near-complete sequence of the virus obtained showed some relatedness with viruses of the species  Rhabdoviridae) have caused losses of percid (perch and pike-perch) fry, juveniles and adults in European countries, mostly on farms and in experimental facilities [Over the past four decades, a group of perhabdoviruses , polymerase-associated phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). For some viruses, additional small open reading frames (ORFs) are encoded, but there are no experimental data illustrating their expression or role in the molecular biology of the virus. The genus Perhabdovirus includes four viral species recognized by the International Committee on Taxonomy of Viruses (ICTV): Perhabdovirus anguilla represented by viruses infecting eels only , Perhabdovirus perca (formerly Perch perhabdovirus) and Perhabdovirus trutta (formerly Sea trout perhabdovirus), both represented by diverse viruses infecting percids as well as a few other fish species , and Perhabdovirus leman (including Leman rhabdovirus (LeRV)), described as virus 18-193 [Perhabdovirus perca. A second cluster includes members of Perhabdovirus trutta. A third cluster is composed of a single virus (LeRV) representing Perhabdovirus leman. A fourth cluster includes a pair of viruses from France (18-203 and 18-206) that were initially thought to represent a new species considering the limited levels of nucleic acid identities of the concatenated genes with the closest virus R6146 (80%). However, considering the new rules of demarcation of ICTV for perhabdoviruses, viruses 18-203 and 18-206 should be classified as members of the Perhabdovirus trutta species since the levels of identity of their amino acid sequences with some members of this taxon are superior to 90%.The s 18-193 . GenomicConsidering this genetic diversity, four sets of primers have previously been designed, each one able to amplify a specific portion of the N gene of all the members of a given genogroup . AlthougRecently, accurate genotyping methods have proved powerful for tracing some of the outbreaks of percid perhabdoviruses in Europe . NeverthPerhabdovirus perca species.In 2019, an outbreak occurred in perch larvae on a farm in Western Europe, reaching a mortality rate of nearly 30%. From sick fish, we isolated a perhabdovirus and obtained a near-complete sequence, which demonstrated the presence of a new genotype hitherto undescribed belonging to the Aeromonas sp., Pseudomonas fluorescens and P. alcaligenes in the kidney, as well as Aeromonas sp., P. fluorescens and Providencia stuartii in the brain. No flavobacteriia were detected and no parasites were observed either in the gills, the skin, the fins or in the intestine. In December 2019, perch juveniles maintained in tanks in freshwater were affected by morbidities and mortalities. The mortality rate was 0.3 to 1.5% daily, reaching about 30% at the end of the episode. A batch of 50 individuals was examined. All fishes had open gills and about 50% exhibited spinal distortion . AbnormaSeven days after inoculation of two cell lines with the supernatant of 10 fish ground together, a cytopathic effect typical of that induced by perhabdoviruses was observed. The cell layer was progressively destructed. After a second passage of the supernatant of these, a strong ECP appeared only 24 h after inoculation. In another assay, after freezing at −80 °C for long-term storage, a second passage led to an ECP as soon as 4.5 dpi with a complete destruction of the cells at 5.5 dpi . ConsidePerhabdovirus perca species produced signals on both nucleic acid extracts, with Ct values between 20.7 and 27.3 from cell culture supernatants and organ extracts, respectively . Furthermore, in 2021, five healthy individual juveniles, from the progeny of genitors originating from the farm, were also tested negative by real-time RT-PCR. Therefore, healthy fish from two different generations were negative for this virus.To genotype the virus, a range of conventional RT-PCR assays targeting the nucleoprotein genes of viruses from the four known genogroups was performed as previously described. Surprisingly, no signal was produced with any of the four RT-PCR assays, suggesting the virus 20-43 was relatively different from the known perhabdoviruses (not shown). However, an experimental generic cPCR targeting conserved portions of the 3′ end of the percid perhabdoviruses and a fragment of the N ORF produced a clear signal of the expected size . A seconPerhabdovirus members. These numerous mismatches explained the absence of amplification signals for all the cRT-PCR primers. However, there were also some mismatches between the N ORF and the oligonucleotides used for real-time RT-PCR: two to three for the primers, and two for the TaqMan probe. It was, therefore, surprising that a signal was produced using real-time RT-PCR, although the maximum fluorescence level of the amplification curve was lower than the one of a positive control perfectly adapted to the probe and primers.To understand why the genome of virus 20-43 was not detected using cRT-PCR with any of the four sets of genogroup-specific primers, the primer sequences were aligned with the sequence of the N ORF newly obtained from virus 20-43. All primers exhibited several mismatches with their homologous sequence in virus 20-43. For instance, there were a total of six mismatches between the N ORF of virus 20-43 and primers oPVP546 and oPVP547 targeting Perhabdovirus perca species and about 150 nt shorter than those of viruses R6146, 18-193 and 18-203.By NGS sequencing the present viral isolate 20-43, a sequence of 11,419 nt was obtained. This sequence did not contain the extremities of the genome but encompassed all the complete virus genes. As previously mentioned, a RT-cPCR assay targeting the 3′ end of the genome led to the production of an amplicon, which once sequenced, added 15 nt to the genomic sequence extending it to 11,434 nt. In comparison with the ends of the genomes of the most related viruses, it was speculated that 3 nt (possibly CGT) were missing at the 5′ end of perhabdovirus 20-43, as well as 20 nt at the 3′ end, leading to an estimated size of 11,457 nt for virus 20-43. If correct, this size is similar to those of the three sequenced members of the Perhabdovirus perca 16-65, 4890 and Perch rhabdovirus (960 nt). However, the P ORF was much shorter than its homologues from more distant viruses of the genus, for instance R6146 (993 nt). The M ORF had exactly the same size (657 nt) as viruses P8350 and 16-65, but it was slightly different from the corresponding ORF of other viruses such as LeRV (660 nt) and R6146 (642 nt). The length of the L ORF was strictly similar to that of the most related viruses from percids.The genetic organization of virus 20-43 was typical of perhabdoviruses, with the five canonical genes N-M-P-G-L . No addiPerhabdovirus. It shared 69 to 81% of its nucleic acid identity with viruses from the genus Perhabdovirus, excluding EVEX which was more distant . The four recognized species of the Perhabdovirus genus were within a clade distinct to another clade grouping rhabdoviruses from dolphin, tortoise or various fish species , we consider virus 20-43 as a member of Perhabdovirus perca, which is the most represented species in the outbreaks over the past decades. However, perhabdovirus 20-43 seems distinct enough to represent a new viral lineage within the species. A virus related to perhabdoviruses was isolated from perch juveniles affected by a mortality event on a European fish farm at the end of year 2019. This virus was replicated in cell culture and was readily detected using a set of generic cRT-PCR assays and a species-specific real-time RT-PCR assay. However, the virus was not detected using more specific assays targeting the four known genogroups of percid perhabdoviruses. This absence of detection was The origin of perhabdovirus 20-43 is unknown, as is its mode of introduction into the farm. The farm is supplied with water from a spring, which cannot have been the source of the virus. Interestingly, several months before this episode, another mortality event affecting perch occurred. It happened just after connecting the water circulation system of tanks containing a few genitors captured in the wild to tanks containing fish from the farm. These latter fish were soon affected by morbidity and mortality. Unfortunately, no analysis was performed on the dead fish from this first episode. Hence, there is a possibility that the wild fish were carriers of the virus and transmitted it horizontally to the perch produced in the farm via the water or fomites.Regarding the mortality observed in December 2019 in this Western European fish farm, it affected fish just after their transfer to new tanks to start a new growing phase. Diverse bacteria were found in the dead fish. However, they are generally considered as opportunistic and, therefore, likely not responsible of the mortality. A hypothesis is that virus 20-43 was at least partly responsible for the disease. The larvae were perhaps already infected with the virus, albeit without any associated pathology. The manifestation of the infection may have been triggered by either a change in rearing conditions that subjected the fish to stress, therefore, making them more vulnerable, or, less likely, a sudden evolution of the virulence of this virus. From other episodes on other farms in Europe, it is known that the weaning of larvae and a change in the feeding regime induces stress that can trigger viral replication. Generally, the pathology of percid perhabdoviruses has been poorly studied and it is not clear why some members cause massive mortalities to larvae and others cause only morbidity to juveniles and adults. More research is needed to understand the virulence mechanisms of this viral genus. A comprehensive overview of the diversity of the perhabdoviruses is needed to prevent their spread across Europe. The present genetic data as well as amplification methods can contribute to the rapid and accurate identification of any new isolate in order to compare it with known isolates, which is crucial for tracing outbreaks. Due to the general lack of active surveillance in Europe, it is likely that known, and yet unknown, genotypes and species will emerge in the next decade . FurtherPerca fluviatilis juveniles  maintained in freshwater at 16–18 °C. The supernatant of 10 fish ground together was used to inoculate two cultured cell lines, BF2 and RTG2 , as already described [Samples were isolated in December 2019 from symptomatic escribed . SeveralPerhabdovirus perca species  with minor modifications as previously described . The pre species . The paiFurther molecular tests were performed using a range of RT-conventional PCRs (RT-cPCRs) combining generic and strain-specific primers . For allAfter RT-PCR, 10 μl of these reactions was loaded on a 2% agarose E-Gel (Invitrogen) and migrated 15 min before observation under UV light. Before sequencing, PCR products were purified using a NucleoSpin PCR clean-up kit (Macherey-Nagel) and subsequently, TA-cloned in a PCR4-TOPO vector (Invitrogen). For each amplicon, three clones were sequenced in both orientations with universal primers using the Sanger method and a 3130 Genetic Analyzer (Applied Biosystems).To obtain the near-complete genome of the virus, next-generation sequencing (NGS) was performed using Illumina sequencing technology as previously described ,13. For DNA sequences obtained from Sanger sequencing were assembled and the consensus sequence edited using VectorNTI11.5 (Invitrogen). The identities levels were obtained by aligning the sequences using the ‘align’ function of VectorNTI and the identity matrix was constructed using the RStudio software. For the phylogenetic analyses, the sequences were aligned with the ClustalW function of MEGA7 or MEGA10 ,15. Maxi


Alcohol measurement in health care settings is an effective intervention for reducing alcohol-related harm. However, in many countries, costs related to alcohol measurement have not yet been transparently assessed, which may hinder its adoption and implementation. Costs of an alcohol measurement programme in three upper-middle-income Latin American countries were assessed via questionnaires and compared, as part of the quasi-experimental SCALA study. Additional to the intervention costs, the costs of three implementation strategies: standard training and clinical package, intensive training and clinical package, and community support, were assessed and subsequently translated into costs per additional alcohol measurement session. Results demonstrated that costs for one alcohol measurement session ranged between Int$ 0.67 and Int$ 1.23 in Colombia, Int$ 1.19 and Int$ 2.57 in Mexico, and Int$ 1.11 and Int$ 2.14 in Peru. Costs were mainly driven by the salaries of the health professionals. Implementation strategies costs per additional alcohol measurement session ranged between Int$ 1.24 and Int$ 6.17. In all three countries, standard training and a clinical package may be a promising implementation strategy with a relatively low cost per additional alcohol measurement session.  Alcohol use is one of the leading preventable risk factors for health and social harms, causing an estimated three million deaths worldwide each year . More thThe World Health Organization’s (WHO) SAFER alcohol control initiative entails five cost-effective strategies to combat alcohol-related harm . One of Substantial evidence indicates that alcohol measurement in health care settings is an (cost-) effective strategy to prevent and manage alcohol-related health harm ,13. HoweOnly a few studies have estimated the costs of alcohol measurement programmes, with some substantial differences among various contexts ,17. A reBased on this, the current paper aims to provide an assessment and comparison of the following costs in three upper-middle-income Latin American countries, namely Colombia, Mexico, and Peru: (1) consultation costs of one alcohol measurement session in primary health care (PHC) settings; and (2) programme costs of three implementation strategies (specified in the next section), including the costs per additional alcohol measurement session.The costs associated with the set-up and implementation of an alcohol measurement programme were collected as part of the “Scale-up of Prevention and Management of Alcohol Use Disorders and Comorbid Depression in Latin America” (SCALA) study . SCALA iwww.scalaproject.eu, accessed on 25 October 2021). No other support materials and activities were offered. Participants in arm 2 received training (consisting of one session) before the implementation, in which they were trained to deliver alcohol measurement and subsequent interventions using the same pathway as in arm 1. Additionally, a booster session of one hour was planned to be given to providers; however, this could not be implemented in all PHCCs, due to the COVID-19 lockdown, and was therefore not included in the current costs assessment. The implementation strategies see  were exePHCCs in arm 3  received the same training and clinical package materials as in arm 2, and community support aimed at helping the adoption and implementation of the intervention. The community support consisted, among others, of regular performance feedback and support given by the project team to the providers during every implementation month see . Communiwww.scalaproject.eu, accessed on 25 October 2021). In arm 4 , PHCCs received the same community support as in arm 3, along with more intensive training and clinical package than arms 2 and 3. The more intensive training consisted of one additional training session of 2 hours in Mexico and Peru, and in Colombia of 30 additional minutes added to the main session, compared to the standard training (arm 3). The more intensive clinical package consisted of administrating the full AUDIT questionnaire (10 items) during the alcohol measurement sessions rather than AUDIT-C  gave information about the providers’ salaries. Providers  gave information about how much time they spent on average delivering alcohol measurement, brief advice, and referral to treatment. For costs valuation, the local prices and costs were converted to International Dollars (Int$), using the purchasing power parity (PPP) exchange rates , to allow for easier inter-country comparison of costs [The measuring of cost units was conducted through three main sources: the local research team, the PHCC managers, and the participating providers. The local research team received a question list to specify the time and costs spent on various activities. The PHCC managers . The identified set-up costs included coordinating PHCCs’ and providers’ participation in the SCALA intervention. Adaptation costs included the costs of adjusting and tailoring the clinical package materials to the local contexts. In SCALA this was conducted in each country with two user panels: one with a group of 10 health care providers and one with 10 patients. Identified costs included user panel coordination, transportation, food and refreshments, printing materials, moderator salary, technical equipment, and materials adaptation coordination. Research-related costs, e.g., the coordination of data collection and survey completion, were not included in the general start-up and adaptation costs, as these do not apply to the actual implementation of the intervention. The costs of the three implementation strategies were identified and assessed as follows. For both (i) standard training and clinical package and (ii) intensive training and clinical package, cost units included coordination of the training, transportation for participants and/or organisers, food and refreshments, training materials, technical equipment, trainer salary, and printing clinical package materials. For community support, the identified cost units include coordination of CAB meetings, food and refreshments, materials, venue rent, transportation, and coordination and implementation of supportive actions. These costs were measured through questionnaires filled in by the three local research teams. The costs of coordinating and/or delivering the abovementioned activities  were assessed by multiplying the average hourly wage of the implementers with the time spent by them in preparing and/or delivering each activity. The costs of printing materials were assessed by multiplying the number of double-sided pages used for a session with the costs for printing one double-sided page. Transportation and venue rent costs were assessed per activity, and food and refreshment costs were assessed per portion. The costs for each implementation strategy were divided by the number of providers participating in the respective study arm, and, subsequently, by the average number of additional alcohol measurement sessions delivered by each provider. The number of additional sessions per implementation strategy was assessed through comparison to the study arm in which the implementation strategy was not implemented, i.e., arm 2 vs. arm 1, for standard training and clinical package; arm 3 vs. arm 2 for community support; and arm 4 vs. arm 3 for intensive training and clinical package.Additionally, we estimated the costs for 10,000 alcohol measurement sessions in each SCALA arm, using the three abovementioned implementation strategies. The number of 10,000 alcohol measurement sessions was identified as a relevant cost indicator for potential policy implementation and scale-up. For these estimations, the period  that would be needed to achieve this number was calculated per SCALA arm, based on the existing alcohol measurement numbers assessed in the five months of implementation. Based on these estimated periods, the number of activities related to each implementation strategy that would have to be implemented was assessed, along with the respective costs see . This alThe statistical significance of differences between countries (within the same arm) and between arms (within the same country) was tested using confidence intervals for two variables assessed at the provider level: (1) the number of delivered alcohol measurement sessions per provider, and (2) the number of minutes spent on these sessions. Confidence intervals were calculated in SPSS 26 with the function ‘explore’, at a 95% confidence level and were compared. Statistical significance of tested differences was indicated by a lack of overlap of the compared confidence intervals. Differences in unit costs and total costs of the programme cost components  were not tested for significance. These costs were assessed at the country level and did not include sufficient variability to allow for statistical tests. In all three countries, providers spent on average between 1.6 and 4.8 min for a standard alcohol measurement session (using AUDIT-C), a brief advice session, and a referral to treatment session. Countries showed differences in the professions of providers who gave the intervention. In Colombia and Mexico, over 60% of the sessions were given by GPs, followed by nurses (in Colombia) and psychologists (in Mexico), whereas in Peru a third of the sessions were given by midwives and a third by psychologists, followed by nurses, nurses technicians, and GPs (each under 5%). The average costs of the three types of sessions , including the costs of the paper tally sheet used to apply the AUDIT-C questionnaire for the alcohol measurement, were, respectively: Int$ 1.19 , Int$ 2.57 , and Int$ 1.84  in Colombia; Int$ 0.67 , Int$ 1.62 , and Int$ 1.23  in Mexico; and Int$ 1.11 , Int$ 2.14 , and Int$ 1.45  in Peru see . The oveSet-up costs  were Int$ 2242.5 in Colombia, Int$ 1711.25 in Mexico, and Int$ 1803.10 in Peru. Adaptation costs of the clinical package materials, including two user panels in each country, were Int$ 1332.15 in Colombia, Int$ 1286.02 in Mexico, and Int$ 1308.45 in Peru  was Int$ 358.50 in Colombia, Int$ 205.35 in Mexico, and Int$ 144.25 in Peru. The higher costs in Colombia are primarily due to the larger amount of hours spent to implement the supportive actions  delivered to the participating PHCCs in the intervention municipality  standard training and clinical package, (2) intensive training and clinical package, and (3) community support, including the costs of these implementation strategies per additional alcohol measurement session. Results indicate that one of the main factors determining the consultation costs is the profession of the providers delivering the intervention. Specifically, when the intervention was largely delivered by GP’s the average salaries were higher than when the intervention was delivered more often by nurses or social workers. Another factor determining the costs is the time spent for certain components of the intervention, such as providing an alcohol measurement session, as also demonstrated in previous studies ,17. For www.scalaproject.eu, accessed on 25 October 2021). Due to the premature stop in data collection as a result of the COVID-19 pandemic, which started in all three countries by end of implementation month 5, we could not assess in more detail the further implementation of the community support. We expect community support activities to have a cumulative effect over months of implementation and in SCALA, the community support activities were designed to be implemented for a period of 18 months, prior to the COVID-19 pandemic. Thus, it could be that the stronger effects of community support in Mexico and Peru would have manifested only after the observed 5 months period, and as such, the costs per additional alcohol measurement session would be smaller than estimated in this paper. Future studies could benefit from collecting data throughout a longer implementation of community support. In addition to impeded program implementation and health care delivery, the COVID-19 pandemic has also been linked to reduced alcohol consumption [Regarding the implementation strategies assessed in the current study, some differences in unit costs in the three countries were noticed. For example, the costs of CAB meetings in Mexico were higher than in Colombia and Peru, e.g., due to larger transportation and materials costs. In addition, noticeably, the community support intervention in Colombia resulted in a higher number of additional alcohol measurement sessions, and, correspondingly, in a lower cost per additional alcohol measurement session. This may be due to a more intensive collaboration with the providers who received community support in Colombia. For example, specific barriers encountered by providers in Colombia were tackled more effectively with community support activities, e.g., through creating video tutorials responding to the specific needs of the providers , which may only pay-off in the long run. Future research may broaden perspectives and include all societal costs relevant to alcohol consumption . In addTo conclude, staff costs are an important component of the costs of an alcohol measurement programme, being largely driven by the type and proportion of the professions of providers delivering the intervention and the time spent on the sessions. Regarding the costs of implementation strategies, the standard training and clinical package is the cheapest of the three analysed implementation strategies and may lead, in all three countries, to additional patients whose alcohol consumption is measured, compared to care as usual. On the other hand, more complex and expensive implementation strategies, such as the intensive training and clinical package, or the provision of community support  may also potentially result in additional alcohol measurement sessions; however, this depends on the country where the strategy is implemented. Therefore, for optimal budgeting efforts of alcohol measurement programmes, increased attention should be paid to the local characteristics of the contexts where the intervention is implemented, thereby contributing to generating evidence for decision making in public health policies.


Bemisia tabaci species complex. However, the molecular mechanisms underlying the transmission of begomoviruses by their whitefly vector are not clear. In this study, we found that B. tabaci vesicle-associated membrane protein 2 (BtVAMP2) interacts with the coat protein (CP) of tomato yellow leaf curl virus (TYLCV), an emergent begomovirus that seriously impacts tomato production globally. After infection with TYLCV, the transcription of BtVAMP2 was increased. When the BtVAMP2 protein was blocked by feeding with a specific BtVAMP2 antibody, the quantity of TYLCV in B. tabaci whole body was significantly reduced. BtVAMP2 was found to be conserved among the B. tabaci species complex and also interacts with the CP of Sri Lankan cassava mosaic virus (SLCMV). When feeding with BtVAMP2 antibody, the acquisition quantity of SLCMV in whitefly whole body was also decreased significantly. Overall, our results demonstrate that BtVAMP2 interacts with the CP of begomoviruses and promotes their acquisition by whitefly.Begomoviruses cause substantial losses to agricultural production, especially in tropical and subtropical regions, and are exclusively transmitted by members of the whitefly  Geminiviridae) are a group of plant viruses characterized by a genome of circular single-stranded DNA encapsidated in twinned virions. These viruses are distributed in tropical and subtropical regions and cause economically important diseases to food and fiber crops worldwide [Geminiviridae is classified into nine genera of which the genus Begomovirus is the largest, including 424 species to date [Manihot esculenta Crantz, Euphorbiaceae) is a staple food of nearly a billion people in 105 countries [Geminiviruses  in a persistent circulative manner. Once acquired orally by whiteflies, begomoviruses follow the sequential path of stylet–midgut–haemolymph before entering the salivary glands; finally, viruses are delivered into plants with the secretion of saliva during the next feeding [B. tabaci is a species complex containing at least 44 cryptic species with distinct genetic structure and biological traits but indistinguishable external morphology [B. tabaci species Asia II 1 efficiently transmits SLCMV and poses a potential threat to cassava production in many regions [Begomoviruses are exclusively transmitted by the whitefly  feeding ,14. It irphology ,16. Diffrphology . Among trphology ,19,20. IB. tabaci depends on the interaction of proteins between the virus and the whitefly vector. Recently, several proteins of B. tabaci have been identified that affect the acquisition and transmission of begomoviruses [B. tabaci via clathrin-mediated endocytosis where they are encapsulated in vesicle structures [B. tabaci midgut. TYLCV CP could bind to BtCubam through the interaction with a specific structure domain of BtCUBN, to facilitate the transport across the midgut barrier via clathrin-mediated endocytosis. However, although vesicle structure is important for virus acquisition and transmission, no vesicle protein has been reported to date that takes part in begomovirus transmission.The successful transmission of begomoviruses by oviruses . The heaoviruses ,23, whiloviruses ,25,26. Pructures ,28,29. Sructures  found thB. tabaci complex [B. tabaci and begomoviruses.The coat protein (CP) of a begomovirus is the only protein that is known to be involved in its transmission by vector(s) in the  complex ,32,33,34 complex ,36. UsinGossypium hirsutum L. cv. Zhemian 1793), tobacco (Nicotiana tabacum L. cv. NC89), and tomato (Solanum lycopersicon L. cv. Hezuo903). All plants were planted in a greenhouse under natural lighting and maintained under controlled conditions of 25 ± 3 °C, 14 h light/10 h darkness.The plants used were cotton (B. tabaci MEAM1 (mtCOI GenBank accession number: GQ332577) and Asia II 1 (mtCOI GenBank accession number: DQ309077) were cultured in cages in an artificial climate chamber on cotton at 26 ± 1 °C, 60% relative humidity, and a cycle of 14 h light/10 h darkness. The purity of the whitefly population was analyzed by mtCOI PCR-RFLP and sequencing according to Qin et al. [Whitefly n et al. .An infectious clone of TYLCV-SH2 (GenBank accession: AM282874) was provided by the Institute of Biotechnology of Zhejiang University, and the construction method of infectious clones of SLCMV (GenBank accession: KT861468 for DNA-A and KT861469 for DNA-B) was as previously reported . TYLCV oB. tabaci were extracted using cytoplasmic extraction buffer . Then, anti-TYLCV CP mouse monoclonal antibody (mAb)  was added into the cytoplasmic proteins at a 1:100 (v/v) dilution and incubated overnight at 4 °C. Controls included were pre-immune serum  or proteins of uninfected whiteflies. Protein G-Sepharose beads  were subsequently added and the mixture incubated at 4 °C for 4 h. The beads were then washed five times with 1x PBS. To elute co-immunoprecipitated proteins, beads were boiled in SDS-PAGE buffer  for 10 min. Finally, the proteins were subjected to SDS-PAGE electrophoresis and the resulting proteins then processed by LC-MS/MS, or by Western blot with anti-BtVAMP2 rabbit polyclonal antibody (pAb), which was produced by GenScript  using synthetic peptides (DSGANLSTGEDGIVG) conjugated to keyhole limpet hemocyanin as antigens. Specificity of the generated anti-BtVAMP2 antibody was tested by Western blot and shown in After a 7 d acquisition on virus-infected plants, the proteins of http://www.whiteflygenomics.org, accessed on 20 January 2020).After Co-IP, Coomassie blue staining, and decoloring procedures, the protein gels were cut down and an LC-MS/MS assay was performed as described previously ,38. FinaE. coli strain BL21 for expression. The expressed proteins were combined with glutathione agarose beads  at 4 °C for 2 h. The beads were then washed five times with 1xPBS in 6 mL affinity chromatography columns . Then, the GST or GST-CP proteins were analyzed by SDS-PAGE electrophoresis and Coomassie blue staining assay after boiling in SDS-PAGE buffer for 10 min. After that, the cytoplasmic proteins of whitefly were added to the beads-bound GST or GST-CP proteins as prey proteins at 4 °C for 4 h. Next, these mixtures were centrifuged and washed with 1xPBS five times, and then the beads-bound proteins were eluted by boiling in SDS-PAGE buffer for 10 min. Finally, the proteins were separated by SDS-PAGE electrophoresis and detected by anti-BtVAMP2 antibody.TYLCV CP and SLCMV CP genes were cloned into pGEX-6p-1 for fusion with glutathione S-transferase (GST), respectively, with primers TYLCV CP-pGEX-6p-1-F (5′-CGCGGATCCATGTCGAAGCGACCAGG-3′), TYLCV CP-pGEX-6p-1-R (5′-CCGGAATTCTTAATTTGATATTGAATCATA-3′), SLCMV CP-pGEX-6p-1-F (5′-CGCGGATCCATGTCGAAGCGACCAGCAGA-3′), and SLCMV CP-pGEX-6p-1-R (5′-CCGGAATTCTTAATTGCTGACCGAATCGT-3′). The recombinant prokaryotic expression vector or no load (negative control) were transferred into B. tabaci were fed on virus-infected plants (or uninfected plants as a negative control) for 0, 24, 48, and 96 h. At each time point, ten females were collected for DNA extraction following the methods of Pan et al. [B. tabaci by quantitative real-time PCR (qPCR). The primers used for TYLCV and SLCMV quantification were TYLCV-qPCR-F (5′-GAAGCGACCAGGCGATATAA-3′), TYLCV-qPCR-R (5′-GGAACATCAGGGCTTCGATA-3′), SLCMV-qPCR-F (5′-ACGCCAGGTCTGAGGCTGTA-3′), and SLCMV-qPCR-R (5′-GTTCAACAGGCCGTGGGACA-3′). Additionally, at each time point, thirty whiteflies were collected for extraction of total RNA using TRIzol , and EVO-M-MLV reverse transcription kit II  for cDNA synthesis. Finally, qPCR was used to analyze the BtVAMP2 transcription using primers VAMP2-qPCR-F (5′-TGCTTAGCATTGGGAGTTGC-3′) and VAMP2-qPCR-R (5′-GTCTGGTGCTGCCATTCTTT-3′). For each analysis, 3–4 replicates were conducted. All qPCR detections were performed using SYBR Green Premix Pro Taq HS qPCR Kit  and the CFX96 Real-time PCR Detection System . β-actin of B. tabaci (β-actin-qPCR-F 5′-TCTTCCAGCCATCCTTCTTG-3′ and β-actin-qPCR-R 5′-CGGTGATTTCCTTCTGCATT-3′) was used as the reference gene for transcript and virus quantification.n et al.  to quantv/v) dilution. At the same time, rabbit pre-immune serum (diluted to 0.713 mg/mL) was added into 15% sucrose as a negative control. Whiteflies were left to feed on the antibody and control solutions for 24 h.The whitefly adults were collected and released into glass tubes of which one end was covered with a double layer of parafilm filled with antibody and the other end with a layer of gauze . BtVAMP2B. tabaci were transferred to infected plants for virus acquisition. After 48 or 96 h, 10 females were collected per sample for DNA extraction and virus quantification as described above.After feeding antibody or pre-immune serum for 24 h, After 24 h antibody feeding and 48 h TYLCV acquisition, 4  MEAM1 adults were transferred in a clip-cage  onto a 2−ΔCT as normalized to whitefly β-actin. For comparing the effect of feeding anti-BtVAMP2 antibody on virus acquisition, the quantity of virus in the whole body of whiteflies feeding with pre-immune serum was set to 1. Statistical analyses were conducted using IBM SPSS Statistics 20 software , and all data were analyzed by independent t-test . For the comparison of transmission efficiency, percentage data were arcsine square root transformed for statistical analysis, and the data presented in the figure are raw data.All qPCR data were calculated using 2Previous studies have shown that, during transportation in their vectors, begomoviruses were enclosed in vesicles ,30. To iNext, we conducted Co-IP, GST pull-down assays to examine further the interaction between BtVAMP2 and TYLCV CP. Co-IP assay showed BtVAMP2 interacted with TYLCV CP in vivo A, and GSBtVAMP2 in the whole body were detected by qPCR. According to the results, with the increase of TYLCV quantity in whiteflies  proteins play an indispensable role in the final membrane fusion steps in eukaryotic membrane traffic and autophagy ,40,41,42nfection , which cTo summarize, we have identified an interaction of VAMP2 with the CP of begomoviruses that promotes their acquisition by whitefly. Thus, our findings present a new role of the protein VAMP2 and may facilitate further studies of the numerous other whitefly–begomovirus interactions that lead to devastating crop yield losses globally.


N-isopropylacrylamide) (PNIPAAm) brush-modified silica beads and poly(3-acrylamidopropyl trimethylammonium chloride) (PAPTAC) brush-modified silica beads. PNIPAAm brush-modified silica beads and PAPTAC brush-modified silica beads were prepared by atom transfer radical polymerization. The beads were then packed into a stainless-steel column in arbitrary compositions. The elution studies evaluated the column performance on hydrophobic, electrostatic, and therapeutic drug samples using steroids, adenosine nucleotide, and antiepileptic drugs as analytes, respectively. Steroids exhibited an increased retention time when the column temperature was increased. The retention of adenosine nucleotides increased with the increasing composition of the PAPTAC-modified beads in the column. The antiepileptic drugs were separated using the prepared mixed-mode columns. An effective separation of antiepileptic drugs was observed on a 10:1 PNIPAAm:PAPTAC column because the balance between the hydrophobic and electrostatic interactions with antiepileptic drugs was optimized for the bead composition. Oligonucleotides were also separated using mixed-mode columns through multiple hydrophobic and electrostatic interactions. These results demonstrate that the developed mixed-mode column can modulate multiple hydrophobic and electrostatic interactions by changing the column temperature and composition of the packed PNIPAAm and PAPTAC beads.In this study, mixed-mode chromatography columns have been investigated using multiple analyte interactions. A mixed-mode chromatography column was developed using poly( For example, reversed-phase chromatography uses a hydrophobic group to separate hydrophobic compounds , and a modified stationary phase aids in retaining the compounds through hydrophobic interactions3. Ion-exchange chromatography employs an ionic-group-modified stationary phase to retain the compounds through electrostatic interactions and to separate the ionic compounds4. These hydrophobic or electrostatic interactions are mainly modulated by changing the composition of the mobile phase, such as the addition of an organic solvent and electrolytes into the mobile phase, respectively.Chromatography is widely used for the separation and purification of compounds. Various types of chromatography, such as reverse-phase and ion-exchange chromatography, have been investigated depending on the properties of the feed compounds7. Poly(N-isopropylacrylamide) (PNIPAAm) is a thermoresponsive polymer that exhibits temperature-dependent hydrophilic and hydrophobic attributes across a phase transition temperature of 32 °C10. Owing to its thermoresponsive properties, PNIPAAm has been used in various applications, such as drug and gene delivery systems16, biosensor and bioimaging systems22, nano-actuators27, cell separation systems34, and cell culture substrates40. In a previous study, a PNIPAAm-modified stationary phase was used in a chromatography system because of its hydrophobicity, which changes with temperature45. The hydrophobic interaction between the stationary phase and analytes can be modulated by changing the column temperature.Temperature-responsive chromatography has been investigated as a new type of chromatography because the properties of the stationary phase can be modulated by changing the temperature using an aqueous isocratic mobile phase48. Chromatography can separate ionic compounds through electrostatic interactions between thermoresponsive ionic polymers and analytes. The electrostatic interactions with analytes can be modulated by changing the composition of the ionic monomer in the PNIPAAm copolymer. However, an excessive incorporation of the ionic monomers into the PNIPAAm copolymer reduces the thermoresponsive properties of the PNIPAAm copolymer50. Thus, electrostatic solid interactions cannot be utilized in thermoresponsive ion-exchange chromatography.To separate ionic compounds using temperature-responsive chromatography, temperature-responsive ion-exchange chromatography has been investigated using thermoresponsive ionic copolymers prepared by the copolymerization of NIPAAm and ionic monomers51. A mixed-mode chromatography column was developed by packing two types of beads in the same column, namely, hydrophobic group immobilized silica beads and ionic group immobilized silica beads. Analyte interacted with the packed beads through multiple hydrophobic and electrostatic interactions, and the interactions can be modulated by changing the beads composition. If the concept of a mixed mode chromatography is applied to temperature-responsive chromatography, effective separation could be performed through temperature-modulated multiple interactions.By contrast, mixed-mode chromatography columns have been investigated for separating analytes with multiple interactionsIn this study, we developed a temperature-responsive mixed-mode chromatography column using PNIPAAm brush-modified beads and a strong cationic polymer poly(3-acrylamidopropyl) trimethylammonium chloride (PAPTAC) brush-modified beads Fig. . The per52.PNIPAAm and PAPTAC brush-modified silica beads were prepared by a silane coupling reaction and subsequent atom transfer radical polymerization (ATRP) of NIPAAm and APTAC Fig. A. In thiThe prepared beads were characterized by using the carbon, hydrogen, and nitrogen (CHN) elemental analysis, attenuated total reflection Fourier-transform infrared spectroscopy (ATR/FT–IR), zeta potential observation, and scanning electron microscopy (SEM).2. This value is relatively small compared to the beads solely modified with CPTMS, as previously reported (~ 5.0 μmol/m2)54. This outcome may be due to the molecular size of GPTMS, which is slightly larger than that of CPTMS, thus lowering the immobilized density of the silane layer.The silica beads were characterized by elemental analysis at each reaction step Table . A large2 and 1.43 μmol/m2, respectively. These values indicated that a relatively large polymer was modified on the silica beads through the ATRP. A larger amount of PNIPAAm was modified on silica beads compared with PAPTAC due to the higher concentration of NIPAAm monomer compared with that of APTAC in the ATRP. Previous reports indicate that the amount of modified polymer on silica beads increased with increasing monomer concentration in ATRP56. In the present study, the monomer concentrations of NIPAAm and APTAC were 1000 mM and 300 mM, respectively. The difference in monomer concentration significantly influenced the quantity of modified polymer on the silica beads.The PNIPAAm- and PAPTAC-brush-modified silica beads exhibited a higher carbon composition than the silane-layer-modified silica beads. This result indicated that PNIPAAm and PAPTAC successfully modified the silica beads through ATRP under the studied reaction conditions. The modified PNIPAAm and PAPTAC were 3.21 μmol/m−1 in the spectrum. By contrast, no peaks were observed in the spectrum of unmodified silica beads and silane-layer-modified beads. The results also indicated that PNIPAAm and PAPTAC were successfully modified on silica beads because the two peaks at 1550 and 1645 cm−1 were attributed to the amide bonds of PNIPAAm and PAPTAC.The prepared beads were characterized by observing the spectrum of ATR/FT–IR Fig. A. The PNThe zeta potentials of the prepared beads at each reaction step were investigated Fig. B. PAPTACThe SEM micrographs of the prepared beads at each reaction step were performed Fig. . SimilarA mixed solvent composed of water:methanol:2-propanol (1:1:1 ratio) was prepared as a suspension of the column packing. The prepared PNIPAAm- and PAPTAC-brush-modified silica beads were suspended in the mixed solvent at weight ratios of 1:0, 20:1, and 10:1. The bead suspension was packed into a stainless-steel column (4.6 mm inner diameter × 50 mm length). The elution behavior of various analytes in the prepared columns was observed.The elution behavior of the hydrophobic steroids 6, two types of oligonucleotides were eluted at one peak on the PNIPAAm brush-modified beads packed column at all column temperatures. The results indicated that hydrophobic interactions between PNIPAAm and oligonucleotides were not adequate for the retention of oligonucleotides.The elution behavior of the oligonucleotides from the prepared mixed-mode column was observed Fig. . The elu5 and d(T)10 from the prepared columns was also observed. On the column packed with PNIPAAm-modified beads, d(T)5 and d(T)10 eluted as one peak at all temperatures because the hydrophobic interaction between PNIPAAm and oligonucleotides was not adequate for retention on the column even at relatively long base oligonucleotides. The PNIPAAm:PAPTAC = 20:1 column and PNIPAAm:PAPTAC = 10:1 column can separate a mixture of oligonucleotides with baseline separation. Longer retention times of oligonucleotides were exhibited on the PNIPAAm:PAPTAC = 10:1 column than on the PNIPAAm:PAPTAC = 20:1 column. This outcome may be due to the electrostatic interaction between PAPTAC and oligonucleotides, which increased with increasing composition of the packed PAPTAC in the column. As the PNIPAAm:PAPTAC = 10:1 column temperature increases, the elution of d(T)10 oligonucleotides decreases, as displayed by the decreased peak areas measured in the chromatogram 10, the oligonucleotides interact strongly with PAPTAC on the beads in PNIPAAm:PAPTAC = 10:1 and PNIPAAm:PAPTAC = 20:1 columns. In addition, the hydrophobic interaction between the oligonucleotides and PNIPAAm increased with increasing temperature, leading to the adsorption of oligonucleotides on the stationary phase at elevated column temperatures. Thus, our results indicate that a low temperature is suitable for the high-recovery separation of oligonucleotides with base lengths similar to d(T)10.On the contrary, on the PNIPAAm:PAPTAC = 20:1 column, the retention times of these oligonucleotides increased, and the peak tops were slightly separated. On the PNIPAAm:PAPTAC = 10:1 column, the oligonucleotides were separated. These results indicated that the electrostatic interaction between PAPTAC and oligonucleotides enhanced the retention of oligonucleotides. The PNIPAAm:PAPTAC = 10:1 column can separate oligonucleotides with different lengths of a single base, 5mer, and 6mer. The elution behavior of oligonucleotides d(T)These results demonstrate that the developed mixed-mode column can modulate multiple hydrophobic and electrostatic interactions simply by changing the column temperature and composition of the packed PNIPAAm and PAPTAC beads. The column can separate antiepileptic drugs and oligonucleotides with all aqueous mobile phases. Thus, the developed mixed-mode columns would be useful for drug monitoring in pharmaceutical therapy and the purification of oligonucleotide therapeutics.A mixed-mode temperature-responsive chromatography column was developed by preparing PNIPAAm brush-modified beads and PAPTAC brush-modified beads through ATRP and then packing these beads in arbitrary compositions. PNIPAAm and PAPTAC were successfully modified on silica beads through ATRP, confirmed by elemental analysis, FT-IR, zeta potential measurement, and SEM. The prepared beads were packed into a stainless-steel column in compositions of PNIPAAm, PNIPAAm:PAPTAC = 20:1, and PNIPAAm:PAPTAC = 10:1. The elution behavior of the steroids from the prepared mixed-mode columns was observed. The retention time of the steroids increased with increasing column temperature, indicating that the hydrophobic interaction between the analytes and PNIPAAm increased with increasing temperature, which was attributed to the dehydration of PNIPAAm. Adenosine nucleotide retention increased with increasing packed PAPTAC composition in the columns, which was attributed to electrostatic interaction between adenosine nucleotides, and PAPTAC increased with the composition of the packed PAPTAC-modified beads in the column. Antiepileptic drugs were separated by increasing the column temperature through multiple hydrophobic and electrostatic interactions. The highest separation efficiency of antiepileptic drugs was observed in the PNIPAAm:PAPTAC = 10:1 column, indicating that an optimal balance between hydrophobic and electrostatic interactions can be obtained at that composition of the beads. Oligonucleotides were separated using PNIPAAm:PAPTAC = 20:1 and PNIPAAm:PAPTAC = 10:1 columns. Further, the PNIPAAm column could not separate oligonucleotides, indicating that multiple hydrophobic and electrostatic interactions are required to separate oligonucleotides. These results indicate that the developed mixed-mode column can modulate multiple hydrophobic and electrostatic interactions by simply varying the column temperature and composition of the packed PNIPAAm and PAPTAC beads. The column can separate antiepileptic drugs and oligonucleotides with all aqueous mobile phases. Thus, the developed mixed-mode columns would be useful for drug monitoring in pharmaceutical therapy, and the purification of oligonucleotide therapeutics.50. The beads were filtered, rinsed with pure water, and dried in a vacuum drying oven  at 150 °C for 16 h, following which the silica beads (5.0 g) were humidified in 500 mL flasks (humidity = 60%) at 25 °C for 4 h61. CPTMS  and GPTMS  were dissolved in toluene (286 mL)63. The prepared reaction solution was poured into the silica beads in the flask and then the silane coupling reaction was performed at 25 °C for 16 h with continuous stirring. Then, the silica beads were filtered, rinsed with acetone, and dried at 110 °C for 3 h34.Packing materials of the temperature-responsive mixed-mode column were prepared by a silane coupling reaction for initiator modification and by the subsequent ATRP for polymer modification Fig. . All mat59. Then, CuCl , CuCl2 , and Me6TREN  were dissolved into the solution under argon gas atmosphere34. The flask was then sealed, and the solution was stirred using a magnetic stirrer at 25 °C for 10 min. The reaction solution and initiator-immobilized silica beads (1.0 g) in a 50 mL glass vessel were placed in a glove bag. The oxygen in the glove bag was removed by repeated vacuuming, and argon gas was flowed thrice59. The reaction solution was poured into the silica beads in the glass vessel, which was then sealed. ATRP was performed with continuous shaking of the bead suspension at 25 °C for 16 h using a shaker . After the ATRP, the beads were poured into two centrifuge tubes (50 mL), and acetone (20 mL) was poured into the tubes. The beads were washed by sonication for 30 min to remove the monomers, followed by centrifugation at 1000 rpm for 3 min64. The supernatant was removed, and a mixture of 50 mM EDTA aqueous solution and methanol (1:1) was added to the centrifuged tubes. The beads were washed by sonication for 30 min to remove the ATRP catalyst, followed by centrifugation at 1000 rpm for 3 min34. The bead suspensions were then filtered, rinsed with pure water and acetone, and dried at 50 °C for 3 h62.PNIPAAm modification was performed following the ATRP. PNIPAAm  was solved in 42.8 mL 2-propanol in a 100 mL flask, and the monomer solution was deoxygenated under flowing argon gas for 30 min65. Then, the same procedure was followed as described for the modification of PNIPAAm.For the PAPTAC modification on silica beads, APTAC  was dissolved in 50 mL of a 2-propanol:water (1:1) mixture solvent. The monomer solution was passed to the inhibitor remover column to remove the polymerization inhibitor in APTACThe prepared beads were characterized by CHN elemental analysis, FT-IR, zeta potential measurement, and SEM.62. The carbon composition was determined using an elemental analyzer 60. The amount of modified silane layer comprising CPTMS and GPTMS was obtained using Eq.  is the calculated carbon percentage of the mixed silane coupling reagents composed of CPTMS and GPTMS (3:1), and S is the surface area of the silica beads (130 m2/g)59. The amount of modified PNIPAAm was obtained using Eq.  is the calculated carbon percentage of NIPAAm34. The amount of modified PNIPAAm was obtained using Eq.  is the calculated carbon percentage of APTAC. The polymer modification of the beads in each reaction step was confirmed by ATR FT–IR 59.The carbon composition of the prepared beads was investigated at each reaction step to determine the amount of silane layer and polymer on the beadssing Eq.  as follond GPTMS :1, and S59. The morphology of the silica beads at each reaction step was observed using SEM 41.The zeta potential of the beads was determined using a zeta potential analyzer . Bead suspensions (0.5 mg/mL) were prepared in a 5 mmol/L potassium chloride solution for zeta potential measurements44. Solely PNIPAAm-modified beads packed column and PNIPAAm-modified beads (0.8 g) were suspended in 20 mL of a 1:1:1 mixed solvent of water:methanol:2-propanol. In the case of the PNIPAAm:PAPTAC = 20:1 column, PNIPAAm-modified beads (0.764 g) and PAPTAC-modified beads (0.0382 g) were suspended in the mixed solvent, while in that of PNIPAAm:PAPTAC = 10:1 column, PNIPAAm-modified beads (0.727 g) and PAPTAC-modified beads (0.0727 g) were suspended in the mixed solvent. The bead suspension was poured into the reservoir of the column packer that was connected to an empty column44. Beads were packed to enable a continuous flow of a mixed solvent with a continuous pressure of 350 kg/cm2 for 1 h43. Then, the column was connected to a high-performance liquid chromatography system , which was rinsed with pure water flowing at 1.0 mL/min at 40 °C for 5 h.A mixed-mode column was prepared by combining PNIPAAm and PAPTAC beads and packing them into an empty column (4.6 inner diameter × 50 mm length)59. Steroid elution from the prepared column was observed at a detection wavelength of 260 nm. The mobile phase was pure water with a flow rate of 1.0 mL/min. The column temperature was controlled using a column oven .To investigate hydrophobic interactions between the prepared stationary phase and the analyte, hydrophobic steroids were used as model analytes. The properties of the steroids are listed in Supplementary Table 59. The solution was filtered using a syringe filter. AMP (500 μL), ADP (500 μL), and ATP (1500 μL) solutions were mixed, and 2.5 mL of 66.7 mM phosphate buffer (pH = 7.0) was added to the solution59. The resultant solution was used as a sample of the adenosine nucleotides. The elution behavior of adenosine nucleotides was observed at a detection wavelength of 260 nm. The mobile phase was 33.3 mM phosphate buffer (pH = 7.0) with a flow rate of 1.0 mL/min.An adenosine nucleotide was used as a model sample to investigate the electrostatic interaction between the developed stationary phase and analytes. The properties of the adenosine nucleotides are summarized in Supplementary Table 3COONH4 solution (pH 6.8). In case of other antiepileptic drugs, the drug was dissolved in 2 mL of methanol, and 10 mM CH3COONH4 solution (pH = 6.8) was added to 5 mL of the solution. The solution was then filtered through a syringe filter, and 500 µL of it was added to 4.5 mL of 10 mM CH3COONH4 solution (pH = 6.8) to adjust the concentration to 100 µg/mL. An antiepileptic drug solution was used as the sample. The elution behavior of the drug was observed in the prepared column at a detection wavelength of 260 nm. The mobile phase was 10 mM CH3COONH4 solution (pH = 6.8) with a flow rate of 1.0 mL/min.The elution behavior of antiepileptic drugs from the prepared column was observed to investigate the availability of the prepared column for therapeutic drug monitoring because it is required by pharmaceutical therapy. The properties of antiepileptic drugs are summarized in Supplementary Table k′, was obtained using Eq.  with a flow rate of 1.0 mL/min for a mixture of d(T)5 and d(T)6 and was a mixture of 66.7 mM phosphate buffer solution (pH = 7.0) and 100 mM NaCl for a mixture of d(T)5 and d(T)10.To investigate the retention behavior on the column, van’t Hoff plots were obtained for the analytes. The retention factor, sing Eq.  as folloSupplementary Information.