Maria-Nefeli Antonopoulou

Richard Whitfield

Nghia P Truong

Dries Wyers

Simon Harrisson

Tanja Junkers

Athina Anastasaki

Concurrent control over sequence and dispersity in multiblock copolymers

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The preparation of synthetic macromolecules with ordered monomer sequences has been a long-standing challenge in polymer chemistry. [START_REF] Lutz | Sequence-controlled polymers[END_REF][START_REF] Ouchi | Single-chain technology using discrete synthetic macromolecules[END_REF][START_REF] Lutz | Defining the Field of Sequence-Controlled Polymers[END_REF][START_REF] De Neve | Sequence-definition from controlled polymerization: the next generation of materials[END_REF][START_REF] Haven | Elements of RAFT navigation: RAFT 20 years later: RAFT-synthesis of uniform, sequence-defined (co) polymers[END_REF][START_REF] Bates | Multiblock polymers: panacea or Pandora's box?[END_REF][START_REF] Parkatzidis | Recent Developments and Future Challenges in Controlled Radical Polymerization: A 2020 Update[END_REF] Although natural polymers such as DNA and proteins are precisely constructed at the cellular level with perfect monomer sequence, synthetically this level of precision remains largely unaccomplished. While solid-state peptide synthesis (Merrifield synthesis) has been a significant breakthrough, [START_REF] Merrifield | Solid phase peptide synthesis. I. The synthesis of a tetrapeptide[END_REF] the limitations of this method (e.g. time-consuming, high cost, low yields, limited scalability, access only to lower molecular weight materials) have forced polymer chemists to seek alternatives in a homogeneous liquid phase. As such, a range of chemical approaches have emerged that can access sequence-controlled or sequence-defined materials at a larger scale including single monomer insertion, [START_REF] Zamfir | Ultra-precise insertion of functional monomers in chain-growth polymerizations[END_REF] segregating templating, [START_REF] Mchale | Biomimetic radical polymerization via cooperative assembly of segregating templates[END_REF][START_REF] Schoonen | Compartmentalization approaches in soft matter science: from nanoreactor development to organelle mimics[END_REF] kinetic control, [START_REF] Pfeifer | A facile procedure for controlling monomer sequence distribution in radical chain polymerizations[END_REF] tandem monomer addition and modification [START_REF] Nakatani | Sequence-regulated copolymers via tandem catalysis of living radical polymerization and in situ transesterification[END_REF] and solution polymerization. [START_REF] Pfeifer | Liquid-phase synthesis of block copolymers containing sequence-ordered segments[END_REF][START_REF] Dong | Sequence-defined multifunctional polyethers via liquid-phase synthesis with molecular sieving[END_REF] Controlled radical polymerization has also made a significant step towards this goal through the synthesis of sequence-controlled multiblock copolymers yielding complex structures with unlimited variations in chain length, composition and encoded information. [START_REF] De Neve | Sequence-definition from controlled polymerization: the next generation of materials[END_REF][START_REF] Beyer | Synthetic approaches for multiblock copolymers[END_REF] Importantly, the synthesis of sequence-controlled multiblocks is scalable, applicable to higher molecular weight polymers and amenable to non-experts, in particular when combined with simple and one pot processes that do not necessitate the use of any specialized equipment. [START_REF] De Neve | Sequence-definition from controlled polymerization: the next generation of materials[END_REF] Such materials have already shown great promise in a range of applications where sequence has been demonstrated to determine fundamental polymer properties and applications (e.g. macromolecular folding, phase separation, antimicrobial materials, etc.). [START_REF] Elacqua | Supramolecular multiblock copolymers featuring complex secondary structures[END_REF][START_REF] Heiler | Photochemically induced folding of single chain polymer nanoparticles in water[END_REF][START_REF] Zhang | Synthesis of sequence-controlled multiblock single chain nanoparticles by a stepwise folding-chain extension-folding process[END_REF][START_REF] Guimarães | Nano-engineered multiblock copolymer nanoparticles via reversible addition-fragmentation chain transfer emulsion polymerization[END_REF][START_REF] Anastasaki | One-Pot Synthesis of ABCDE Multiblock Copolymers with Hydrophobic, Hydrophilic, and Semi-Fluorinated Segments[END_REF][START_REF] Barbon | Elucidating the effect of sequence and degree of polymerization on antimicrobial properties for block copolymers[END_REF][START_REF] Kuroki | Sequence control as a powerful tool for improving the selectivity of antimicrobial polymers[END_REF][START_REF] Judzewitsch | Towards Sequence-Controlled Antimicrobial Polymers: Effect of Polymer Block Order on Antimicrobial Activity[END_REF][START_REF] Zhang | Effect of hydrophilic monomer distribution on self-assembly of a pH-responsive copolymer: Spheres, worms and vesicles from a single copolymer composition[END_REF] Owing to these advantages, the groups of Hawker, Matyjaszewski, Boyer, Whittaker, Haddleton, Junkers and Perrier have reported the remarkable synthesis of sequence-controlled multiblocks. [START_REF] Anastasaki | One-Pot Synthesis of ABCDE Multiblock Copolymers with Hydrophobic, Hydrophilic, and Semi-Fluorinated Segments[END_REF][START_REF] Chin | Synthesis of triblock and multiblock methacrylate polymers and self-assembly of stimuli responsive triblock polymers[END_REF][START_REF] Shanmugam | Stereo-, temporal and chemical control through photoactivation of living radical polymerization: synthesis of block and gradient copolymers[END_REF][START_REF] Xu | A robust and versatile photoinduced living polymerization of conjugated and unconjugated monomers and its oxygen tolerance[END_REF][START_REF] Soeriyadi | High-order multiblock copolymers via iterative Cu (0)-mediated radical polymerizations (SET-LRP): toward biological precision[END_REF][START_REF] Boyer | Synthesis of multi-block copolymer stars using a simple iterative Cu (0)-mediated radical polymerization technique[END_REF][31][START_REF] Engelis | Sequence-controlled methacrylic multiblock copolymers via sulfur-free RAFT emulsion polymerization[END_REF][START_REF] Anastasaki | Photoinduced sequence-control via one pot living radical polymerization of acrylates[END_REF][START_REF] Zhang | Sequence-controlled multi-block glycopolymers to inhibit DC-SIGN-gp120 binding[END_REF][START_REF] Chuang | Photoinduced sequence-controlled coppermediated polymerization: synthesis of decablock copolymers[END_REF][START_REF] Wenn | Efficient multiblock star polymer synthesis from photo-induced copper-mediated polymerization with up to 21 arms[END_REF][START_REF] Debuigne | Straightforward synthesis of symmetrical multiblock copolymers by simultaneous block extension and radical coupling reactions[END_REF][START_REF] Gody | Rapid and quantitative one-pot synthesis of sequence-controlled polymers by radical polymerization[END_REF][START_REF] Gody | Ultrafast RAFT polymerization: multiblock copolymers within minutes[END_REF][START_REF] Clothier | Exploitation of the nanoreactor concept for efficient synthesis of multiblock copolymers via macroRAFT-mediated emulsion polymerization[END_REF] For instance, Whittaker and co-workers first reported the synthesis of high-order multiblocks via iterative Cu(0)-mediated radical polymerization while omitting the need for purification between the iterative block formation steps. [START_REF] Soeriyadi | High-order multiblock copolymers via iterative Cu (0)-mediated radical polymerizations (SET-LRP): toward biological precision[END_REF] The Junkers group showed that photo-ATRP (atom transfer radical polymerization) could also enable the synthesis of well-defined multiblocks in the absence of dye sensitizers or photoinitiators. [START_REF] Chuang | Photoinduced sequence-controlled coppermediated polymerization: synthesis of decablock copolymers[END_REF] Perrier and co-workers revolutionized RAFT (reversible addition-fragmentation chain-transfer) polymerization by demonstrating that a large number of multifunctional multiblocks could be possible if high livingness could be ensured during each block addition. [START_REF] Gody | Rapid and quantitative one-pot synthesis of sequence-controlled polymers by radical polymerization[END_REF][START_REF] Gody | Pushing the limit of the RAFT process: multiblock copolymers by one-pot rapid multiple chain extensions at full monomer conversion[END_REF] However, a significant limitation of all previous strategies is that they are incapable of simultaneously controlling both polymer sequence and dispersity. This is due to gradually increasing dispersities in multiblock copolymer synthesis as a result of the accumulation of dead chains and loss of the end-group fidelity. Indeed, in both ATRP and RAFT methodologies, although the first block can be synthesized with low dispersity, low and high molecular weight tailing/shoulders gradually appear during subsequent monomer additions, thus yielding multiblocks with much higher dispersity values and loss of control over both dispersity and livingness. 31,[START_REF] Chuang | Photoinduced sequence-controlled coppermediated polymerization: synthesis of decablock copolymers[END_REF] This phenomenon is further accentuated during the synthesis of higher molecular weight multiblocks (DP ~100 per block) which are accompanied by an even greater loss of control over dispersity and end-groups. [START_REF] Gody | Pushing the limit of the RAFT process: multiblock copolymers by one-pot rapid multiple chain extensions at full monomer conversion[END_REF][START_REF] Anastasaki | Photoinduced synthesis of α, ω-telechelic sequence-controlled multiblock copolymers[END_REF] Therefore, on demand tuning dispersity of sequence-controlled multiblock copolymers remains unattainable. However, dispersity is a key parameter in polymer design and can significantly influence material properties (e.g. bulk and solution self-assembly, mechanical and rheological behaviour, etc.). [START_REF] Gentekos | Controlling polymer properties through the shape of the molecular-weight distribution[END_REF][START_REF] Whitfield | Tailoring polymer dispersity and shape of molecular weight distributions: methods and applications[END_REF][START_REF] Junkers | Polymers in the Blender[END_REF] In fact, high and low dispersity polymers exhibit unique properties and functions, which has encouraged many groups to develop strategies that can precisely control dispersity. [START_REF] Liu | Modulating Polymer Dispersity with Light: Cationic Polymerization of Vinyl Ethers Using Photochromic Initiators[END_REF][START_REF] Liu | Polymer Dispersity Control by Organocatalyzed Living Radical Polymerization[END_REF] For instance, the Fors group elegantly exploited a modular strategy which controls the temporal regulation of initiation during chain growth polymerization, [START_REF] Gentekos | Beyond dispersity: Deterministic control of polymer molecular weight distribution[END_REF][START_REF] Gentekos | Exploiting molecular weight distribution shape to tune domain spacing in block copolymer thin films[END_REF][START_REF] Kottisch | Shaping" the Future of Molecular Weight Distributions in Anionic Polymerization[END_REF][START_REF] Nadgorny | Manipulation of Molecular Weight Distribution Shape as a New Strategy to Control Processing Parameters[END_REF] while Matyjaszewski and co-workers leveraged the catalyst concentration to govern the molecular weight distributions. [START_REF] Wang | Control of Dispersity and Grafting Density of Particle Brushes by Variation of ATRP Catalyst Concentration[END_REF][START_REF] Plichta | Tuning dispersity in diblock copolymers using ARGET ATRP[END_REF] Despite the tremendous importance of dispersity in polymer science, the synthesis of sequence-controlled multiblocks with tuneable dispersity remains a formidable challenge and is far from being realized. This is because high dispersity polymers are often associated with increased terminations and side reactions which prevent the efficient addition of subsequent blocks. A few recent breakthroughs have allowed the synthesis of high dispersity polymers with minimal termination, thus promoting the preparation of diblock copolymers. [START_REF] Liu | Modulating Polymer Dispersity with Light: Cationic Polymerization of Vinyl Ethers Using Photochromic Initiators[END_REF][START_REF] Liu | Polymer Dispersity Control by Organocatalyzed Living Radical Polymerization[END_REF] However, full control over the dispersity even in diblock copolymers remains severely restricted due to inherent limitations of the reported synthesis approaches. For example, the use of mixtures of RAFT agents can accurately control the dispersity of homopolymers but this level of control cannot be extended to diblock copolymers. [START_REF] Whitfield | Tailoring Polymer Dispersity by RAFT Polymerization: A Versatile Approach[END_REF][START_REF] Parkatzidis | Tailoring polymer dispersity by mixing chain transfer agents in PET-RAFT polymerization[END_REF] In a similar vein, using low deactivator concentrations in ATRP enabled the synthesis of polymers with variable dispersities, but even a low to high dispersity diblock is currently unattainable. [START_REF] Rolland | Tailoring Polymer Dispersity in Photoinduced Iron-Catalyzed ATRP[END_REF][START_REF] Whitfield | Tuning Dispersity by Photoinduced Atom Transfer Radical Polymerisation: Monomodal Distributions with ppm Copper Concentration[END_REF] Collectively, these constraints hinder the development of sequence-controlled multiblock copolymers with on demand control over dispersity.

Results and Discussion

Tailoring Polymer Dispersity in Homopolymers using a Switchable RAFT Agent

We envisioned that the use of a single RAFT agent that can on-demand switch its activity (e.g. from low to high and vice versa) would enable the synthesis of sequence-controlled multiblocks where the dispersity can be controlled throughout the addition of each block. Switchable RAFT agents were developed as universal RAFT agents to provide access to low dispersity materials from either more or less activated monomers. [START_REF] Benaglia | Polystyrene-block-poly (vinyl acetate) through the Use of a Switchable RAFT Agent[END_REF][START_REF] Benaglia | Universal (switchable) RAFT agents[END_REF][60][START_REF] Keddie | Switchable reversible addition-fragmentation chain transfer (RAFT) polymerization in aqueous solution, N, Ndimethylacrylamide[END_REF] In contrast, to realize our concept, we hypothesized that, for a certain monomer class, switchable RAFT agents can transition "insitu" from a low to a high activity RAFT agent (or vice versa) by simply adjusting the amount of added acid (or base), thus enabling full control over the dispersity in highly ordered sequence-controlled multiblock copolymers (Figure 1 & Supplementary Scheme 1).

Figure 1. Schematic representation of our proposed approach for the preparation of multiblocks with tuneable dispersity. In a, the addition of base results in gradual deprotonation of the chain transfer agent yielding multiblock copolymers with incremental increases in dispersity. In b, the addition of acid results in a gradual protonation of the CTA yielding multiblock copolymers with incremental decreases in dispersity. In c, the addition of acid and base yields multiblock copolymers with alternating high and low dispersities .

To demonstrate this concept, we used dimethyl acrylamide (DMA) as a model monomer, methyl 2-[methyl(4-pyridinyl)carbamothioylthio]propionate as the switchable RAFT agent, and 2,2'-azobis(2-methylpropionitrile) (AIBN) as the free radical initiator (0.1 equivalents with respect to the RAFT agent) in DMF. In the absence of added acid (i.e. fully deprotonated RAFT agent), a broad, yet monomodal, molecular weight distribution (Mn=17500, Ɖ=1.67) was obtained when targeting DP=200 (Supplementary Table 1 Entry 1 & Supplementary Figure 1). Importantly, and despite the high dispersity, good agreement between the theoretical and experimental molecular weights was observed at high conversions (Supplementary Figure 2, NMR was used in calculating theoretical Mn, depicted as Mn(Theo), and SEC was employed to determine the experimental Mn, depicted as Mn (SEC)). This is in contrast to previous methodologies, where higher Ɖ polymers were accompanied by very low conversions (~20%) and a significant deviation between theoretical and experimental molecular weight values, as a result of slow initiation. [START_REF] Whitfield | Tuning Dispersity by Photoinduced Atom Transfer Radical Polymerisation: Monomodal Distributions with ppm Copper Concentration[END_REF] By repeating the experiment, albeit in the presence of a small amount of sulphuric acid (H2SO4, 1 µL, 18M, 0.5 equiv.), the Ɖ was decreased to 1.50 while maintaining a unimodal shape (Supplementary Table 1 Entry 2 & Supplementary Figure 1).

Further increase in the amount of added H2SO4 (2 µL (1 equiv.), 3.5 µL (1.74 equiv.) and 8 µL (4 equiv.), 18M), led to significantly lower Ɖs yielding PDMA with a Ɖ of 1.42, 1.29 and 1.18, respectively (Supplementary Table 1 Entries 3-5 & Supplementary Figure 1). As a result, a range of targeted monomodal molecular weight distributions (Mn ~17200-17500, Ɖ=1. 18-1.67) at very high monomer conversions could be obtained. An important feature of this technique is that the polymers possess identical end-groups (either in the protonated or deprotonated form), regardless of their dispersity. We noted that to achieve the lowest possible Ɖ, higher than stoichiometric amounts of acid were required. This is because the RAFT agent is a weak base in very dilute solution and the presence of the organic solvent and monomer can also reduce the polarity of the solution, thus lowering the protonation of the RAFT agent. [START_REF] Fraenkel | Protonation of Amides1[END_REF][START_REF] Farona | Acrylamide and N, N-dimethylacrylamide complexes. II. N-protonation and N-bonding to some transition metal perchlorates and tetrafluouroborates[END_REF] Therefore, even more acid was added to drive the equilibrium to the protonated form. This is confirmed when the target DP of the polymerization or the reaction medium are changed, whereby different equivalents of acid are required so to obtain the lowest possible dispersity (Supplementary Tables 23& Supplementary Figures 34). Next, we reversed the phenomenon (i.e. the control increase of Ɖ in homopolymers) by starting with a polymer of low Ɖ (through the addition of acid) and subsequently repeated the polymerization in the presence of gradually increased sodium hydroxide (NaOH) concentrations (Supplementary Table 4 & Supplementary Figure 5). It should be highlighted that even higher amounts of NaOH (i.e. 6 equivalents or higher) must be avoided as this would lead to the degradation of the RAFT agent and an uncontrolled polymerization, while under the conditions utilized (i.e. 4-5 equivalents) no RAFT agent degradation is observed even after 10 hours (Supplementary Table 5 & Supplementary Figure 6789). This is a significant improvement over previous strategies where employing RAFT agent mixtures led to vastly different end-groups or the addition of comonomers and termination agents led to adulterated polymer chains and chains without functional end-groups, respectively. [START_REF] Liu | Polymer Dispersity Control by Organocatalyzed Living Radical Polymerization[END_REF][START_REF] Whitfield | Tailoring Polymer Dispersity by RAFT Polymerization: A Versatile Approach[END_REF][START_REF] Parkatzidis | Tailoring polymer dispersity by mixing chain transfer agents in PET-RAFT polymerization[END_REF][START_REF] Yadav | Dispersity control in atom transfer radical polymerizations through addition of phenylhydrazine[END_REF] These results confirm that the activity of the RAFT agent used can be modulated by increasing or decreasing the electron withdrawing capacity of the pyridine group via protonation or deprotonation of the aromatic amine, respectively (Figure 1 & Supplementary Scheme 1).

Tailoring Polymer Dispersity in Pentablock Copolymers

These initial data encouraged us to demonstrate the concept of tailoring polymer dispersity in sequence-controlled multiblocks by modulating the activity of the RAFT agent in each iterative block formation step. For subsequent polymerizations a target of DP=100 per block was selected to minimize the number of defective chains, i.e. chains where one or more block is not incorporated. Previously, Harrisson and co-workers illustrated that for low dispersity multiblock copolymers DP=6 was required per block to avoid defective chains, but for the synthesis of high dispersity materials, there is a higher chance of defects, so higher DP blocks must be prepared. [START_REF] Harrisson | The Chain Length Distribution of an Ideal Reversible Deactivation Radical Polymerization[END_REF][START_REF] Gody | The limits of precision monomer placement in chain growth polymerization[END_REF] By targeting DP=100 we minimize the number of defective chains, with target dispersities of 1.1 and 1.7, giving ~0% and ~0.2% of defective chains respectively. The percentage of defective chains for each dispersity value can be found in Supplementary Table 6 & Supplementary Figures 1011). We optimized an initial ratio of [RAFT Agent]:[VA-044 (2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride)] at 1:0.034 and experiments were performed at 70 C in aqueous solutions with the addition of organic solvent (DMF, 20% v/v) to allow for the complete solubility of the RAFT agent. It is noted that an increased water content in the solvent composition was selected to accelerate the polymerization rate, so lower amounts of free radical initiator are required and the number of terminated chains reduced. To investigate end-group fidelity, two low molecular weight PDMA polymers were synthesized; the first one in the absence of added acid (yielding higher Ɖ) and the second one using an excess of acid (yielding lower Ɖ). The two polymers (without further purification) were then characterized by electrospray ionization mass-spectrometry (ESI-MS). The generated ESI data with full peak assignments can be found in the SI (Supplementary Tables 78& Supplementary Figures 1213141516). Pleasingly, even in the absence of acid, the major polymer peak distributions correspond to the expected PDMA with the RAFT end-group attached. Importantly, negligible termination, if any, could be detected which confirms that our selected conditions yield high end-group fidelity polymers. Matrix assisted desorption ionization time-of-flight massspectrometry (MALDI-ToF-MS) was also conducted for both a low and a high dispersity polymer and further supported the high livingness observed in our system (Supplementary Figures 1718). Another note is that we use poly(methyl methacrylate) standards in our SEC so to confirm the good agreement between theoretical and experimental molecular weight we have also calculated the experimental molecular weight for both low and high dispersity polymers via [START_REF] Lutz | Sequence-controlled polymers[END_REF] H NMR (by comparing the protons from the RAFT agent to the protons of the repeat unit for both the purified and the unpurified polymers) and found a very good agreement (Supplementary Figures 192021222324). The water solubility of the RAFT agent was also considered (Supplementary Figure 24b). After all these considerations, our initial target was a sequence-controlled pentablock copolymer with gradually descending Ɖs via sequential additions of individual aliquots from a palette of three different acrylamide-based monomers. First, a poly(4-acryloylmorpholine) (PNAM) block was obtained at near quantitative conversion (98%) as characterized by [START_REF] Lutz | Sequence-controlled polymers[END_REF] H NMR yielding a homopolymer with a broad molecular weight distribution (Mn=12000, Ɖ=1.66) and good agreement between experimental and theoretical molecular weight (Figures 2a-c & Supplementary Table 9 Entry 1). Upon addition of the second monomer (DMA) together with a small amount of H2SO4 (0.65 µL, 18M, 0.41 equiv.), a gradual decrease in the Ɖ of the diblock (Mn=22200, Ɖ=1.50) was evident. This was accompanied by a clear shift to higher molecular weights. (Figure 2a-c & Supplementary Table 9 Entry 2). The decrease in the Ɖ was attributed to the addition of the acid and the partial protonation of the RAFT agent, as in the absence of acid an equally high Ɖ could be maintained for a diblock or even a pentablock (Supplementary Table 10 & Supplementary Figures 252627). When full conversion was reached, another aliquot of acid (1.5 µL, 0.95 equiv.) was subsequently injected together with the third monomer diethylacrylamide (DEA) yielding a triblock with a further decrease in the dispersity (Mn=33600, Ɖ=1.41, Supplementary Table 9 Entry 3). This process was repeated two more times while gradually increasing the amount of the added acid alongside the sequential monomer additions, yielding tetrablock (2.0 µL H2SO4, 1.26 equiv. Mn=45600) and pentablock (3.8 µL H2SO4, 3.75 equiv., Mn=65300) copolymers with Ɖ=1.34 and Ɖ=1. 22, respectively (Figures 2a-c, Supplementary Table 9 Entries 4-5 & Supplementary Scheme 2a). This is the first example of sequence-controlled multiblock copolymers with gradually descending Ɖs and contrasts all previously reported examples where a gradual increase in the dispersity could only be achieved as a result of the gradual accumulation of dead polymer chains often resulting in the loss of the unimodal shape. 31,[START_REF] Engelis | Sequence-controlled methacrylic multiblock copolymers via sulfur-free RAFT emulsion polymerization[END_REF][START_REF] Chuang | Photoinduced sequence-controlled coppermediated polymerization: synthesis of decablock copolymers[END_REF][START_REF] Gody | Pushing the limit of the RAFT process: multiblock copolymers by one-pot rapid multiple chain extensions at full monomer conversion[END_REF] However, in our system very little termination is observed, and we also maintain monomodal distributions. In particular, a high livingness of the final pentablock was obtained (92.5%) as expected from theory (see SI for RAFT livingness equation). Importantly, due to the high DP of each block, defective chains are minimized. The reverse scenario was then attempted aiming at a sequence-controlled multiblock with gradually ascending Ɖs. For this purpose, PDMA with a narrow molecular weight distribution was first prepared (Mn=11500, Ɖ=1.16, Figures 2d-f & Supplementary Table 11 Entry 1) by employing an excess of sulphuric acid. To incrementally increase the Ɖ, four subsequent monomer additions were performed where the amount of added base (NaOH) was slowly increased. [START_REF] Lutz | Sequence-controlled polymers[END_REF] H NMR spectroscopy confirmed high monomer conversions in all steps yielding higher ordered multiblocks with continuously increased Ɖs (ƉHomopolymer=1.16, ƉDiblock=1.29, ƉTriblock=1.41, ƉTetrablock=1.52 and ƉPentablock=1.60, Figures 2d-f, Supplementary Table 11 Entries 2-5 & Supplementary Scheme 2b). Importantly, the pentablock copolymer retained a monomodal shape in contrast to previous work where the increase in Ɖ was accompanied by low and high molecular weight shoulders.

The next challenging step towards the synthesis of sequence-controlled multiblocks with tailored Ɖs was to achieve full control over the Ɖ in multiblock copolymers where any Ɖ combination could be feasible. To achieve this ambitious target, we first aimed to synthesize three diblock extremes with; (i) high Ɖ for both the homopolymer and the diblock, (ii) high Ɖ for the homopolymer and low Ɖ for the diblock and (iii), low Ɖ for the homopolymer and high Ɖ for the diblock, with the latter one being particularly challenging for any reported methodology (Figure 3a). [START_REF] Whitfield | Tailoring polymer dispersity and shape of molecular weight distributions: methods and applications[END_REF] For all the diblock experiments, DMA and NAM were used as the monomers. A crucial factor was to ensure very high monomer conversions (99%) for the first block in order to assess the suitability of our system to afford the synthesis of "in-situ" diblocks. PNAM with Ɖ=1.60 was initially synthesized as previously stated through the use of the deprotonated RAFT agent (no acid was added, Figure 3b & Supplementary Table 12 Entry 1).

Once >99% polymerization conversion had been reached, DMA was added yielding a welldefined diblock copolymer with a comparably high dispersity (Mn=12000 Ɖ=1.66, Figure 3b & Supplementary Table 12 Entry 2). This data indicates that despite the high Ɖ of the first block (resembling values typically acquired in free radical polymerization), high end-group fidelity is maintained allowing for the in-situ formation of block copolymers. Moreover, when the second monomer was added together with an excess of H2SO4 (to ensure full protonation of the RAFT agent), the final diblock copolymer presented a much lower Ɖ of 1.19 while fully shifting to higher molecular weights, further emphasizing the high chain end fidelity maintained in our system (Figure 3c & Supplementary Table 12 Entries 3-4). Finally, PDMA with low dispersity (Ɖ=1.16) was also synthesized by complete protonation of the RAFT agent. This time, an excess of base (NaOH) was injected together with the second monomer, yielding P(DMA-b-NAM) with Ɖ=1.58 (Figure 3d & Supplementary Table 12 Entries 5-6). It should be highlighted that such a low-to-high dispersity diblock has never been synthesized by any controlled radical polymerization methodology and would be fundamentally incompatible with existing strategies such as the use of a mixture of RAFT agents or when employing ppm concentrations of copper in ATRP. [START_REF] Whitfield | Tailoring Polymer Dispersity by RAFT Polymerization: A Versatile Approach[END_REF][START_REF] Whitfield | Tuning Dispersity by Photoinduced Atom Transfer Radical Polymerisation: Monomodal Distributions with ppm Copper Concentration[END_REF] We also note that all the chain extensions were performed in-situ at near-quantitative conversion, in contrast to previous methods where the isolation of the macroinitiator was essential. [START_REF] Gentekos | Beyond dispersity: Deterministic control of polymer molecular weight distribution[END_REF][START_REF] Whitfield | Tuning Dispersity by Photoinduced Atom Transfer Radical Polymerisation: Monomodal Distributions with ppm Copper Concentration[END_REF] Altogether, our proposed approach allows full control over dispersity of both homopolymers and diblock copolymers. To demonstrate the versatility of our approach and achieve the ambitious goal of on-demand control over the dispersity of multiblocks, a considerably challenging multiblock copolymer was designed. In particular, an "extreme" pentablock copolymer was set as the goal, where low and high Ɖ values would alternate throughout the iterative monomer additions. In the presence of an excess of sulphuric acid, a low Ɖ PDMA homopolymer was prepared (Mn=5200, Ɖ=1.17, Figures 4a-c & Supplementary Table 13 Entry 1). When near-quantitative conversion was achieved (>96%), the second monomer and an excess of NaOH were subsequently injected in order to obtain a much higher Ɖ diblock (Mn=11500, Ɖ=1.61, Figures 4a-c & Supplementary Table 13 Entry 2). The inclusion of the third monomer in the reaction mixture was accompanied with a high dosage of acid and as a result the triblock copolymer reached a low dispersity value (Mn=25900, Ɖ=1.24, Figures 4a-c & Supplementary Table 13 Entry 3). By alternating the addition of base and acid together with the subsequent monomer injections, tetrablock and pentablock copolymers could easily be prepared with Ɖ=1.70 (Mn=45400) and Ɖ=1.26, (Mn=88400), respectively (Figures 4a-c, Supplementary Table 13 Entries 4-5 & Supplementary Scheme 2c). With these results, we next wanted to investigate further the reactivity switch of the RAFT agent upon addition of base and acid and thus performed Predici simulations.

Modelling was performed by assuming typical parameters for a thermal acrylamide RAFT polymerization at 70⁰C, incorporating our experimental conditions (i.e. [CTA]:[VA-044] = 1:0.01 and a switch in the chain transfer coefficient of the CTA on the addition of either acid (CT1=15) or base (CT2=1) for each block (Supplementary Scheme 3). Pleasingly, the molecular weight distributions nicely switched from low dispersity to high dispersity (and vice versa) on each block addition with a variation in dispersity between roughly 1.2 and 1.6, replicating what was observed experimentally (Supplementary Figure 28). Furthermore, the simulation allowed the amount of terminated polymer compared to the full polymer sample to be analyzed throughout the chain extensions. Importantly, neither, the full variation in the value of the chain transfer constant or the switching of the reactivity of the chain transfer agent, by addition of acid or base, had any significant impact on the amount of termination. (This is in line with what one would expect from the RAFT livingness equation). It is also clearly visible that the amount of termination levels off prior to the addition of fresh monomer, indicating that full conversion is reached for each block. Termination data for a range of chain transfer constants is illustrated in Supplementary Figure 29, demonstrating a high retention of livingness in all cases, independent of what dispersity block is created during each chain extension.

We next prepared the reverse multiblock copolymer by switching between fully basic and fully acidic conditions throughout the monomer additions (ƉHomopolymer=1.52, ƉDiblock=1.21, ƉTriblock=1.52 and ƉTetrablock=1.25) and the final pentablock copolymer presented a dispersity of 1.52 (Mn=74600) and a fairly unimodal shape (Figures 4d-f, Supplementary Table 14 Entries 1-5 & Supplementary Scheme 2d). Our experiments confirm that we can achieve on demand control over the dispersity in sequence-controlled multiblock copolymers. Another important highlight of our work is that we can prepare such complex materials while at the same time reaching relatively high molecular weights. It is widely accepted within the polymer community that the higher the molecular weight of the final multiblock copolymer, the harder it is to control the dispersity while maintaining a monomodal molecular weight distribution; yet our system presents fairly small amounts of termination and maintains monomodality. the first block was designed to possess high dispersity and this could be achieved in the absence of acid (Ɖ=1.61, Figures 5d-f & Supplementary Table 17 Entry 1). The subsequent five blocks were designed to have gradually descending dispersities. To achieve this, an increase amount of acid was injected together with the subsequent monomer addition thus eventually producing a hexablock copolymer with Ɖ=1.24 (Full dispersity evolution: ƉHomopolymer=1.61, ƉDiblock=1.52, ƉTriblock=1.43, ƉTetrablock=1.37, ƉPentablock=1.32 and ƉHexablock=1.24, Figures 5d-f & Supplementary Table 17 Entries 2-6). Low dispersity could be maintained for subsequent blocks by neither addition of acid nor base (Supplementary Table 18, Supplementary Figure 31 & Supplementary Scheme 2f) but our goal was to gradually increase the dispersity for the next three blocks. By adding a gradually increasing amount of base along with the monomer injections, heptablock, octablock and nonablock copolymers with gradually increasing dispersities could be prepared (ƉHeptablock=1.29, ƉOctablock=1.38 and ƉNonablock=1.52, Figures 5df & Supplementary Table 17 Entries 7-9). As a final goal, we applied acidic conditions when the final batch of monomer was added, in order to again decrease the dispersity to 1.26 for the decablock copolymer (Figures 5d-f, Supplementary Table 17 Entry 10 & Supplementary Scheme 2h). Taken all together, complex decablock copolymers can be rigorously synthesized with control over the dispersity achieved in all addition steps. Again, high monomer conversions were maintained throughout the syntheses. By carefully adjusting the amount of acid and base, the dispersity of every block can be targeted on demand, thus significantly increasing the range of accessible multiblock copolymers. Impressively, the calculated livingness found for our decablock copolymers was very high (94.5% and 95.2%) as such small amounts of free radical initiator were employed. In block 1, a high dispersity polymer is prepared, followed by targeted decreases and increases in the dispersity of subsequent blocks on addition of acid or base. b, The controlled evolution of dispersity during this multiblock synthesis. c, [START_REF] Lutz | Sequence-controlled polymers[END_REF] H NMR spectra for each cycle, illustrating the monomer conversion for each block (≥95% in all cases). d, SEC traces illustrating the molecular weight distributions for consecutive cycles during the synthesis of a decablock copolymer. In block 1, a low dispersity polymer is prepared, followed by targeted increases and decreases in the dispersity of subsequent blocks on addition of acid or base. e, The controlled evolution of dispersity during this multiblock synthesis. f, 1 H NMR spectra for each cycle, illustrating the monomer conversion for each block (≥95% in all cases).

Expanding the Monomer Scope

We finally sought to demonstrate the compatibility of our methodology with another polymer class (i.e. polyacrylates by efficiently controlling the Ɖ in homopolymers, diblock, triblock and tetrablock copolymers. We first used methyl acrylate (MA) as a model monomer and conducted the polymerization in pure organic media (DMF). In the absence of acid, a high Ɖ PMA was observed (Ɖ=1.70) while the addition of a small amount of acid (0.30 equivalents with respect to the RAFT agent) led to a lower Ɖ (Ɖ=1.62) (Supplementary Figure 32 & Supplementary Table 19 Entries 1-2). Upon continuing to increase the acid concentration, the dispersity was gradually decreased to 1.41, 1.33 and 1.16 (Supplementary Figure 32 & Supplementary Table 19 Entries 3-5). Monomodal molecular weight distributions were observed in all cases with good agreement between experimental and theoretical molecular little termination could be observed in the SEC chromatographs while near-quantitative conversions could be achieved throughout the iterative block formations along with a nearperfect match between theoretical and experimental molecular weights was observed. The experimental method can be applied to a number of different monomer classes and is fully supported by Predici simulations. We believe that this new concept will open the door for the synthesis of advanced sequence-controlled materials that can enable structure-property relationship studies and be used in various applications and fields including phase-separation, solution self-assembly, rheology, macromolecular folding and drug delivery.

Online content

Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/xxxx 0.36 mL of N-acrylomorpholine (0.40 g, 2.85 mmol, 100 equiv., 20% solution v/v in 80:20 H2O:DMF), 0.65 mL of distilled water and a stirrer bar were added. The vial was then sealed with a septum, prior to deoxygenation by nitrogen bubbling for 15 minutes. Polymerization was conducted in an oil bath at 70°C for 2 h with a 200-rpm stirring rate to reach a conversion of >95% by 1 H NMR. In a separate vial, 0.30 ml of DMA (0.29 g, 2.85 mmol, 100 equiv., 20% solution v/v in 80:20 H2O:DMF), 0.65 µL of sulphuric acid (11.7 µmol, 18 M, 0.41 equiv.) and 175 µL of initiator solution (0.11 mg, 0.34 µmol, 0.0118 equiv.) were mixed and degassed together for 15 minutes prior to addition to the polymerization mixture via a nitrogen purged syringe. Polymerization was again conducted for 2 h, yielding a lower dispersity second block. This process was repeated for all subsequent block to gradually decrease the dispersity. In block 3, 1.5 µL (27.1 µmol, 18 M, 0.95 equiv.) of sulphuric acid solution was added along with 0.39 mL of DEA (0.36 g, 2.85 mmol, 100 equiv., 20% solution v/v in 80:20 H2O:DMF) and 160 µL of initiator solution (0.10 mg, 0.31 µmol, 0.0108 equiv.). In block 4, 0.36 mL of NAM (0.40g, 2.85 mmol, 100 equiv., 20% solution v/v in 80:20 H2O:DMF), 2 µL (35.9 µmol, 18 M, 1.26 equiv.) of sulphuric acid solution and 130 µL (0.08 mg, 0.25 µmol, 0.0088 equiv.) of initiator solution were added and for block 5, 0.6 mL of DMA (0.58 g, 5.7 mmol, 200 equiv., 20% solution v/v in 80:20 H2O:DMF), 3.75 µL of sulphuric acid solution (67.8 µmol, 18 M, 2.37 equiv.) and 210 µL (0.13 mg, 0.40 µmol, 0.0141 equiv.) of initiator stock solution were added to the reaction. This procedure corresponds to the data in Figures 2a-2c and Supplementary Table 9.

Synthesis of a decablock copolymer from a low Ð first block

Into a 10 mL glass vial, 7.70 mg of methyl 2-[methyl(4-pyridinyl)carbamothioylthio]propionate (CTA, 28.5 μmol, 1 equiv.) were dissolved in 0.15 mL of DMF. A stock solution of VA-044 (6.2 mg) was prepared in 10 mL of water, and 0.15 mL of this solution (0.093 mg, 0.288 μmol, 0.0102 equiv.) was transferred to the vial. Subsequently, 0.15 mL (0.14 g, 1.43 mmol, 50 equiv.) of DMA, 2.5 µL (46 µmol, 18 M, 1.60 equiv.) of sulphuric acid, 0.4 mL of distilled water and a stirrer bar were added. The vial was then sealed with a septum, prior to deoxygenation by nitrogen bubbling for 15 minutes. Polymerization was conducted in an oil bath at 70°C for 1.5 h with a 200-rpm stirring rate to reach a conversion >95% measured by [START_REF] Lutz | Sequence-controlled polymers[END_REF] H NMR (Ð = 1.15). In a separate vial, 0.18 mL of NAM (0.20 g, 1.43 mmol, 50 equiv., 20% solution v/v in 80:20 H2O:DMF), 163 µL (0.101 mg, 0.31 µmol, 0.0110 equiv.) of initiator solution and 32.5 µL of sodium hydroxide (55.3 μmol, 1.7 M, 1.94 equiv.) were mixed and degassed together for 15 minutes prior to addition to the polymerization mixture via a nitrogen purged syringe.

Polymerization was again conducted for 1.5 h, yielding a high dispersity second block (Ð = 1.62). This process was repeated for all subsequent block additions. First the dispersity was incrementally lowered between blocks 3 and 5. In block 3, 1.5 µL (27.1 µmol, 18M, 0.95 equiv.) of sulphuric acid, 62.5 µL (0.039 mg, 0.12 µmol, 0.0042

Figure 2 .

 2 Figure 2. Pentablock copolymers with gradually evolving dispersity. a, SEC traces illustrating the molecular weight distributions for consecutive cycles during the synthesis of a descending dispersity pentablock copolymer. b Evolution of dispersity (red) and the measured molecular weight (light blue) compared to theoretical (dark blue) during this multiblock synthesis. c, 1 H NMR spectra for each cycle, illustrating the monomer conversion for each block (≥ 96% in all cases). d, SEC traces illustrating the molecular weight distributions for consecutive cycles during the synthesis of an ascending dispersity pentablock copolymer. e, Evolution of dispersity (red) and the measured molecular weight (light blue) compared to theoretical (dark blue) during this multiblock synthesis. f, 1 H NMR spectra for each cycle, illustrating the monomer conversion for each block (≥ 96% in all cases).

Figure 3 .

 3 Figure 3. Diblock copolymers synthesized with dispersity value extremes by switching the activity of the RAFT agent. a, scheme illustrating the preparation of high to high dispersity, high to low dispersity and low to high dispersity diblocks directly corresponding to the SEC traces in b, c and d.

Figure 4 .

 4 Figure 4. Pentablock copolymers synthesized with dispersity extremes. a, SEC traces illustrating the molecular weight distributions for consecutive cycles during the synthesis of an alternating high and low dispersity pentablock copolymer. b, Dispersity evolution during this multiblock synthesis. c, SEC traces illustrating the molecular weight distributions for consecutive cycles during the synthesis of an alternating low and high dispersity pentablock copolymer. d, Dispersity evolution during this multiblock synthesis.

Figure 5 .

 5 Figure 5. Decablock copolymers synthesized with tunable dispersity. a, SEC traces illustrating the molecular weight distributions for consecutive cycles during the synthesis of a decablock copolymer. In block 1, a high dispersity polymer is prepared, followed by targeted decreases and increases in the dispersity of subsequent blocks on addition of acid or base. b, The controlled evolution of dispersity during this multiblock synthesis. c,[START_REF] Lutz | Sequence-controlled polymers[END_REF] H NMR spectra for each cycle, illustrating the monomer conversion for each block (≥95% in all cases). d, SEC traces illustrating the molecular weight distributions for consecutive cycles during the synthesis of a decablock copolymer. In block 1, a low dispersity polymer is prepared, followed by targeted increases and decreases in the dispersity of subsequent blocks on addition of acid or base. e, The controlled evolution of dispersity during this multiblock synthesis. f, 1 H NMR spectra for each cycle, illustrating the monomer conversion for each block (≥95% in all cases).

Acknowledgements A.A. gratefully acknowledges ETH Zurich (Switzerland) for financial support. N.P.T. acknowledges the award of a DECRA Fellowship from the ARC (DE180100076). We acknowledge Manon Rolland for her artistic contribution in the conceptual figure. Finally, we acknowledge Laurent Bigler and Urs Stadler (University of Zurich) for MALDI-TOF-MS access.

Tailoring Polymer Dispersity in Higher-Order Multiblock Copolymers

Excited by this data, we then targeted even higher-ordered multiblock copolymers consisting of further variations in dispersity and also a larger number of blocks. First, we targeted a decablock copolymer. Under acidic conditions, the first block was synthesized with a low initial Ɖ value (Ɖ=1.15, Figures 5a-c & Supplementary Table 15 Entry 1). By switching to basic conditions together with the next monomer addition (deprotonated RAFT agent), the chain extension yielded a diblock copolymer with a clear shift to higher molecular weight and a much higher dispersity value (Ɖ=1.62, Figures 5a-c & Supplementary Table 15 Entry 2). For the next three monomer additions, a small amount of acid was injected thus leading to the preparation of triblock, tetrablock and pentablock copolymers with lower dispersities (ƉTriblock=1.49, ƉTetablock=1.40 and ƉPentarablock=1. 35, respectively, Figures 5a-c & Supplementary Table 15 Entries 3-5). We then aimed to hold the dispersity at ~1.35 and to do so, no additional acid or base was added for the next four blocks. Indeed, the hexablock, heptablock, octablock and nonablock copolymers presented a value at around ~1.35 (ƉHexablock=1. 35, ƉHeptablock=1.33, ƉOctablock=1.35 and ƉNonablock=1.37 respectively, Figures 5a-c & Supplementary Table 15 Entries 6-9). For the last block, a protonation of the RAFT agent was attempted leading to a decablock copolymer with a final dispersity of 1.27 (Figures 5a-c the versatility of our approach to operate not only with another polymer class but also in organic media. We finally wanted to demonstrate the applicability of our method with low kp monomers such as styrene. To do so, we attempted to in-situ chain extend a PEA macroinitiator (Ɖ ~ 1.58)

with styrene while adding various amounts of acid thus targeting P(EA-b-S) diblock copolymers with tailored dispersity. In the first diblock copolymer, when PEA reached high monomer conversion (~97%), styrene together with 5% of acid with respect to the RAFT agent were added leading to a diblock copolymer with a dispersity of 1.68 (Supplementary Figure 36a & Supplementary Table 23). In a second diblock, PEA was chain extended with styrene along with a higher amount of acid (20% with respect to the RAFT agent) yielding a diblock copolymer with a dispersity of 1.44 (Supplementary Figure 36b & Supplementary Table 23).

In a final diblock copolymer, PEA was chain extended with styrene using an excess of acid, yielding a well-defined diblock copolymer with a dispersity of 1.26 (Supplementary Figure 36c & Supplementary Table 23). We also attempted to control the dispersity during the 3839). We could also synthesize triblock copolymers with the dispersity of the first two blocks composed of acrylates and acrylamides could be fully controlled, followed by a third unconjugated block (ƉPEA=1.18, ƉP(EA-b-NAM)=1. 55 and ƉP(EA-b-NAM-b-NVK)=1.37, Suppplementary Table 28 & Supplementary Figures 40). Taken all together, our method can operate for both low and high kp monomers and unconjugated moieties can also be incorporated even though their dispersity cannot be fully controlled.

Conclusions

To summarize, we have reported a new generation of sequence-controlled multiblock copolymers with on demand control over dispersity. A number of multiblock copolymers could be efficiently synthesized with gradual increasing and gradual decreasing dispersities, alternating dispersities or any combination thereof. This breakthrough was achieved by regulating the chain transfer activity of a RAFT agent through the simple addition of acid or base together with the new monomer additions. Despite full control over the dispersity, very

Methods

See supplementary information for detailed methods and protocols

Synthesis of a pentablock copolymer from a high Ð first block (descending in dispersity)

Into a 4 mL glass vial, 7.70 mg of methyl 2-[methyl(4-pyridinyl)carbamothioylthio]propionate (CTA, 28.5 μmol, 1 equiv.) were dissolved in 0.29 mL of DMF. A stock solution of VA-044 (6.2 mg) was prepared in 10 mL of water, and 500 μL of this solution (0.32 mg, 0.96 μmol, 0.034 equiv.) were transferred to the vial. Subsequently,

Author contributions

Competing Interests

The authors declare no competing interests

Additional Information

Supplementary information is available for this paper at https://doi.org/xxx.

Correspondence and requests for materials should be addressed to A.A (athina.anastasaki@mat.ethz.ch).

Reprints and permissions information is available at www.nature.com/reprints.


Alexandra M Bitty-Anderson
email: alexandra.bitty-anderson@u-bordeaux.fr

Fifonsi A Gbeasor-Komlanvi

Akila W Bakoubayi

Martin K Tchankoni

Arnold J Sadio

Mounerou Salou

Didier Claver A Dagnra

Koumavi Ekouevi

Patrick A Coffie

Manisha Yadav

Sheila Araujo Teles

Didier K Ekouevi

Patrick A Co

High burden of self-reported sexually transmitted infections among female sex workers in Togo in 2021

Keywords: sexually transmitted infections (STI), female sex workers (FSW), key populations, sub-Saharan Africa, STI symptoms

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background: Sexually Transmitted Infections (STI) remain a public health challenge, especially for Female Sex Workers (FSW) who are particularly vulnerable. In sub-Saharan Africa where the overlapping epidemics of STI and HIV are concentrated among key populations, epidemiological data are needed in order to better understand STI trends in this population. The aim of this study was to assess the prevalence of self-reported STI symptoms and their risk factors among FSW in Togo in .

Methods: A cross-sectional study was completed in June and July among FSW in two cities of Togo: in Lomé (capital city, south of Togo) and in Kara (north of Togo). A snowball sampling method was used and after consent, a standardized questionnaire was administered by trained research sta to collect information on STI. A multivariate logistic regression model was used to identify factors associated with self-reported STI.

Results: A total of FSW, in Lomé and in Kara participated in this study. Median age was [IQR: -] and STI symptoms in the previous months were reported by FSW [ . %, % CI: ( . -. )], among whom ( . %) sought medical care. The most common reported symptoms were abnormal vaginal discharge (n = ;

. %) and vaginal itching (n = ; . %). Current STI symptoms (at the time of the survey) were reported by FSW ( . %). In multivariable logistic regression, selfreported STI symptoms in the previous months was negatively associated with living in the Lomé and older age and positively associated with having more than clients in the previous week and being victim of violence.

Introduction

Sexually Transmitted Infections (STIs) are an important public health challenge worldwide, with an estimated 357 million new cases of four curable STIs (chlamydia, gonorrhea, trichomoniasis and syphilis) among 15-49 year olds, 417 million cases of herpes simplex type 2 (HSV2), and 291 million cases of human papillomavirus (HPV) among women each year (1). In sub-Saharan Africa (SSA), the incidence of STI was estimated at 63 million per year making it one of the most affected area in the World, sharing an estimated 40% of the global STI burden (1)[START_REF]Global health sector strategy on sexually transmitted infections 2016-2021: implementation framework for the African Region[END_REF][START_REF] Stewart | Sexually transmitted infections among African women: an opportunity for combination sexually transmitted infection/HIV prevention[END_REF].

The epidemics of STIs have considerable repercussions on morbidity, mortality and the overall quality of life and wellbeing of individuals. STIs are among the main causes of infertility worldwide, are responsible for fetal and neonatal deaths (300 000 fetal and neonatal deaths each year), could lead to pelvic inflammatory disease and cervical cancer with HPV infection responsible for 530 000 cases of cervical cancer and 264 000 cervical cancer deaths each year (1,[START_REF] Unemo | Sexually transmitted infections: challenges ahead[END_REF]. Furthermore, STIs could cause cellular changes preceding cancers, and are recognized for facilitating HIV transmission (1). As such, key populations and female sex workers (FSW) in particular are among the most vulnerable population due to the social, economic and structural challenges they face. In fact, HIV prevalence among FSW in SSA is estimated at 36.9% compared to a 13.5% prevalence among women of reproductive age [START_REF] Beyrer | An action agenda for HIV and sex work[END_REF]. The burden of STIs in this population is similarly high in SSA: 50 to 66% of FSW typically have a curable STI at any one time, 10% have active genital ulcer, 30% have syphilis, more than 30% have gonorrhea and chlamydia and 60% have HSV2 [START_REF]Preventing HIV among sex workers in sub-Saharan Africa: A literature review[END_REF]. In Togo, most recent estimates indicate an overall prevalence of STI among FSW ranging from 4.2% for gonorrhea to 32.9% for HPV in 2017, based on biological testing [START_REF] Ferré | Prevalence of human papillomavirus, human immunodeficiency virus and other sexually transmitted infections among female sex workers in Togo: a national cross-sectional survey[END_REF][START_REF] Tchankoni | Prevalence and factors associated with trichomonas vaginalis infection among female sex workers in Togo, 2017[END_REF].

In Togo and in most countries of SSA and low-and-middleincome countries (LMIC), the syndromic management of STI is recommended and syndromic management flow charts and algorithms remain the standard of care for the prevention and control of STI [START_REF]Guidelines for the management of symptomatic sexually transmitted infections[END_REF]. The establishment of an etiologic diagnosis of STI, though ideal, could be difficult and unfeasible in those settings for several reasons including the lack of established laboratories that could delay same-day diagnosis results treatment or the lack of qualified human resources. In those cases, as is the case in Togo, a symptom-based approach to STI treatment has been recommended and simplified algorithms for each symptom have been developed by the World Health Organization (WHO) since 1984 and regularly updated since [START_REF]Guidelines for the management of symptomatic sexually transmitted infections[END_REF]. Hence, in Togo screening and treatment of STIs primarily rely on self-reported symptoms. Based on this strategy and directly addressing this first pillar of the WHO global health sector strategy on STI of providing an update on the trends on STI prevalence, the aim of this study was to document the prevalence of self-reported STI and their risk factors among FSW in the two main cities of Togo (1).

Materials and methods

Study design and setting

A cross-sectional study, using a snowball sampling method, was conducted among FSW in June and July 2021 in the two most populous cities of Togo: Lomé, the capital city and Kara. Togo is a country of West Africa, with a population of 7.6 million inhabitants in 2018, covering 57,000 square kilometers with an average density of 133 inhabitants per square kilometers, an infant mortality of 45.2/1,000 and an estimated life expectancy of 64.5 years old. Lomé is located on the coast of the Atlantic Ocean, in the far south-western corner of Togo. Kara, the second most populous city of the country is located 413 km north of the capital city. Based on previous mapping and size estimation studies among key populations including FSW in Togo (9), Lomé and Kara were selected as study sites.

Sample size and study population

FSW were eligible to participate in the study if they were 18 years and older; self-identified as sex workers (defined as engaging in transactional sex at least in the past 6 months; having sex in exchange for money or goods with clients as opposed to emotional partners or boyfriends); and were in the possession of a recruitment coupon. The sample size was determined based on STI prevalence among FSW in Togo in 2017 hovering at 6% [START_REF] Ferré | Prevalence of human papillomavirus, human immunodeficiency virus and other sexually transmitted infections among female sex workers in Togo: a national cross-sectional survey[END_REF][START_REF] Tchankoni | Prevalence and factors associated with trichomonas vaginalis infection among female sex workers in Togo, 2017[END_REF]. With a precision of 3%, a confidence interval of 95% and an assumption of 10% missing data/non-response rate, the estimated minimum sample size was 264.

Steps were taken prior to the beginning of the study to identify and establish, with leaders from local FSW nongovernmental organizations, the "hotspots" for FSWs, including brothels and bars. A total of 10 sites were selected across Lomé and 03 sites in Kara. A snowball sampling method was used to identify potential participants who then recruited other participants until the required sample size was reached at each site. Leaders were identified in each location, contacted and recruited into the study. Each leader received recruitment coupons to recruit other peers. A total of 2 leaders of FSW, who were also the first "seeds" were required per site (identified "hotspots"). The leaders from each site collaborated with the research team to arrange a space within or near the site that would protect the privacy of each participant. Leaders were informed of the research process and notified prior to the arrival of the research team. At each site, the research team was accompanied by a peer educator from a community-based FSW NGO. Questionnaires were pre-tested among three randomly selected FSW in Kara and Lomé.

Data collection

After verifying eligibility and obtaining written consent, trained research staff (medical students) administered a faceto-face structured questionnaire in participant's language of choice (French, Ewe, Kabyè or English) using Google Forms. Data were collected using a questionnaire adapted from the guidelines on repeated behavioral surveys in population at risk of HIV [START_REF] Amon | Guidelines for Repeated Behavioral Surveys in Populations at Risk of HIV[END_REF] and based on an extended literature review on the subject. The questionnaire included questions on six main themes: (1) socio-demographic characteristics; (2) sexual history (types of partners, condom use); (3) STI and other sexual and reproductive health history; (4) Pre-exposure Prophylaxis; and (5) violence. Each questionnaire took approximately twenty minutes to complete. No personal identifiers were collected and each participant was assigned an identification number.

In addition, participants presenting STI symptoms at the time of the survey were offered the opportunity to be treated on the spot by a medical professional, based on the syndromic approach and STI guidelines currently in vigor in Togo [START_REF]Guidelines for the management of symptomatic sexually transmitted infections[END_REF]. Another written consent was obtained for this part of the study, for those accepting to be treated. Participants were requested to provide a telephone contact information for followup on their symptoms and future referral in case of symptoms persistence. Patients were informed that their information will be kept confidential. Each participant received male condoms, lubricants and an amount of XOF 2,000 (around 4 US dollars) for their participation in the study.

Measurements

Based on the syndromic management of STI, self-reported STI symptoms were considered a proxy for possible STI [START_REF]Guidelines for the management of symptomatic sexually transmitted infections[END_REF][START_REF] Amon | Guidelines for Repeated Behavioral Surveys in Populations at Risk of HIV[END_REF][START_REF] Boothe | High burden of self-reported sexually transmitted infections among key populations in Mozambique: the urgent need for an integrated surveillance system[END_REF]. Hence, self-reported STI during the past 12 months was assessed using the question: "During the past 12 months, did you have any STI symptoms?"; Medical consultations for those STI symptoms was assessed using the question: "During the past 12 months, did you have a consultation in a health care service for those STI symptoms?". For symptoms of STI, participants were asked: "Which of the following symptoms did you have during the past 12 months?". Choices of answers included abnormal vaginal discharge (odorous and/or colored), vaginal itching, genital ulceration, pain while urinating, abdominal pain. For current symptoms, the question asked was: "Do you currently have STI symptoms?". The definition of STI was explained to participants prior to the beginning of the survey. Each measure was thoroughly explained to participants in case they could not understand. STI treatment was based on the WHO guidelines for the management of symptomatic STI and currently implemented in Togo. This guidelines provides specific treatment options and algorithm for each STI syndrome [START_REF]Guidelines for the management of symptomatic sexually transmitted infections[END_REF].

Statistical methods

Data was collected on Google Forms, which provided directly a Microsoft Excel output. Analyses were performed using STATA software (STATA TM 12.0 College Station, Texas, USA). Results of descriptive analysis were presented as median and interquartile range (IQR) for quantitative variables and frequency and proportions for categorical variables. The prevalence of STI, calculated by dividing the number of TS who had at least one episode of STI by the total number of TS, and its 95% Confidence interval (95% CI) were calculated. Univariate and multivariate logistic regression were performed with a stepwise-descending selection procedure to identify factors associated with STI-self-report in the previous 12 months. The selection of covariates for multivariate analysis was based on the univariate analyses with factors associated with a p-value <0.20. We deemed a p-value <0.05 as statically significant for all analyses. Associations in the regression model were expressed as adjusted odds ratio (aOR).

Ethical considerations

This study was approved by the Bioethics committee for Health research of the Ministry of Health (no. 11/2020) in Togo. Prior to the beginning of the study, participants were told about study purpose, procedures, protection of privacy and confidentiality and provided written consent. No identifiers were collected, hence no information that could reveal their identity was entered.

Results

Sociodemographic characteristics and sexual history

A total of 447 FSW were recruited, 300 in Lomé and 147 in Kara. Median age was 30 years old [Interquartile range (IQR): 24-38] and 63.8 % (n = 285) were single. Most FSW had reached at least a secondary school level of education (n = 218; 48.8%) and had other income generating activities aside from sex work (n = 262; 58.6%), mainly in trade and services (hairdressing, tailoring, housemaid, bar hostess) (Table 1A).

Sexual history

Median age at first sex was 18 [IQR: [START_REF] Ingabire | Female sex workers in Kigali, Rwanda: a key population at risk of HIV, sexually transmitted infections, and unplanned pregnancy[END_REF][START_REF] Tounkara | Prevalence and factors associated with HIV and sexually transmitted infections among female sex workers in Bamako, Mali[END_REF][START_REF] Diabaté | Gonorrhea, Chlamydia and HIV incidence among female sex workers in Cotonou, Benin: a longitudinal study[END_REF][START_REF] Torrone | Prevalence of sexually transmitted infections and bacterial vaginosis among women in sub-Saharan Africa: an individual participant data meta-analysis of 18 HIV prevention studies[END_REF][START_REF] Newman | Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012based on systematic review and global reporting[END_REF] and median age at first transactional sex was 23 [IQR: [START_REF] Torrone | Prevalence of sexually transmitted infections and bacterial vaginosis among women in sub-Saharan Africa: an individual participant data meta-analysis of 18 HIV prevention studies[END_REF][START_REF] Newman | Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012based on systematic review and global reporting[END_REF][START_REF] Turner | Baseline infection with a sexually transmitted disease is highly predictive of reinfection during follow-up in malagasy sex workers[END_REF][START_REF] Das | High rates of reinfection and incidence of bacterial sexually transmitted infections in a cohort of female sex workers from two Indian cities: need for different STI control strategies?[END_REF][START_REF] Wi | Diagnosing sexually transmitted infections in resource-constrained settings: challenges and ways forward[END_REF][START_REF] Vickerman | Using mathematical modelling to estimate the impact of periodic presumptive treatment on the transmission of sexually transmitted infections and HIV among female sex workers[END_REF][START_REF] Labbé | Periodical antibiotic treatment for the control of gonococcal and chlamydial infections among sex workers in Benin and Ghana: a cluster-randomized placebo-controlled trial[END_REF][START_REF] Shahmanesh | Effectiveness of interventions for the prevention of HIV and other sexually transmitted infections in female sex workers in resource poor setting: a systematic review[END_REF][START_REF] Ferguson | A comprehensive review of HIV/STI prevention and sexual and reproductive health services among sex Workers in Conflict-Affected Settings: call for an evidence-and rights-based approach in the humanitarian response[END_REF][START_REF] Decker | Gender-based violence against female sex workers in Cameroon: prevalence and associations with sexual HIV risk and access to health services and justice[END_REF][START_REF] Decker | Estimating the impact of reducing violence against female sex workers on HIV epidemics in Kenya and Ukraine: a policy modeling exercise[END_REF][START_REF] Awungafac | Systematic review of sex work interventions in sub-Saharan Africa: examining combination prevention approaches[END_REF][START_REF]Condoms are a critical part of combination prevention[END_REF]. Participants had a median of six clients [IQR: 4-10] in the preceding 7 days in Lomé, while in Kara, they had a median of four clients [IQR: 2-6] with an overall median earning between XOF 10,000 (17.29 USD) and XOF 25,000 (43.22 USD) in those preceding 7 days (Table 1B).

A total of 259 (57.5%) FSW confirmed having a non0paying partner (defined as a boyfriend; emotional partner as opposed to clients, paying partner), and only 32.0% (n = 83) indicated using condoms with those partners during the last sexual intercourse. In regards to the use of condoms with clients, 95.7% (n = 428) indicated using condoms with their clients during their last sexual intercourse, and 87.7% (n = 392) indicated using condoms systematically (every time) with their clients in the previous month. Nearly a fifth of the sample (19.7%; n = 88) indicated having experienced condom breakage or slippage in the previous month. Almost three-fourth of the sample (n = 313; 70.0%) indicated having done an HIV test in the past 12 months and 21 of them (6.7%) reported being HIV positive (Table 1B).

Self-reported STI

A total of 191 [42.7%, 95% CI: (38.1-47.5)] participants reported having STI symptoms in the 12 months preceding ] reported current STI symptoms (at the time of the survey). The most commons symptoms reported at the time of the survey were abnormal vaginal discharge (n = 52; 59.1%) and vaginal itching (n = 41; 46.6%). Abnormal vaginal discharge was mostly reported by FSW in Lomé (n = 40; 59.7%); while abdominal pain was mostly reported in Kara (n = 15; 71.4%) with a significant difference between the two cities (n = 18; 26.9; p < 0.001) (Figure 1). The median number of current symptoms reported by each participant was 2 [IQR: (1-2)].

Fourteen women (15.9%) with current STI symptoms (at the time of the survey) indicated having consulted a medical professional for those symptoms. The 74 remaining women with at least one symptom at the time of the survey were treated according the WHO guidelines for the management of symptomatic STI, currently in vigor in Togo. A total of 31 FSW agreed to be contacted to document the disappearance or persistence of symptoms. Of these, 20 (64.5%) reported no symptoms after 10days follow-up, two (6.5%) reported persisting symptoms (vaginal discharge and vaginal itching) and nine (29.0%) were lost to follow-up.

Factors associated with self-reported STI

In multivariate logistic regression, living in Lomé [aOR = 0.56; 95% CI: (0.36-0.88, p = 0.01) and being 35 years old and older [aOR = 0.53; 95% CI: (0.31-0.90), p = 0.04] were negatively associated with self-reported STI symptoms. In addition, having more than 15 clients in the previous week [aOR = 2.30; 95% CI: (1.20-4.38), p = 0.02], and being a victim of violence (emotional, physical or sexual) [aOR = 1.61; 95% CI: (1.61-2.45), p = 0.03] increased the odds of self-reported STI symptoms (Table 3). Second analyses were performed using current STI self-report (n = 88) as dependent variable and no factors were associated with this variable.

Discussion

This study sought to assess the prevalence of self-reported STI symptoms and its associated factors among FSW in the two main cities of Togo. The overall prevalence of STI self-reported symptoms over the previous 12 months was 42.7%, and among 60.7% of them who sought medical care for those symptoms, the most common STI symptoms were abnormal vaginal discharge (odorous and/or colored) and vaginal itching, despite quite high proportions of condom use with clients (95.7%).

This prevalence of self-reported STI symptoms in the previous year is high, but slightly comparable to those found in the region among key populations using the same method, with rates of 33.5% among FSW in Mozambique [START_REF] Boothe | High burden of self-reported sexually transmitted infections among key populations in Mozambique: the urgent need for an integrated surveillance system[END_REF], and 35.6% in Ethiopia [START_REF] Hailemariam | Prevalence of self-reported symptoms of sexually transmitted infection among establishment-based female sex workers in Ethiopia[END_REF]. Also, the current STI symptoms at the time of the survey was relatively high (19.7%), despite the self-reported high rate of condom use with clients during the last sexual intercourse (89.7%) and during the last 30 days (89.7%) in this population. Similar proportions of condom use have been observed in previous studies carried out in the region [START_REF] Kakisingi | Prevalence and characteristics of HIV infection among female sex workers in Lubumbashi, Democratic Republic of Congo[END_REF][START_REF] Patel | HIV prevalence, risk factors for infection, and uptake of prevention, testing, and treatment among female sex workers in Namibia[END_REF][START_REF] Becquet | Sexual health needs of female sex workers in Côte d'Ivoire: a mixed-methods study to prepare the future implementation of pre-exposure prophylaxis (PrEP) for HIV prevention[END_REF]. Studies using biological methods for STI confirmation also confirm this high burden of STI among FSW with prevalence estimated at 43% for syphilis among HIV-positive FSW in Rwanda ( 16) and 35.1% of FSW having at least one STI in a study in Mali [START_REF] Tounkara | Prevalence and factors associated with HIV and sexually transmitted infections among female sex workers in Bamako, Mali[END_REF]. In Togo, the highest prevalence found was that of hrHPV of 48.5% among HIV-positive FSW and 31.1% among HIV-negative FSW [START_REF] Ferré | Prevalence of human papillomavirus, human immunodeficiency virus and other sexually transmitted infections among female sex workers in Togo: a national cross-sectional survey[END_REF]. The data on the incidence is rare in this population. In a survey conducted in Benin, incidence of STI was 10.8 event/personyears among FSW [START_REF] Diabaté | Gonorrhea, Chlamydia and HIV incidence among female sex workers in Cotonou, Benin: a longitudinal study[END_REF]. All of these studies confirm high rate of STI among FSW. In comparison with data in the general population, studies among women indicate rates at least a third lower than that of FSW. For example, STI prevalence was assessed among women in the general population in Southern Africa, Southeast Africa and Eastern Africa and the overall prevalence of chlamydia, gonorrhea and syphilis among women aged 25 to 49 ranged from 0.9% to a maximum of 7.5% [START_REF] Torrone | Prevalence of sexually transmitted infections and bacterial vaginosis among women in sub-Saharan Africa: an individual participant data meta-analysis of 18 HIV prevention studies[END_REF].

Another study found a global prevalence of 4.2% for chlamydia, 0.8% for gonorrhea and 5.0% for trichomoniasis among women 15 to 49 years old [START_REF] Newman | Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012based on systematic review and global reporting[END_REF]. A better understanding of the dynamic of STI is needed in a context of high condom use among FSW, in order to develop more appropriate and adapted, tailored interventions for this population. Among FSW who consulted for their STI symptoms, more than a third of the sample consulted more than twice throughout the year for STI symptoms, evoking either a STI reinfection or STI symptoms persistence. The high frequency of STI reinfection among FSW has been confirmed by other studies. In a study among Malagasy FSW, those who had an STI at baseline were 2-4 times more likely to have an STI during follow-up [START_REF] Turner | Baseline infection with a sexually transmitted disease is highly predictive of reinfection during follow-up in malagasy sex workers[END_REF]. In India, a study among FSW revealed high rates of STI persistence, despite an STI care package intervention [START_REF] Das | High rates of reinfection and incidence of bacterial sexually transmitted infections in a cohort of female sex workers from two Indian cities: need for different STI control strategies?[END_REF]. This study also demonstrated an increasing trend toward asymptomatic STI [START_REF] Das | High rates of reinfection and incidence of bacterial sexually transmitted infections in a cohort of female sex workers from two Indian cities: need for different STI control strategies?[END_REF]. This, coupled to the low accuracy of syndromic diagnosis of STI could lead to overtreatment, under treatment, missed treatment or incorrect treatment of STI [START_REF] Wi | Diagnosing sexually transmitted infections in resource-constrained settings: challenges and ways forward[END_REF]. Advocacy could be directed toward additional effort for investment in simpler and more affordable laboratory diagnostics or the implementation of new and innovative strategies for STI diagnosis. Indeed, recent studies have suggested innovative strategies such as pointof-care tests (POCTS) under-development for the etiologic diagnosis of STI that could take into account the challenges and barriers faced in resource-limited settings and by FSW [START_REF] Stewart | Sexually transmitted infections among African women: an opportunity for combination sexually transmitted infection/HIV prevention[END_REF][START_REF] Wi | Diagnosing sexually transmitted infections in resource-constrained settings: challenges and ways forward[END_REF]. Other biomedical interventions include periodical antibiotic treatment or periodic presumptive treatment, although both methods have inconclusive results in terms of efficacy for STI prevention [START_REF] Stewart | Sexually transmitted infections among African women: an opportunity for combination sexually transmitted infection/HIV prevention[END_REF][START_REF] Vickerman | Using mathematical modelling to estimate the impact of periodic presumptive treatment on the transmission of sexually transmitted infections and HIV among female sex workers[END_REF][START_REF] Labbé | Periodical antibiotic treatment for the control of gonococcal and chlamydial infections among sex workers in Benin and Ghana: a cluster-randomized placebo-controlled trial[END_REF]. Nevertheless, biomedical strategies should be combined with innovative behavioral and structural interventions that take into account the needs of this population [START_REF] Shahmanesh | Effectiveness of interventions for the prevention of HIV and other sexually transmitted infections in female sex workers in resource poor setting: a systematic review[END_REF]. A paradigm-shift from strictly biomedical strategies for STI prevention among FSW to innovative publichealth driven solutions that take into account structural, economic, social and political factors is an imperative [START_REF] Beyrer | An action agenda for HIV and sex work[END_REF][START_REF] Ferguson | A comprehensive review of HIV/STI prevention and sexual and reproductive health services among sex Workers in Conflict-Affected Settings: call for an evidence-and rights-based approach in the humanitarian response[END_REF].

Several factors were associated with self-reported STI symptoms among FSW. FSW victim of violence were more than twice as likely to report STI. This result is consistent with previous studies that confirmed that violence is associated with sexual HIV risk and fear of accessing health care services [START_REF] Decker | Gender-based violence against female sex workers in Cameroon: prevalence and associations with sexual HIV risk and access to health services and justice[END_REF]. Similarly, a reduction of 25% in HIV incident cases was observed when physical and sexual violence was reduced in Ukraine and Kenya [START_REF] Decker | Estimating the impact of reducing violence against female sex workers on HIV epidemics in Kenya and Ukraine: a policy modeling exercise[END_REF]. Lifting barriers to access to care by providing stigma-free environment for this population, as well as addressing structural factors such as prevention of genderbased violence and the promotion of human rights should be a priority. The use of peer-educators and communitybased organizations has been proven effective in the context of SSA [START_REF] Awungafac | Systematic review of sex work interventions in sub-Saharan Africa: examining combination prevention approaches[END_REF]. In addition to these, interventions comparing a differentiated model of care, tailored specifically to FSW and gearing only to them and an integrated model of care with trained healthcare professionals capable of catering to the specific needs of FSW, should be tested. Services that could be part of a differentiated model of care could include in addition to medical aspects of STI prevention, psychological and legal assistance. This study also found that self-reported STI symptoms was significantly associated with having more than 15 clients per week. Although a prevention message of limiting the number of clients would be difficult to achieve as this is related to income and thus livelihood of FSW, the consistent and correct use of condoms remains the only appropriate strategy. Condom use is an essential element of STI and HIV prevention with simulations indicating that condom use scale-up since 1990 has averted 117 million of new infections, half of them in SSA [START_REF]Condoms are a critical part of combination prevention[END_REF]. However, promotion of condom use cannot be used as a stand-alone strategy as other factors such as access to condoms, condom use negotiation and economic and personal factors (i.e., partner's trust) influence the use of condoms in this population [START_REF] Becquet | Sexual health needs of female sex workers in Côte d'Ivoire: a mixed-methods study to prepare the future implementation of pre-exposure prophylaxis (PrEP) for HIV prevention[END_REF][START_REF] Decker | Inconsistent condom use among female sex workers: partner-specific influences of substance use, violence, and condom coercion[END_REF]. Combination prevention with access to condoms, education and continuous campaigns for raising awareness on the importance of the use of condoms with partners and clients, the involvement of partners and clients into STI prevention, are strategies that could be used to offset the burden of STI in this population [START_REF]Condoms are a critical part of combination prevention[END_REF]. The involvement of partners in STI prevention strategies and as targets of STI prevention effort is of utmost importance, particularly since this study found that 32.0% of FSW who had a partner did not use condoms with them during their last sexual intercourse. Furthermore, older age was protective of STI self-reported symptoms. This finding should be further explored as the opposite trend has been observed in other studies, especially with HIV infection with older age associated with HIV infection [START_REF] Kakisingi | Prevalence and characteristics of HIV infection among female sex workers in Lubumbashi, Democratic Republic of Congo[END_REF][START_REF] Ingabire | Female sex workers in Kigali, Rwanda: a key population at risk of HIV, sexually transmitted infections, and unplanned pregnancy[END_REF][START_REF] Musyoki | Prevalence of HIV, sexually transmitted infections, and risk behaviours among female sex workers in Nairobi, Kenya: results of a respondent driven sampling study[END_REF]. However, one hypothesis could be that young FSW lack the experience necessary to protect them against STI, such as inexperience in dealing with clients or in negotiating condom use [START_REF] Crankshaw | Age matters: determinants of sexual and reproductive health vulnerabilities amongst young women who sell sex (16-24 years) in Zimbabwe[END_REF]. Young FSW face particular vulnerabilities putting them at heightened risk for STI and HIV, which should be specifically addressed in national strategies and policies of HIV and STI control.

In our study, some significant city differences were noted. FSW from Lomé had lower odds of STI self-reported symptoms compared to their counterparts in Kara. This finding could be expected when assuming the STI burden among FSW follows the same trend as the HIV pandemic in the same population. As such, previous data of HIV prevalence among FSW in Togo has indicated the highest prevalence of HIV among FSW in Kara (26.8%) [START_REF]Conseil National de Lutte contre le SIDA et les Infections Sexuellement Transmissibles. Plan Stratégique National de Lutte contre le VIH et le SIDA: 2021-2025: Accélerer le mouvement[END_REF]. Despite higher vulnerabilities among FSW in Lomé (lower level of education, higher number of clients), more FSW in Lomé use condoms consistently with their clients compared to those in Kara which also could explain the lower odds of STI symptoms among FSW in Lomé. In addition, another hypothesis could be that FSW in Lomé, living in the capital with may have greater access to condoms in general and to sensitization campaigns on the importance of condom use with clients [START_REF]Programme national de lutte contre l'infection à VIH/Sida, les hépatites virales et les infections sexuellement transmissibles (PNLS-HV-IST)[END_REF]. There is a need for additional research on the STI prevalence across regions, and specifically in Kara to elucidate factors associated with high prevalence of STI and to develop targeted interventions specific to that region.

There are several limitations to this study. First, some symptoms declared might not directly be related to STI and in the same sense, asymptomatic STI might have been missed, which could have underestimated or overestimated the overall estimation of STI prevalence. Second a cross-sectional study was conducted, thus the temporal link between STI-self report and other factors could not be assessed. Also, as is the case with non-random sampling methods, the use of the snowball sampling method does not guarantee representation and could have introduced some bias. Finally, the use of a self-administered questionnaire could have generated biases, including social desirability bias (i.e., self-report of the use of condoms), memory bias and prevarication bias. Despite those limitations, this study is among the first in Togo to explore STI burden based on syndromic management and to show a relatively high prevalence of STI symptoms among FSW as demonstrated by other studies with STI diagnosis confirmed by laboratory analyses [START_REF] Tounkara | Prevalence and factors associated with HIV and sexually transmitted infections among female sex workers in Bamako, Mali[END_REF]. Results from this study suggest a heavy burden of STI among FSW, and as such those findings could serve as the basis for the development of policies and actions toward a better management of STI among FSW, as a public health priority.

Further research should couple STI self-reported symptoms with confirmation of etiological diagnosis of STI among FSW, in order to assess the effectiveness of syndromic management of STI in this population. Although the syndromic approach to STI management has had a significant impact on the burden of STI over the years, a review of its continued performance should be considered, especially among most-at-risk populations, such as FSW. In addition, trials on STI interventions focusing on different methods for prevention of STI in this population should be done. Directly observed treatment (DOT) or fielddelivered therapy for STI as experienced in this study seems an interesting approach that has proven to be successful for the management of STI [START_REF] Steiner | Fielddelivered therapy increases treatment for chlamydia and gonorrhea[END_REF]. However, the results observed should be confirmed. To our knowledge limited data are available using this approach for vulnerable populations, specifically FSW and for STI treatment, for comparison and lessons learned. Qualitative studies should also explore more deeply barriers to access to care and reasons for nonconsultation among FSW, as solutions to reduce the gap between FSW and utilization of health care services should be a priority.

Conclusion

Self-reported STI is highly prevalent among FSW, with most common symptoms abnormal vaginal discharge and vaginal itching and risk factors for STI symptoms including a high number of clients (more than 15) and being victim of violence. On the other hand, living in the capital city and being older (35 years old and older) were protective of self-reported STI. Additional research is needed in the area of STI prevention, treatment and management among FSW, including improvements in diagnosis, innovative interventions such as directly-observedtreatment as experimented in this study. Policy advocacy for structural improvements taking into account genderbased violence and access to care could also contribute to reducing the burden of STI and HIV among FSW in Togo.

FIGURE

  FIGURESelf-reported current STI symptoms among FSW in Togo in (n = ).

TABLE A

 A Sociodemographic characteristics of female sex workers in Togo in (N =).

	Characteristic	Total	Lomé	Kara
		(n = 447)	(n = 300)	(n = 147)
		N (%)	n (%)	n (%)
	Age (years)
	Median [IQR]*	30 (24-38)	30 (24-39)	29 (23-36)
	<30	221 48.5	147 49.0	74 50.3
	≥30	226 51.5	153 51.0	73 49.7
	Level of education
	None	88 19.7	76 25.4	12 8.2
	Primary	141 31.5	100 33.3	41 27.9
	Secondary	194 43.4	108 36.0	86 58.5
	University	24 5.4	16 5.3	8 5.4
	Marital status
	Living with a husband/a partner	46 10.3	30 10.0	16 10.9
	Divorced/separated/widowed	116 25.9	79 26.3	37 25.2
	Single	285 63.8	191 63.7	94 63.9
	Nationality
	Togolese	302 67.5	162 54.0	140 95.2
	Nigerian	84 18.8	79 26.3	5 3.4
	Ghanaian	41 9.2	41 13.7	0 0.0
	Other**	20 4.5	18 6.0	2 1.4
	Other income generating activities
	Yes	262 58.6	178 59.3	84 57.1
	No	185 41.4	122 40.7	63 42.9

*IQR, Interquartile Range. **Côte d'Ivoire, Mali, Bénin, Burkina Faso. Frontiers in Public Health frontiersin.org

TABLE B

 B Sexual history of female sex workers in Togo in (N =).

	Characteristic	Total	Lomé	Kara
		(n = 447)	(n = 300)	(n = 147)
		N (%)	n (%)	n (%)
	Age at first sex (years)
	Median [IQR]	18 (16-20)	18 (16-20)	18 (16-20)
	<18	201 45.0	135 45.0	66 44.9
	≥18	246 55.0	165 55.0	81 55.1
	Age at first transactional	23 (19-30)	24 (19-32)	22 (18-30)
	sex, Median [IQR]
	Number of years in sex work
	Median [IQR]	5 (3-11)	5 (3-11)	5 (3-11)
	<1	62 13.9	45 15.0	17 11.6
	Between 1 and 3	156 34.9	108 36.0	48 32.6
	>3	229 51.2	147 49.0	82 55.8
	Number of clients in the
	previous 7 days
	Median [IQR]	5 (3-10)	6 (4-10)	4 (2-6)
	≤7	300 67.1	176 58.7	124 84.4
	Between 8 and 14	95 21.3	81 27.0	14 9.5
	≥15	52 11.6	43 14.3	9 6.1
	Income earned in the last
	7 days (XOF) §
	Less than 10,000	128 28.6	63 21.0	65 44.2
	10,000-25,000	185 41.4	134 44.7	51 34.7
	25,000-100,000	113 25.3	96 32.0	17 11.6
	More than 100,000	4 0.9	3 1.0	1 0.7
	Does not know/would	17 3.8	4 1.3	13 8.8
	not like to answer
	Use of condom during
	last sexual intercourse
	with a client
	Yes	428 95.7	290 96.7	138 93.9
	No	19 4.3	10 3.3	9 6.1
	Consistent condom use
	with a client
	(use of condoms during
	last intercourse AND
	during last 30 days)
	Yes	384 89.7	265 88.3	119 81.0
	No	63 10.3	35 11.7	28 19.0
	Condom accident
	(breakage or slippage)
	Yes	88 19.7	71 23.7	17 11.6
	No	359 80.3	229 76.3	130 88.4
	§ 1 USD = 581.2 XOF and 1 e = 657 XOF.
	Frontiers in Public Health			frontiersin.org

TABLE Self -

 Self reported sexually transmitted infections symptoms during the previous months among female sex workers in Togo in (n = ).

		Total		Lomé		Kara		P
		(n = 447)		(n = 300)		(n = 147)
		n	%	n	%	n	%
	STI symptoms self-report	191	42.7	122	40.7	69	46.9	0.200
	Medical check-up for STI symptoms (yes)	116	60.7	57	46.7	59	85.5	<0.001
	Self-reported STI symptoms
	Abnormal vaginal discharge	78	67.2	33	57.9	45	76.3	0.03
	Abdominal pain	50	43.1	21	36.8	29	49.2	0.181
	Genital ulcer	32	27.6	26	45.6	6	10.2	<0.001
	Pain while urinating	28	24.1	11	19.3	17	28.8	0.231
	Vaginal itching	68	58.6	24	42.1	44	74.6	<0.001
	The bold values are significant values.

the survey. Among them, 116 (60.7%) reported consulting a medical professional for these symptoms. The most common STI symptoms reported among them were abnormal vaginal discharge (n = 78; 67.2%) and vaginal itching (n = 68; 58.6%), both significantly higher in Kara than in Lomé. Conversely, genital ulcer was significantly higher in Lomé than in Kara (45.6 vs. 10.2% p < 0.001) (Table

2

). The median number of reported symptoms per participant over the preceding year was 2 [IQR: 2-3], so was the median number of consultations in a health care service for those symptoms over the year [IQR:

[1][START_REF]Global health sector strategy on sexually transmitted infections 2016-2021: implementation framework for the African Region[END_REF][START_REF] Stewart | Sexually transmitted infections among African women: an opportunity for combination sexually transmitted infection/HIV prevention[END_REF]

. More than a quarter (n = 33; 28.4%) reported going more than twice for consultations in a health care service for those STI symptoms.

Eighty-eight participants [19.7%; 95% CI: (16.09-23.68)

Use of condoms during last sexual intercourse with a partner

  TABLE Factors associated with self-reported STI symptoms during the previous months among FSW in Togo in .

	Variables	Univariable analysis		Multivariable analysis
		OR (95% CI)	p	aOR (95% CI)	p
	City
	Kara	1		1
	Lomé	0.77 (0.52-1.15)	0.20	0.62 (0.40-0.95)	0.03
	Age (years)
	<25	1		1
	25-35	0.82 (0.52-1.29)	0.40	0.74 (0.46-1.19)	0.20
	>35	0.57(0.34-0.93)	0.02	0.58 (0.35-0.97)	0.04
	Level of education
	None/primary	1
	Secondary/university	0.89 (0.61-1.29)	0.54
	Marital status
	Married or living with a partner	1
	Divorced/separated/widowed	1.35 (0.67-2.71)	0.39
	Single	1.11 (0.58-2.10)	0.73
	HIV testing in the previous 12 months
	No	1
	Yes	0.81 (0.54-1.22)	0.32
	Condom breakage
	No	1
	Yes	1.52 (0.95-2.43)	0.07
	Duration of sex work (years)
	<1	1
	Between 1 and 3	1.00 (0.66-1.51)	0.99
	>3	1.12 (0.62-2.03)	0.70
	Number of clients in the previous 7
	days
	≤7	1		1
	Between 8 and 14	1.29 (0.81-2.06)	0.27	1.32 (0.81-2.18)	0.24
	≥15	2.30 (1.26-4.20)	0.006	2.15 (1.13-4.05)	0.02
	No	1
	Yes	0.59 (0.52-1.44)	0.59
	Non applicable (no partner)	0.86 (0.63-1.47)	0.86
	Health care seeking for non-STI
	reasons
	No	1
	Yes	0.67 (0.46-0.99)	0.04
	Victim of violence (emotional,
	physical or sexual)
	No	1		1
	Yes	1.61 (1.09-2.39)	0.01	1.60 (1.06-2.42)	0.03
	The bold values are significant values.
	Frontiers in Public Health				frontiersin.org

Frontiers in Public Health frontiersin.org

Acknowledgments

The authors would like to thank all the women who participated in this study. We would also like to acknowledge the ANRS|Maladies infectieuses émergentes for the doctoral scholarship provided for the first author of this work.

Funding

This work was supported by the African Research Center on Epidemiology and Public Health (CARESP).

Data availability statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

Ethics statement

The studies involving human participants were reviewed and approved by Bioethics Committee for Health Research, Ministry of Health, Togo (No. 11/2020). The patients/participants provided their written informed consent to participate in this study.

Author contributions

PC, DE, AB-A, and FG-K conceived and planned the study. AB-A, AB, AS, MS, and CD implemented the study and collected the data. AB-A, MT, and AS analyzed the data. AB-A wrote the original draft with input from PC, DE, and FG-K. All authors reviewed the results and approved the final version of the manuscript.

Conflict of interest

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.

Publisher's note

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