Michèle Chevrier

Gérald Lopez

Wojciech Zajaczkowski

Jurgen Kesters

Ruben Lenaerts

Mathieu Surin

Julien De Winter

Sébastien Richeter

Wojciech Pisula

Ahmad Mehdi

Pascal Gerbaux

Roberto Lazzaroni

Philippe Dubois

Wouter Maes

Bruno Ameduri
email: bruno.ameduri@enscm.fr

Sébastien Clément
email: sebastien.clement1@umontpellier.fr

Synthesis and properties of a P3HT-based ABA triblock copolymer containing a perfluoropolyether central segment

Keywords: block copolymer, poly(3-hexylthiophene), perfluoropolyether, bulk heterojunction organic solar cells

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

Conjugated semiconducting polymers have become unavoidable materials in active layers in organic electronics such as organic photovoltaics (OPVs), organic light-emitting diodes (OLEDs) and organic thin-film transistors (OTFTs). [START_REF] Heeger | Semiconducting polymers: the Third Generation[END_REF][START_REF] Grimsdale | Synthesis of Light-Emitting Conjugated Polymers for Applications in Electroluminescent Devices[END_REF][START_REF] Wang | Semiconducting π-Conjugated Systems in Field-Effect Transistors: A Material Odyssey of Organic Electronics[END_REF][START_REF] Guo | Designing π-conjugated polymers for organic electronics[END_REF] Among these materials, poly(3hexylthiophene) (P3HT) is certainly the most studied due to its well-balanced properties in terms of solubility, environmental/thermal stabilities and charge carrier transport, but also and above all, its synthetic availability. [START_REF] Marrocchi | Poly(3-hexylthiophene): synthetic methodologies and properties in bulk heterojunction solar cells[END_REF][START_REF] Kleinschmidt | Poly(3-hexylthiophene) (P3HT): fruit fly or outlier in organic solar cell research?[END_REF][START_REF] Chevrier | Functionalization of P3HT-Based Hybrid Materials for Photovoltaic Applications[END_REF][START_REF] Wadsworth | Progress in Poly (3-Hexylthiophene) Organic Solar Cells and the Influence of Its Molecular Weight on Device Performance[END_REF] Indeed, P3HTs with multiple architectures (homopolymers, random/block copolymers) and an excellent control over the molecular weight (Mw), molecular weight distribution (Ð) and regioregularity (RR) can readily be obtained via catalyst-transfer polymerization. [START_REF] Marrocchi | Poly(3-hexylthiophene): synthetic methodologies and properties in bulk heterojunction solar cells[END_REF][START_REF] Yokozawa | Catalyst-transfer condensation polymerization for precision synthesis of π-conjugated polymers[END_REF] Many studies have shown that the molecular packing and nanoscale morphology of P3HT determine its optoelectronic properties and thus, strongly influence the corresponding electronic applications. [START_REF] Agbolaghi | A comprehensive review on poly(3-alkylthiophene)-based crystalline structures, protocols and electronic applications[END_REF][START_REF] Koch | The impact of molecular weight on microstructure and charge transport in semicrystalline polymer semiconductors-poly(3-hexylthiophene), a model study[END_REF] Therefore, the control of the P3HT nanostructuration in the solid state have been pursued to achieve optimal device performance.

Block copolymers are interesting materials to generate suitable morphologies for optoelectronic applications due to their ability to self-assemble into ordered, periodic structures at the length scale of the individual polymer chains. [START_REF] Matsen | Unifying Weak-and Strong-Segregation Block Copolymer Theories[END_REF][START_REF] Bates | Block Copolymer Thermodynamics: Theory and Experiment[END_REF] In this respect, many block copolymers containing P3HT segment(s) were developed in particular, those combining a non-conjugated block (coil) and a P3HT (rod) block. To date, a wide variety of "coil-like" segments such as poly(styrene), [START_REF] Liu | Tuning the Electrical Conductivity and Self-Assembly of Regioregular Polythiophene by Block Copolymerization: Nanowire Morphologies in New Di-and Triblock Copolymers[END_REF][START_REF] Kaul | Synthesis of a Bifunctional Initiator for Controlled Kumada Catalyst-Transfer Polycondensation/Nitroxide-Mediated Polymerization and Preparation of Poly(3-hexylthiophene)-Polystyrene Block Copolymer Therefrom[END_REF][START_REF] Britze | Synthesis of Blockcopolymers P3HT-b-PS Using a Combination of Grignard-Metathesis and Nitroxide-Mediated Radical Polymerization[END_REF] poly(allene), [START_REF] Yu | Polyallene-block-polythiophene-blockpolyallene Copolymers: One-Pot Synthesis, Helical Assembly, and Multiresponsiveness[END_REF][START_REF] Ge | One-pot synthesized ABA tri-block copolymers for high-performance organic field-effect transistors[END_REF] poly(urethane), [START_REF] Liu | Tuning the Electrical Conductivity and Self-Assembly of Regioregular Polythiophene by Block Copolymerization: Nanowire Morphologies in New Di-and Triblock Copolymers[END_REF] polyphthalaldehyde, [START_REF] Pessoni | Synthesis of Polyphthalaldehyde-Based Block Copolymers: Utilization of a Thermo-Sacrificial Segment for an Easy Access to Fine-Tuned Poly(3-hexylthiophene) Nanostructured Films[END_REF] poly(methyl methacrylate), [START_REF] Moon | Facile Synthetic Route for Well-Defined Poly(3hexylthiophene)-block-poly(methyl methacrylate) Copolymer by Anionic Coupling Reaction[END_REF][START_REF] Gilroy | Fiber-like Micelles via the Crystallization-Driven Solution Self-Assembly of Poly(3-hexylthiophene)-block-Poly(methyl methacrylate) Copolymers[END_REF][START_REF] Ji | Self-Assembly of Ionizable "Clicked" P3HT-b-PMMA Copolymers: Ionic Bonding Group/Counterion Effects on Morphology[END_REF] poly(4-vinylpyridine), [START_REF] Lohwasser | Synthesis of Amphiphilic Rod-Coil P3HT-b-P4VP Carrying a Long Conjugated Block Using NMRP and Click Chemistry[END_REF][START_REF] Gernigon | Microstructure and Optoelectronic Properties of P3HT-b-P4VP/PCBM Blends: Impact of PCBM on the Copolymer Self-Assembly[END_REF] poly(ε-caprolactone), [START_REF] Surin | Regioregular poly(3-hexylthiophene)-poly(ε-caprolactone) block copolymers: Controlled synthesis, microscopic morphology, and charge transport properties[END_REF] polylactide, [START_REF] Grancharov | Stereocomplexed Materials Based on Poly(3-hexylthiophene)-b-poly(lactide) Block Copolymers: Synthesis by Organic Catalysis, Thermal Properties, and Microscopic Morphology[END_REF] poly(dimethoxysilane) [START_REF] Tsuchiya | Synthesis and characterization of poly(3hexylthiophene)-block-poly(dimethylsiloxane) for photovoltaic application[END_REF] and poly(ethylene glycol) [START_REF] Reichstein | Influence of Composition of Amphiphilic Double-Crystalline P3HT-b-PEG Block Copolymers on Structure Formation in Aqueous Solution[END_REF][START_REF] Erothu | Facile synthesis of poly(3-hexylthiophene)-block-poly(ethylene oxide) copolymers via Steglich esterification[END_REF] have been exploited to tune the crystallinity of the materials and their film morphology. Among rod-coil block copolymers, ABA-type rod-coil-rod block copolymers have rarely been investigated although they can provide new opportunities for directing selfassembly through inter-and intrachain interactions. [START_REF] Ge | One-pot synthesized ABA tri-block copolymers for high-performance organic field-effect transistors[END_REF][START_REF] Kang | Synthesis of poly(3-hexylthiophene)-block-poly(ethylene)block-poly(3-hexylthiophene) via a combination of ring-opening olefin metathesis polymerization and grignard metathesis polymerization[END_REF][START_REF] Brazard | Mimicking Conjugated Polymer Thin-Film Photophysics with a Well-Defined Triblock Copolymer in Solution[END_REF] Such P3HT triblock copolymers can also possess improved mechanical properties, similar to ABA triblock copolymer thermoplastic elastomers (TPEs), such as polystyrene-block-polybutadiene-blockpolystyrene (SBS). [START_REF] Peng | An ABA triblock copolymer strategy for intrinsically stretchable semiconductors[END_REF] Introducing a fluorinated segment as the coil block in a rod-coil block copolymer also allows manipulating the solid-state organization. Indeed, the immiscibility with hydrocarbon groups and the F• • •π, F• • •H and F• • •S inter-and intramolecular interactions may have a significant role in the self-assembly of the block copolymers in the solid-state. [START_REF] Babudri | Fluorinated organic materials for electronic and optoelectronic applications: the role of the fluorine atom[END_REF][START_REF] Clément | Synthesis and Supramolecular Organization of Regioregular Polythiophene Block Oligomers[END_REF][START_REF] Voet | Well-defined copolymers based on poly(vinylidene fluoride): From preparation and phase separation to application[END_REF] Incorporated as the coil segment into rod-coil block copolymers, fluorinated moieties may impart their unique properties (e.g. chemical inertness, hydrophobicity, and high thermal and oxidation stability). [START_REF] Meyer | Fluorinated conjugated polymers in organic bulk heterojunction photovoltaic solar cells[END_REF][START_REF] Friesen | Outstanding telechelic perfluoropolyalkylethers and applications therefrom[END_REF] Along that line, Mc Cullough et al. have shown that the attachment of a hydrophobic poly(fluorinated alkyl methacrylate) coil allows to significantly improve the charge carrier transport properties of regioregular poly(3-hexylthiophene) (rr-P3HT). [START_REF] Liu | Self Encapsulated Poly(3hexylthiophene)-poly(fluorinated alkyl methacrylate) Rod-Coil Block Copolymers with High Field Effect Mobilities on Bare SiO2[END_REF] The presence of fluorinated chains may also have a positive impact on the interfacial area between the donor and the acceptor as well as provide bicontinuous networks of donor and acceptor domains in bulk heterojunction organic solar cells, leading to enhanced percolation pathways for charge transport and thus, better power conversion efficiencies. [START_REF] Lim | A morphology controller for high-efficiency bulkheterojunction polymer solar cells[END_REF][START_REF] Kim | High-Efficiency Organic Solar Cells Based on End-Functional-Group-Modified Poly(3-hexylthiophene)[END_REF][START_REF] Jia | Boosting the efficiency and stability of polymer solar cells using poly(3-hexylthiophene)-based all-conjugated diblock copolymers containing pentafluorophenyl groups[END_REF] In this work, a telechelic bisazide fluorinated polyether is covalently attached by copper(I)catalyzed alkyne-azide cycloaddition (CuAAC) to an ethynyl end-functionalized regioregular P3HT, forming a rod-coil-rod triblock copolymer (P3HT-b-PFPE-b-P3HT). The thin film microstructure of the P3HT-b-PFPE-b-P3HT copolymers is investigated using grazing incidence wide-angle X-ray scattering (GIWAXS) and atomic force microscopy (AFM).

P3HT rod blocks self-assemble into highly ordered lamellar configurations, similar to rr-P3HT homopolymers. Device evaluation of photovoltaic cells based on P3HT-b-PFPE-b-P3HT blended with [START_REF] Kleinschmidt | Poly(3-hexylthiophene) (P3HT): fruit fly or outlier in organic solar cell research?[END_REF][START_REF] Kleinschmidt | Poly(3-hexylthiophene) (P3HT): fruit fly or outlier in organic solar cell research?[END_REF]-phenyl-C61-butyric acid methyl ester (PC61BM) reveals triblock copolymer only slightly underperforms as compared to commercial P3HT.

Materials and experimental method 2.1. Materials

All reactions were carried out under an atmosphere of dry argon. THF was dried using a solvent purification system PureSolve MD5 from Innovative Technology with alumina drying columns. 2-Bromo-3-hexyl-5-iodothiophene, [START_REF] Zhang | Synthesis of All-Conjugated Diblock Copolymers by Quasi-Living Polymerization and Observation of Their Microphase Separation[END_REF] (o-tolyl)Ni(PPh3)2Br, [START_REF] Beckmann | Square-Planar 2-Toluenido(triphenylphosphane)nickel(II) Complexes Containing Bidentate N,O Ligands: An Example of Planar Chirality[END_REF][START_REF] Park | Development of an Air-Stable Nickel Precatalyst for the Amination of Aryl Chlorides, Sulfamates, Mesylates, and Triflates[END_REF] telechelic PFPE-bisazide were synthesized following reported procedures. [START_REF] Lopez | An amphiphilic PEG-b-PFPE-b-PEG triblock copolymer: synthesis by CuAAC click chemistry and self-assembly in water[END_REF][START_REF] Lopez | A perfluoropolyether-based elastomers library with ondemand thermorheological features[END_REF] Isopropylmagnesium chloride (2.0M in THF), 1,3-bis(diphenylphosphino)propane (dppp, 97%), ethynylmagnesium chloride (0.5M in THF), copper(I) bromide (98%) and N,N,N′,N′,N′′pentamethyldiethylenetriamine (PMDETA, 99%) were purchased from Aldrich. P3HT (Mn = 25 000 g mol -1 , Mw = 31 000 g.mol -1 , Ð = 1.24) was prepared as previously described. [START_REF] Chevrier | Expanding the light absorption of poly(3-hexylthiophene) by end-functionalization with πextended porphyrins[END_REF][START_REF] Boon | Synthesis and Characterization of Nanocomposites Based on Functional Regioregular Poly(3-hexylthiophene) and Multiwall Carbon Nanotubes[END_REF] Commercial P3HT (Mw = 50 000 -70 000 g.mol -1 ) was obtained from Solarmer. [START_REF] Kleinschmidt | Poly(3-hexylthiophene) (P3HT): fruit fly or outlier in organic solar cell research?[END_REF][START_REF] Kleinschmidt | Poly(3-hexylthiophene) (P3HT): fruit fly or outlier in organic solar cell research?[END_REF]phenyl-C61-butyric acid methyl ester (PC61BM) (99%) was purchased from Solenne BV.

Reagent Plus grade methanol (>99%, Aldrich), ACS reagent grade chlorobenzene (99.5%, Aldrich), reagent grade xylene (98.5%, Aldrich) and HPLC grade o-dichlorobenzene (o-DCB) (99%, Aldrich) were used without further purification. Deuterated chloroform (CDCl3), used for NMR spectroscopy, was purchased from Euriso-top (purity >99.8%).

Techniques

The NMR spectra of the polymers were collected at 25 °C on a Bruker Avance III 400-MHz spectrometer equipped with two independent broadband ( 15 N-31 P and 15 N- 19 F, 300 W) and a high band ( 1 H, 100 W) rf channel. A 5 mm 1 H/ 19 F/ 13 C TXO triple resonance pulsed field gradient probe for which 13 C and 19 F are on the inner coil and 1 H on the outer coil is used for three-channel experiments. This triple resonance 1 H/ 19 F/ 13 C probe has a lower background 19 F signal compared to standard dual-channel probes, and is capable of producing short 90° pulses (6.5 μs for 19 F, 9.5 μs for 13 C and 9.2 μs for 1 H). Diffusion-ordered NMR spectroscopy (DOSY) experiments were performed as previously described. [START_REF] Lopez | An amphiphilic PEG-b-PFPE-b-PEG triblock copolymer: synthesis by CuAAC click chemistry and self-assembly in water[END_REF] Size exclusion chromatography (SEC) measurements were performed on a Polymer Laboratories liquid chromatograph equipped with a PL-DG802 degasser, an isocratic HPLC pump LC 1120 (flow rate of THF of 1 mL min -1 ), a Marathon autosampler (loop volume of 200 mL, solution concentration of 1 mg mL -1 ), a PL-DRI refractive index detector, and three columns: a PL gel 10 mm guard column and two PL gel Mixed-B 10 mm columns (linear columns for the separation of polystyrene standards with molecular weight ranging from 500 to 10 6 Da). Polystyrene was used as standard for calibration.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectra were recorded using a Water QToF Premier mass spectrometer equipped with a Nd:YAG (third harmonic) operating at 355 nm with a maximum output of 65 µJ delivered to the sample in 2.2 ns pulses at 50 Hz repeating rate. Time-of-flight mass analyses were carried out in the reflectron mode at a resolution of about 10 000. All samples were analyzed using trans-2-[3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB) as matrix, which was prepared as a 40 mg mL -1 solution in CHCl3. [START_REF] Winter | MALDI-ToF analysis of polythiophene: use of trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile-DCTB-as matrix[END_REF] The matrix solution (1 mL) was applied to a stainless steel target and air dried. Polymer samples were dissolved in CHCl3 to obtain 1 mg mL -1 solutions. 1 µL aliquots of these solutions were applied onto the target area already bearing the matrix crystals and air dried. For the recording of the single-stage MS spectra, the quadrupole (rf-only mode) was set to pass ions from m/z 500 to m/z 15 000, and all ions were transmitted into the pusher region of the time-of-flight analyzer where they were massanalyzed with 1 s integration time.

Thermogravimetric measurements were performed on a TA Instruments TGA 51 thermogravimetric analyzer in air, at a heating rate of 10 °C min -1 from room temperature up to a maximum of 600 °C. Differential scanning calorimetry (DSC) measurements (TA instruments Q100) were performed under an atmosphere of nitrogen at a heating/cooling rate of 10 °C min -1 . The sample size varied between 10 and 15 mg. UV/vis absorption spectra were recorded with a JASCO V-650 UV-visible spectrophotometer.

The atomic force microscopy (AFM) measurements were performed on an Icon Bruker microscope in tapping mode. The samples were drop casted from xylene solution (0.1 mg mL - 1 ) of the polymer onto mica substrates and then, placed under saturation in saturated xylene vapor for 16 h.

(GIWAXS) measurements of the thin films were made using a custom setup consisting of copper solid-anode X-ray tube (Bruker AXS Krystalloflex 760, operated at 35 kV and 30 mA), Osmic confocal MaxFlux optics and a three pin-hole collimation system. The X-ray beam size has 1.0 mm radius, and samples were irradiated just below the critical angle for total reflection with respect to the incoming X-ray beam (∼0.1°). The scattering intensity was detected on a 2D image plate (MAR-345) with a pixel size of 100 μm (2345 × 2345 pixels), and the detector was placed 300 mm from the sample center. Scattering data are expressed as a function of the scattering vector: q=4π/λ sin(Θ), where Θ is a half the scattering angle and λ=0.154 Å is the wavelength of the incident radiation. Here, qxy (qz) is a component of the scattering vector in plane (out-of-plane) to the sample surface. All X-ray scattering measurements were performed under vacuum (~1 mbar) to reduce air scattering and beam damage to the sample. All GIWAXS data processing and analysis were performed by using the software package Datasqueeze (http://www.datasqueezesoftware.com).

Solar cell fabrication and characterization

Bulk heterojunction organic solar cells were prepared using the conventional architecture consisting of glass/ITO/PEDOT:PSS/active layer/Ca/Al. Before device processing, the indium tin oxide (ITO, Kintec, 100 nm, 20 Ω sq -1 ) coated substrates were subjected to a standard cleaning procedure using soap, demineralized water, acetone, isopropanol, and a UV/O3 treatment. PEDOT:PSS [poly (3,4-ethylenedioxythiophene):poly(styrenesulfonic acid),

Heraeus Clevios] was then deposited via spin-coating to obtain a layer thickness of ~30 nm, followed by a thermal treatment of 15 min at 130 °C to remove any residual water. Further processing was performed in a nitrogen-filled glove box (O2 /H2O < 0.1 ppm). The photoactive polymer:PC61BM blend in a 1.2:1 (wt:wt%) ratio was then spin-coated from a 40 mg mL -1 solution in ortho-dichlorobenzene. The photoactive layers were annealed at 140 °C for 5 min after spin-coating. To complete the fabrication of the devices, Ca and Al electrodes were deposited with a thickness of ~30 and ~80 nm, respectively. The I-V characteristics were determined using a Newport Class A solar simulator (model 91195A), calibrated with a silicon solar cell to give an AM 1.5G spectrum. External quantum efficiencies (EQEs) were determined by recording the monochromated (Newport Cornerstone 130 with sorting filters) output of a xenon lamp (100 W, Newport 6257) by a lock-in amplifier (Stanford Research Systems SR830). The recorded values were calibrated with an FDS-100 calibrated silicon photodiode.

Synthesis

Synthesis of the ethynyl end-functionalized P3HT

Dry THF (15.0 mL) was added via a syringe into a flask containing 2-bromo-3-hexyl-5-iodothiophene (1.90 g, 5.11 mmol) under a nitrogen atmosphere, and the mixture was stirred at 0 °C. iPrMgCl (2.0 M solution in THF, 2.55 mL, 5.11 mmol) was added via a syringe, and the mixture was stirred at 0 °C for 0.5 h. At the same time, a suspension of (otolyl)Ni(PPh3)2Br (27.40 mg, 0.04 mmol) and dppp (30.05 mg, 0.08 mol) in THF (15.0 mL) was stirred at room temperature for 15 min. To this suspension, the monomer solution was added and the mixture was stirred at 0 °C for 3 h. Ethynylmagnesium bromide (25 mol %) was injected all at once and the mixture was stirred for another 5 min. The mixture was then injected into methanol and the suspension was stirred for 30 min. The resulting product was filtered, washed with MeOH and dried under vacuum yielding a purple solid. Yield: 71%. 1 H NMR (400 MHz, CDCl3): δ = 6.98 (s, 47H), 6.97 (d, 3 JH-H = 8.0 Hz, 2H), 6.90 (d, 3 JH-H = 8.0 Hz, 2H), 3.53 (s, 1H, C≡CH), 2.80 (t, 3 JH-H = 8.0 Hz, 94H), 2.50 (s, 3H, CH3), 1.71 (quint, 3 JH-H = 8.0 Hz, 94H), 1.45-1.27 (m, 282H), 0.91 (t, 3 JH-H = 6.0 Hz, 141H) ppm; SEC analysis (THF) : Mn = 13150 g/mol; Ð = 1.16.

Synthesis of the P3HT-b-PFPE-b-P3HT triblock copolymer

Telechelic PFPE-bisazide (70 mg, 0.035 mmol, 1 eq.) and ethynyl end-functionalized P3HT (500 mg, 0.07 mmol, 2 eq.) were suspended in DMF (10 mL). The mixture was degassed by bubbling nitrogen for 30 min. Then, copper(I) bromide (CuBr, 0.50 mg, 0.0035 mmol, 0.1 eq.) and N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA, 0.60 mg, 0.0035 mmol, 0.1 eq.) were added into the reaction mixture. After stirring at room temperature for 24 h, the reaction mixture was added dropwise into cold methanol. After filtration, the slightly green powder was dried under vacuum until constant weight. Yield: 95%. 1 H NMR (400 MHz, CDCl3): δ = 7.75 (s, 2H), 6.98 (s, 128H), 6.97 (d, 3 JH-H = 8.0 Hz, 4H), 6.90 (d, 3 JH-H = 8.0 Hz, 4H), 4.68-4.60 (m, 4H), 4.15-3.28 (m, 14H, CH2), 2.80 (t, 3 JH-H = 8.0 Hz, 256H), 2.50 (s, 6H, CH3), 1.71 (quint, 3 JH-H = 8.0 Hz, 256H), 1.45-1.27 (m, 768H), 0.91 (t, 3 JH-H = 6.0 Hz, 384H) ppm; 19 F NMR (376.4 MHz, CDCl3): δ = -53 (OCF2), -78 (OCF2CH2), -90 (OCF2CF2) ppm; 13

Results and discussion

Synthesis and characterization of P3HT-b-PFPE-b-P3HT

Ethynyl end-functionalized regioregular P3HT was first synthesized through the externally-initiated Kumada catalyst transfer polymerization (KCTP) of 2-bromo-3-hexyl-5iodothiophene using a σ-aryl nickel initiator ((2-tolyl)Ni(PPh3)2Br) [START_REF] Bronstein | Externally Initiated Regioregular P3HT with Controlled Molecular Weight and Narrow Polydispersity[END_REF][START_REF] Schiefer | All-conjugated P3HT donor PCDTBT acceptor graft copolymers synthesised via a grafting through approach[END_REF] and its quenching with ethynylmagnesium chloride (Scheme 1).

Scheme 1. Synthesis of the P3HT-b-PFPE-b-P3HT triblock copolymer from the Huisgen reaction between an ethynyl end-functionalized P3HT and a telechelic bis-azido perfluoropolyether (PFPE).

To avoid end-group degradation, no Soxhlet extraction was performed to remove lowmolecular weight fractions. [START_REF] Pessoni | Synthesis of Polyphthalaldehyde-Based Block Copolymers: Utilization of a Thermo-Sacrificial Segment for an Easy Access to Fine-Tuned Poly(3-hexylthiophene) Nanostructured Films[END_REF][START_REF] Li | Synthesis and self-assembly of poly(3-hexylthiophene)block-poly(acrylic acid)[END_REF] As such, a number-averaged molecular weight (Mn) of 13150 g mol -1 and a dispersity (Ð) of 1.16 are obtained from the monomodal trace of the ethynyl end-functionalized P3HT polymer in the SEC analysis (Figure S1 in the Supporting Information). To identify polymer end-groups, MALDI-ToF mass spectrometry was used (Figure S2 in the Supporting Information). The polymer shows a major distribution of otolyl/acetylene end groups and few o-tolyl/Br end groups (not involved in the envisaged click reaction). An average molecular weight of 9000 g mol -1 is approximated by MALDI. Such a difference between the molecular weights obtained by SEC and MALDI is not unusual since SEC analysis using PS calibration usually overestimates the molecular weight of rigid-rod P3HT by a factor of 1.2-2.3. [START_REF] Winter | MALDI-ToF analysis of polythiophene: use of trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile-DCTB-as matrix[END_REF][START_REF] Liu | Employing MALDI-MS on Poly(alkylthiophenes): Analysis of Molecular Weights, Molecular Weight Distributions, End-Group Structures, and End-Group Modifications[END_REF] A singlet at δ = 3.53 ppm, corresponding to the alkyne proton (-C≡CH) (Figure S3 in the Supporting Information) is also observed by 1 H NMR spectroscopy. The molecular weight of the end-functionalized P3HT was also estimated from its 1 H NMR spectrum (Figure S3 in the Supporting Information). The peaks observed at δ = 2.80 and 2.50 ppm are assigned to the resonance of α-methylene group of the P3HT side chains and the methyl group of the o-tolyl initiator, respectively. From the integration of these two peaks, the number of 3HT units was found to be 40, corresponding to an average molecular weight of ~ 7000 g mol -1 , that confirms the mass range estimated by MALDI mass spectrometry. The fluorinated coil of the ABA triblock copolymer was obtained by using fluorolink E10H a telechelic PFPE-diol, abbreviated E10H (~ 1 800 g.mol -1 ), marketed by Acota Company that was chemically modified by tosylation then azidation, as previously described. [START_REF] Lopez | A perfluoropolyether-based elastomers library with ondemand thermorheological features[END_REF] PFPEbisazide and ethynyl end-functionalized P3HT were then covalently coupled via click chemistry to afford the P3HT-b-PFPE-b-P3HT triblock copolymer (Scheme 1). To ensure the complete consumption of the reactive species, copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) was performed using two equivalents of P3HT-alkyne compared to PFPE-bisazide.

Copper(I) bromide (0.1 mol eq.) and N,N,N′,N′,N′′-pentamethyldiethylenetriamine (PMDETA, 0.1 mol eq.) were employed as the catalyst and the ligand, respectively. After one day-reaction at room temperature, the P3HT-b-PFPE-b-P3HT triblock copolymer was recovered by precipitation in cold methanol (95 wt.% yield). The appearance of the triazole protons that resonate at δ = 7.75 ppm in the 1 H NMR spectrum of the triblock copolymer (Figure 1 those attributed to the PFPE segment are too weak to be identified. SEC analysis of the isolated material reveals a monomodal trace recorded at lower elution volume (higher Mn) than the ethynyl end-functionalized P3HT (Figure S1 in the Supporting Information), indicating a successful coupling reaction. The measured Mn of the triblock copolymer is 27 000 g mol -1 , which is very close to the sum (Mn = 28 100 g mol -1 ) of the Mn of the ethynyl end-functionalized P3HT precursor (Mn = 13 150 g mol -1 ) and the PFPE-bisazide (Mn = 1 800 g mol -1 ). The formation of the triblock copolymer is also confirmed by diffusion-ordered 1 H NMR spectroscopy ( 1 H DOSY) (Figure 2). As expected, the lower molar mass ethynyl endfunctionalized P3HT precursor has a higher diffusion coefficient (2.83 x 10 -10 m 2 s -1 ) whereas the higher triblock copolymer has a lower diffusion coefficient (2.56 x 10 -10 m 2 s -1 ).

Thermal, optical and morphological properties of P3HT-b-PFPE-b-P3HT

The thermal properties of the P3HT-b-PFPE-b-P3HT triblock copolymer were assessed by thermogravimetric analysis (TGA) in air and differential scanning calorimetry (DSC) measurements. The P3HT-b-PFPE-b-P3HT triblock copolymer displays a multiple-stage decomposition behavior in air (Figure S6 in the Supporting Information) as previously observed for P3HT-based polymers. [START_REF] Rodrigues | Thermal stability of P3HT and P3HT:PCBM blends in the molten state[END_REF][START_REF] Ramani | Application of model-free kinetics to the thermal and thermooxidative degradation of poly(3-hexyl thiophene)[END_REF] The profile of the decomposition behavior of P3HT-b-PFPE-b-P3HT is quite similar as that of the P3HT precursor. The onset decomposition temperature of P3HT-b-PFPE-b-P3HT for a 5% weight loss (Td,5%) is 320 °C, which is somewhat lower than the ethynyl end-functionalized P3HT precursor (Figure S6 in the Supporting Information). This is probably due to the presence of the PFPE block, which is expected to show a lower stability. The DSC thermogram of the triblock copolymer (Figure S7 in the Supporting Information) exhibits a Tg of -50 °C due to the soft PFPE segments and a melting peak arising from P3HT at 236 °C [START_REF] Kohn | On the Role of Single Regiodefects and Polydispersity in Regioregular Poly(3-hexylthiophene): Defect Distribution, Synthesis of Defect-Free Chains, and a Simple Model for the Determination of Crystallinity[END_REF][START_REF] Yuan | Polymorphism and Structural Transition around 54 °C in Regioregular Poly(3-hexylthiophene) with High Crystallinity As Revealed by Infrared Spectroscopy[END_REF]. Another transition takes place at higher temperatures (~ 25 °C); such a phenomenon is also noted in the DSC thermogram of commercially available Fluorolink E10H. This transition is likely to be due either to a mixed phase or to an interphase region, containing both fluorinated and hydrogenated segments. [START_REF] Priola | UV-curable systems containing perfluoropolyether structures: Synthesis and characterisation[END_REF] The optical properties of the P3HT-b-PFPE-b-P3HT triblock copolymer were studied by UV-Visible (UV-Vis) absorption spectroscopy. The UV-Vis absorption spectrum of the P3HT-b-PFPE-b-P3HT films prepared by drop-casting a 0.5 mg mL -1 polymer solution exhibits a similar absorption profile to that of pure P3HT, with an absorption maximum at 558 nm as well as a shoulder at 605 nm (Figure S8 in the Supporting Information). [START_REF] Oosterbaan | Efficient formation, isolation and characterization of poly(3-alkylthiophene) nanofibres: probing order as a function of side-chain length[END_REF] The presence of this shoulder is attributed to the vibronic progression of the C=C stretching mode (∆E = 0.15 eV). [START_REF] Sundberg | Optical absorption of poly(3alkylthiophenes) at low temperatures[END_REF] This vibronic structure also supports the formation of the P3HT crystalline domains in the film.

In order to further investigate the organization of the P3HT-b-PFPE-b-P3HT triblock copolymer in thin films, grazing incidence wide-angle X-ray scattering (GIWAXS) studies were performed. The polymer was drop-cast from a 0.15 mg mL -1 chlorobenzene solution on a Si/SiO2 substrate. The solvent was evaporated over 4 hours at room temperature. Afterwards the sample was stored at 50 °C overnight to remove residual solvent from the film. The GIWAXS pattern of P3HT-b-PFPE-b-P3HT in Figure 3 indicates a typical lamellar organization on the surface as observed for P3HT. The out-of-plane h00 reflections are related to the lamellar structure with an interlayer distance of 1.63 nm, which is similar to the value for pristine P3HT. [START_REF] Duong | The chemical and structural origin of efficient p-type doping in P3HT[END_REF][START_REF] Lim | Thermoelectric Properties of Poly(3hexylthiophene) (P3HT) Doped with 2[END_REF] Reflections up to 3rd order indicate long-range arrangement in the out-of-plane direction, but the large azimuthal distribution of the scattering intensities is characteristic for certain misalignment of the domains towards the surface. The wide-angle inplane 010 reflection is assigned to the π-stacking distance of 0.39 nm. [START_REF] Lin | Lamellar and liquid crystal ordering in solventannealed all-conjugated block copolymers[END_REF] The position of the h00 and 0k0 reflections on specific planes of the pattern is characteristic for an edge-on arrangement of the conjugated polymer backbone on the surface. Tapping mode height and phase AFM images of the topography of P3HT-b-PFPE-b-P3HT thin films obtained by drop-casting from a 0.1 mg mL -1 xylene solution onto mica substrates further support the periodic P3HT nanofibrillar structure in thin films (Figure 4). This morphological feature is often encountered in regioregular P3HT homopolymers, [START_REF] Yang | Effect of Mesoscale Crystalline Structure on the Field-Effect Mobility of Regioregular Poly(3-hexyl thiophene) in Thin-Film Transistors[END_REF][START_REF] Surin | Relationship between the microscopic morphology and the charge transport properties in poly(3hexylthiophene) field-effect transistors[END_REF][START_REF] Berson | Poly(3-hexylthiophene) Fibers for Photovoltaic Applications[END_REF] and copolymers, suggesting that the organization of P3HT-b-PFPE-b-P3HT is governed by the P3HT blocks. (wt/wt) ratio were spin-coated from o-dichlorobenzene. A commercial batch of P3HT (Solarmer) was taken along as well for proper comparison. As summarized in Table 1 and Figure S9 in the Supporting Information, the triblock copolymer performs significantly better than the synthesized P3HT material (which underwent the same type of purification), almost up to the level of commercial P3HT. The lower power conversion efficiency (PCE) for synthesized P3HT than commercial one may be in part explained by the presence of metal residues, which act as charge carrier traps and consequently, reduce the photovoltaic performance. [START_REF] Usluer | Metal Residues in Semiconducting Polymers: Impact on the Performance of Organic Electronic Devices[END_REF] External quantum efficiencies (EQEs) of the top performing devices were also measured (Figure S10 in the Supporting Information). The shape of the spectra recorded for all three polythiophene materials is quite similar (absence of additional absorption at a different wavelength) and only the absolute EQE values in the range of 450 to 650 nm differ.

Conclusions

for P3HT:PC61BM and P3HT-b-PFPE-b-P3HT:PC61BM solar cell devices. Supplementary material related to this article can be found, in the online version, at doi:…

  C{ 1 H} NMR (100.6 MHz, CDCl3): δ = 140.1, 133.1, 130.6, 128.4, 31.8, 30.4, 29.4, 29.4, 22.8, 14.3 ppm; SEC analysis (THF) : Mn = 27000 g/mol; Ð = 1.36.

Fig. 1 .

 1 Fig. 1. 1 H NMR spectrum of the P3HT-b-PFPE-b-P3HT triblock copolymer in CDCl3 at 25°C.

  , signal c) provides evidence of a successful click reaction. The characteristic signals of PFPE[45] (i.e., O-CH2CH2-(Figure 1, signal b) and CF2-CH2-O (Figure 1, signal a) are also present along with those of P3HT. The 19 F NMR spectrum of the P3HT-b-PFPE-b-P3HT triblock copolymer (Figure S4 in the Supporting Information) displays the signals typically attributed to PFPEs (i.e. OCF2, OCF2CH2 and OCF2CF2 centered at δ = -53, -78 and -90 ppm, respectively). The 13 C{ 1 H} NMR spectrum of the P3HT-b-PFPE-b-P3HT triblock copolymer (Figure S5 in the Supporting Information) exhibits the characteristic signals of P3HT, whereas

Fig. 2 .

 2 Fig. 2. 1 H NMR-DOSY spectra of the P3HT-b-PFPE-b-P3HT triblock copolymer (blue) and ethynyl end-functionalized P3HT precursor (red) recorded in CDCl3 at 25 °C.

Fig. 3 .

 3 Fig. 3. GIWAXS pattern of the P3HT-b-PFPE-b-P3HT film. Reflections are assigned by using Miller indexes. (*) Artefact.

Fig. 4 .

 4 Fig. 4. 1.5x1.5 µm 2 height (a) and phase (b) AFM images of a thin film of P3HT-b-PFPE-b-P3HT cast from a xylene solution.

3. 3 .

 3 Photovoltaic properties of the P3HT-b-PFPE-b-P3HT triblock copolymer Preliminary photovoltaic studies were then performed to investigate the behavior of the P3HT-b-PFPE-b-P3HT triblock copolymer as electron donor material in bulk heterojunction polymer solar cells. Standard architecture devices consisting of glass/ITO/PEDOT:PSS/active layer/Ca/Al were prepared. Photoactive layers consisting of P3HT-b-PFPE-b-P3HT or synthesized P3HT (Mn = 25000 g mol -1 , Ð = 1.24) as a reference with PC61BM in a 1.2:1

  ) and P3HT-b-PFPE-b-P3HT when employed as active layer component (Voc: open-circuit voltage, Jsc: short-circuit current density, FF: fill factor and PCE: power conversion efficiency). Average PCEs were taken over 5-6 cells.

Table 1 .

 1 Photovoltaic performance of P3HT (synthesized in this work and a commercial batch from Solarmer

	Active layer	Mw of the polymer (g.mol -1 )	VOC (V) JSC (mA cm -²)	FF	Average PCE (%)	Best PCE (%)
	P3HT(synth):PC61BM	31 000	0.48	5.98 ± 0.32	0.65 1.85 ± 0.13	2.05
	P3HT-b-PFPE-b-	36 600	0.52	7.69 ± 0.25	0.70 2.79 ± 0.14	2.99
	P3HT:PC61BM
	P3HT(Sol.):PC61BM	50 000-70 000	0.55	7.80 ± 0.39	0.73 3.14 ± 0.18	3.29

Acknowledgments

The authors thank the CNRS and the Université de Montpellier for financial support.

Research in

Mons is supported by the Excellence of Science program (Precision and 2Dto3D projects, FNRS-FRFC and the 'Actions de Recherche Concertée' program (MADSSCELLS project). M.S. is a research associate of the FNRS. The FNRS is also acknowledged for a visiting professor grant for S.C. The UMons MS laboratory acknowledges the F.R.S.-FNRS for the acquisition of the Waters QToF Premier mass spectrometer and for continuing support. W.P. acknowledges the National Science Centre, Poland, through the grant UMO-2015/18/E/ST3/00322. The UHasselt co-authors thank the Research Foundation -Flanders (FWO -Vlaanderen) and Hasselt University for continuous financial support.

A ABA triblock copolymer based on π-conjugated P3HT segments and a fluorinated nonconjugated sequence was successfully synthesized. First, P3HT functionalized with an ethynyl end group was prepared using Kumada catalyst-transfer condensative polymerization.

Then, this ethynyl end-functionalized P3HT was covalently coupled to a telechelic bisazido perfluopolyether (PFPE) via Huisgen click chemistry to afford the P3HT-b-PFPE-b-P3HT triblock copolymer. The copolymer thin films show a periodic P3HT nanofibril morphology with crystallites with "edge-on" orientation of the polymer chains. Modifying P3HT with the attachment of a soft PFPE segment allows to (almost) retain the favorable photovoltaic parameters when blended with PC61BM. To go further, the impact of the molecular weight and the weight ratio of the PFPE segment on the solid-state morphologies and electrical properties will be systematically investigated. This work is currently in progress.

Declaration of interest

All author declare no conflict of interest.


J Fawaz

M Dormoi

Barthélémy Madamet

Pradines

M Gendrot

R Fawaz

J Dormoi

M Madamet

B Pradines
email: bruno.pradines@gmail.com

Genetic diversity and deletion of Plasmodium falciparum histidine-rich protein 2 and 3: a threat to diagnosis of P. falciparum malaria

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

sequences. Sequencing analysis revealed high-level genetic variation in the pfhrp2 and pfhrp3 genes (above 90% of variation in Madagascar, Nigeria or Senegal) both within and between countries. However, genetic variation of PfHRP2 and PfHRP3 does not seem to be a major cause of false-negative results. The countries which showed the highest proportions of pfhrp2negative parasites were Peru (20 to 100%) and Guyana (41%) in South America, Ghana (36%) and Rwanda (23%) in Africa. High prevalence of pfhrp2 deletion causes a high rate of false-negatives results.

Implications: Presence of parasites lacking pfhrp2 gene may pose a major threat to malaria control programs because P. falciparum infected patients are not diagnosed and properly treated.

In 2016, approximately 3.2 billion people, which is nearly half of the population of the world, were at risk of contracting malaria [1]. According to the latest World Health Organization (WHO) estimates, there were 216 million cases of malaria in 2016 and 445 000 deaths. Sub-Saharan Africa continues to carry a disproportionately high share of the global malaria burden, with 90% of malaria cases and 90% of malaria deaths, mostly in young children. The WHO recommends diagnostic confirmation of P. falciparum in all patients with suspected malaria before treatment with antimalarial drugs. This parasitological confirmation can be done by microscopy or malaria rapid diagnostic tests (RDTs) [START_REF]Guidelines for the treatment of malaria[END_REF]. The sale of 221 million malaria RDTs across the globe in 2016 is a proof of their utility [1].

Malaria RDTs are flow devices based on the detection of parasite proteins by immunochromatography. Currently, more than 200 different malaria RDTs are commercially available. Most of them are designed to identify Plasmodium falciparum-specific proteins and target either the P. falciparum histidine-rich protein 2 (PfHRP2) or the P. falciparum lactate dehydrogenase (PfLDH). RDTs can detect other pan-malaria (all species) antigens, such as pLDH or pALD (aldolase). PfHRP2-based RDTs are more sensitive for the detection of P. falciparum and heat-stable than PfLDH-based RDTs [START_REF]Malaria rapid diagnostic test performance. Results of WHO product testing of malaria RDTs: round 7 (2015-2016)[END_REF][START_REF] Chiodini | The heat stability of Plasmodium lactate dehydrogenase-based and histidine-rich protein 2-based malaria rapid diagnostic tests[END_REF]. However, most of the antibodies used in RDTs for the detection of PfHRP2 also detect the P. falciparum histidine-rich protein 3 (PfHRP3) due to structural homology [START_REF] Wellems | Homologous genes encode two distinct histidine-rich proteins in a cloned isolate of Plasmodium falciparum[END_REF].

False negative RDT results were reported with PfHRP2-based RDTs [START_REF] Berhane | Rapid diagnostic tests failing to detect Plasmodium falciparum infections in Eritrea: an investigation of reported false negative RDT results[END_REF]. Numerous factors are implicated, leading to decreases in the performance of the RDTs. The first factor is operator dependent (errors in performing the test and interpreting the result) and can be reduced by training [START_REF]Good practices for selecting and procuring rapid diagnostic tests for malaria[END_REF]. Second, poor product design or manufacturing quality may be involved and can be eliminated by using only good quality RDTs that have been verified, tested and qualified by the WHO. In this context, the WHO has created two programmes, the Foundation for Innovative New Diagnostics (FIND) malaria RDT quality assurance programme and the WHO-FIND malaria RDT lot testing programme [START_REF]WHO-FIND malaria RDT evaluation programme: product testing round 7[END_REF]. Another factor in the false negative result is antibody degradation due to poor thermal stability, as well as incorrect transport or storage conditions. Parasite density can also lead to false negative results. Very low parasite densities may not be detected [START_REF] Wurtz | Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests[END_REF][START_REF] Kyabayinze | HRP2 and pLDH-based rapid diagnostic tests, expert microscopy, and PCR for detection of malaria infection during pregnancy and delivery in areas of varied transmission: A prospective cohort study in Burkina Faso and Uganda[END_REF][START_REF] Natama | Diagnosing congenital malaria in high-transmission setting: clinical relevance and usefulness of P. falciparum HRP2-based testing[END_REF]. Conversely, a false negative result can occur due to a prozone-like effect in high parasite densities [START_REF] Luchavez | Laboratory demonstration of a prozone-like effect in HRP2-detecting malaria rapid diagnostic tests: implications for clinical management[END_REF][START_REF] Gillet | Prozone in malaria rapid diagnostic tests: how many cases are missed?[END_REF]. Finally, misdiagnosis can also be due to the genetic variation of the pfhrp2 gene leading to a modified protein PfHRP2 with a new epitope that is not recognized by RDT antigens or parasites lacking the PfHRP2 protein because of pfhrp2 gene deletion [START_REF] Wurtz | Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests[END_REF][START_REF] Wurtz | Retard de diagnostic d'un accès palustre à Plasmodium falciparum chez un militaire en Ouganda: négativité des tests de diagnostic rapide associée à un polymorphisme de l'antigène HRP2[END_REF][START_REF] Houzé | Combined deletions of pfhrp2 and pfhrp3 genes result in Plasmodium falciparum malaria false-negative rapid diagnostic test[END_REF]. In the absence of the PfHRP2 protein, most of the antibodies used in RDTs for the detection of PfHRP2 also detect the PfHRP3 due to structural homology [START_REF] Wellems | Homologous genes encode two distinct histidine-rich proteins in a cloned isolate of Plasmodium falciparum[END_REF]. However, more P. falciparum parasites lacking pfhrp3 gene were observed across the globe than parasites lacking pfhrp2 gene [START_REF] Wurtz | Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests[END_REF].

Additionally, most of the parasites lacking pfhrp2 gene also lost pfhrp3 gene [START_REF] Wurtz | Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests[END_REF][START_REF] Houzé | Combined deletions of pfhrp2 and pfhrp3 genes result in Plasmodium falciparum malaria false-negative rapid diagnostic test[END_REF]. This review summarizes the publications that reported pfhrp2 and pfhrp3 genetic variation or deletion and describe their implications on RDT use and clinical management. Information sources were identified using PubMed, Google Scholar and Google. Searches were performed in these electronic databases using the search term 'PfHRP2' or 'PfHRP3' alone or in combination with 'rapid diagnostic test', 'RDT', 'deletion', 'genetic variation' or 'polymorphism'. A total of 120 and 73 publications were identified using PubMed using the search term 'PfHRP2' alone and in combination with the other terms, respectively. Only 41 publications were interested to pfhrp2 or pfhrp3 gene deletion or genetic variation. Two of these 41 publications were in Chinese and excluded.

Genetic variation in the pfhrp2 and pfhrp3 genes

The first hypothesis was that the accuracy of PfHRP2-based RDTs can be affected by the pfhrp2 and pfhrp3 genetic variation of P. falciparum parasites. Pfhrp2 and pfhrp3 genes are located on chromosome 8 and 13, respectively, and both include two exons and one intron.

Intron 2 of pfhrp2 (PF3D7_0831800) and pfhrp3 (PF3D7_1372200) consists of a repeated amino acid pattern [START_REF] Baker | Genetic diversity of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and its effect on the performance of PfHRP2-based rapid diagnostic tests[END_REF]. Theoretically, the presence or absence, as well as the numbers of repeats could affect the binding affinity of the antibodies used in RDTs to the parasites antigen. Fourteen repeated amino acid sequences characterized into 18 types were identified.

Type 2 and type 7 were described as potential epitopes targeted by monoclonal antibodies used in PfHRP2-based RDTs [START_REF] Baker | Genetic diversity of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and its effect on the performance of PfHRP2-based rapid diagnostic tests[END_REF][START_REF] Lee | Effect of sequence variation in Plasmodium falciparum histidine-rich protein 2 on binding of specific monoclonal antibodies: implications for rapid diagnostic tests for malaria[END_REF][START_REF] Baker | Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests[END_REF]. There is no published information available on the specific target epitopes of the antibodies used in any RDTs or on the monoclonal antibodies that are directed against PfHRP2. Because of the limited supplies of monoclonal antibodies, it is very likely that most of the PfHRP2-based RDTs use similar antibodies. Additionally, it is likely that either some or all of the epitopes detected by antibodies used in PfHRP2-based RDTs also exist in PfHRP3 Genetic variations in the pfhrp2 gene have been investigated in the Asia-Pacific region [START_REF] Baker | Genetic diversity of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and its effect on the performance of PfHRP2-based rapid diagnostic tests[END_REF][START_REF] Baker | Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests[END_REF], India [START_REF] Kumar | Genetic variation in histidine rich proteins among Indian Plasmodium falciparum population: possible cause of variable sensitivity of malaria rapid diagnostic tests[END_REF][START_REF] Bharti | Sequence variation in Plasmodium falciparum histidine-rich proteins 2 and 3 in Indian isolates: implications for malaria rapid diagnostic test performance[END_REF][START_REF] Bharti | Prevalence of pfhrp2 and/or pfhrp3 gene deletion in Plasmodium falciparum population in eight highly endemic states in India[END_REF], Yemen [START_REF] Atroosh | Genetic variation of Plasmodium falciparum isolates from Yemen and the performance of HRP2-based malaria rapid diagnostic test[END_REF], Madagascar [START_REF] Mariette | Country-wide assessment of the genetic polymorphism in Plasmodium falciparum and Plasmodium vivax antigens detected with rapid diagnostic tests for malaria[END_REF], Senegal [START_REF] Wurtz | Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests[END_REF][START_REF] Deme | Analysis of pfhrp2 genetic diversity in Senegal and implications for use of rapid diagnostic tests[END_REF], Mali [START_REF] Deme | Analysis of pfhrp2 genetic diversity in Senegal and implications for use of rapid diagnostic tests[END_REF], Uganda [START_REF] Deme | Analysis of pfhrp2 genetic diversity in Senegal and implications for use of rapid diagnostic tests[END_REF],

French Guiana [START_REF] Trouvay | High performance of histidine-rich protein 2 base rapid diagnostic tests in French Guiana are explained by the absence of pfhrp2 gene deletion in P. falciparum[END_REF], Colombia [START_REF] Dorado | Genetic characterisation of Plasmodium falciparum isolates with deletion of the pfhrp2 and/or pfhrp3 genes in Colombia: The Amazon Region, a challenge for malaria diagnosis and control[END_REF] and in the China-Myanmar border area [START_REF] Li | Genetic diversity of Plasmodium falciparum histidine-rich protein 2 in the China-Manmar border area[END_REF].

The PfHRP2 protein conformation was highly diverse in parasite isolates from the same area [START_REF] Wurtz | Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests[END_REF][START_REF] Li | Genetic diversity of Plasmodium falciparum histidine-rich protein 2 in the China-Manmar border area[END_REF] or the same country [START_REF] Kumar | Genetic variation in histidine rich proteins among Indian Plasmodium falciparum population: possible cause of variable sensitivity of malaria rapid diagnostic tests[END_REF]. Significant differences were seen depending on the geographic localization. First, the average length of the pfhrp2 amplicons was shorter in the Asia-Pacific region than what was reported in Mozambique and Tanzania [START_REF] Ramutton | Sequence variation does not confound the measurement of plasma PfHRP2 concentration in African children presenting with severe malaria[END_REF] but longer than in Vanuatu [START_REF] Baker | Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests[END_REF]. Sequencing analysis revealed high-level genetic variation in pfhrp2: 34 different sequences (87% of genetic variation) were detected in Tanzania [START_REF] Baker | Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests[END_REF], 74 (93%) in Nigeria [START_REF] Baker | Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests[END_REF], 120 (98%) in Senegal [START_REF] Wurtz | Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests[END_REF] or 221 (97%) in Madagascar [START_REF] Mariette | Country-wide assessment of the genetic polymorphism in Plasmodium falciparum and Plasmodium vivax antigens detected with rapid diagnostic tests for malaria[END_REF]. Then, there was a significant difference between the number of repeats for type 5 and type 6 in the isolates from south, centre and north Madagascar [START_REF] Mariette | Country-wide assessment of the genetic polymorphism in Plasmodium falciparum and Plasmodium vivax antigens detected with rapid diagnostic tests for malaria[END_REF]. Type 4 was rarely observed in Dakar (16% in 2009-2012) and was mainly identified in isolates from Pikine (a suburban area of Dakar approximately 15 km from Dakar), Thies (70 km from Dakar) or Velingara (565 km from Dakar in southern Senegal) (96.3%) [START_REF] Wurtz | Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests[END_REF][START_REF] Deme | Analysis of pfhrp2 genetic diversity in Senegal and implications for use of rapid diagnostic tests[END_REF]. Sequencing analysis revealed high-level genetic diversity in the pfhrp2 and pfhrp3 genes both within and between countries. Some studies showed that type 2 and type 7 were predictive of the sensitivity of PfHRP2based RDT detection at low parasite densities [START_REF] Wurtz | Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests[END_REF][START_REF] Baker | Genetic diversity of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and its effect on the performance of PfHRP2-based rapid diagnostic tests[END_REF][START_REF] Mariette | Country-wide assessment of the genetic polymorphism in Plasmodium falciparum and Plasmodium vivax antigens detected with rapid diagnostic tests for malaria[END_REF]. A PfHRP2-based RDT false negative sample was reported in a soldier in Uganda, certainly due to the low number of type 2 × type 7 repeats (40 repeats) [START_REF] Wurtz | Retard de diagnostic d'un accès palustre à Plasmodium falciparum chez un militaire en Ouganda: négativité des tests de diagnostic rapide associée à un polymorphisme de l'antigène HRP2[END_REF]. According to the predictive Baker's model, this isolate belonged to the group C of non-sensitive parasites to PfHRP2-based RDTs, confirming that the numbers of repeats could affect the binding affinity of the antibodies used in RDTs to the parasites antigen and so the performance of the PfHRP2-based RDTs.

However, in other studies, no correlation was observed between variations in the size of repeat types 2 and 7 in Pfhrp2, nor between any combination of repeat motifs, and performance of the PfHRP2-based RDTs at low parasitemia [START_REF] Baker | Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests[END_REF][START_REF] Bharti | Sequence variation in Plasmodium falciparum histidine-rich proteins 2 and 3 in Indian isolates: implications for malaria rapid diagnostic test performance[END_REF][START_REF] Li | Genetic diversity of Plasmodium falciparum histidine-rich protein 2 in the China-Manmar border area[END_REF]. The genetic variation of PfHRP2 does not seem to be a major cause of false negative results.

Deletion of pfhrp2 and pfhrp3 genes

The presence or absence of pfhrp2 and pfhrp3 genes can be characterized by amplifying the full lengh of exon 2 and a region across exon 1 and exon 2 of both genes. Similarly, genes flancking pfhrp2 (PF3D7_0831900 and PF3D7_0831700) and pfhrp3 (PF3D7_1372100 and PF3D7_1372400) genes can be amplified to characterize the extent of the chromosomal deletions in the parasites lacking the pfhrp2 and pfhrp3 genes [9,15,17.26,27].

The results of the studies that have reported P. falciparum parasites lacking pfhrp2 and/or pfhrp3 are summarized in Table 1. The prevalence of P. falciparum parasites lacking pfhrp2 and/or pfhrp3 per country are presented in Figure 1 and Figure 2, respectively.

South America was the first region to report the occurrence of false negative PfHRP2based RDTs, due to parasites lacking pfhrp2 gene [START_REF] Dorado | Genetic characterisation of Plasmodium falciparum isolates with deletion of the pfhrp2 and/or pfhrp3 genes in Colombia: The Amazon Region, a challenge for malaria diagnosis and control[END_REF][START_REF] Viana | Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia[END_REF][START_REF] Solano | Deletion of Plasmodium falciparum histidine-rich protein 2 (pfhrp2) and histidine-rich protein 3 (pfhrp3) genes in Colombian parasites[END_REF][START_REF] Saenz | Clonal population expansion in an outbreak of Plasmodium falciparum on the northwest coast of Ecuador[END_REF][START_REF] Okoth | Variation in Plasmodium falciparum histidine-rich protein 2 (Pfhrp2) and Plasmodium falciparum histidine-rich protein 3 (Pfhrp3) gene deletions in Guyana and Suriname[END_REF][START_REF] Abdallah | Prevalence of pfhrp2 and pfhrp3 gene deletions in Puerto Lempira, Honduras[END_REF][START_REF] Akinyi | Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites in Peru[END_REF][START_REF] Baldeviano | Molecular epidemiology of Plasmodium falciparum malaria outbreaks, Tumbes, Peru, 2010-2012[END_REF][START_REF] Gamboa | A large proportion of P. falciparum isolates in the Amazon Region of Peru lack pfhrp2 and pfhrp3: implications for malaria rapid diagnostic tests[END_REF][START_REF] Maltha | Rapid diagnostic tests for malaria diagnosis in the Peruvian Amazon: Impact of pfhrp2 genes deletions and cross-reactions[END_REF]. The first publication on P. falciparum parasites lacking pfhrp2 reported data from Peru in 2010 [START_REF] Gamboa | A large proportion of P. falciparum isolates in the Amazon Region of Peru lack pfhrp2 and pfhrp3: implications for malaria rapid diagnostic tests[END_REF]. This retrospective study showed that 41% and 70% of the samples collected from different areas of the Amazon [START_REF] Baldeviano | Molecular epidemiology of Plasmodium falciparum malaria outbreaks, Tumbes, Peru, 2010-2012[END_REF]. Analysis in South America revealed high-level differences in the deletion of the pfhrp2 gene both within and between countries. In 2010, 41% of parasites in Guyana and 15% in Suriname in 2009-2011 lacked the pfhrp2 gene [START_REF] Okoth | Variation in Plasmodium falciparum histidine-rich protein 2 (Pfhrp2) and Plasmodium falciparum histidine-rich protein 3 (Pfhrp3) gene deletions in Guyana and Suriname[END_REF] while very low proportions (ranging from 0 to 4%) were found in Honduras from 2008-2009 [START_REF] Abdallah | Prevalence of pfhrp2 and pfhrp3 gene deletions in Puerto Lempira, Honduras[END_REF], in French Guiana in 2009 [START_REF] Trouvay | High performance of histidine-rich protein 2 base rapid diagnostic tests in French Guiana are explained by the absence of pfhrp2 gene deletion in P. falciparum[END_REF], in Ecuador from 2012-2013 [START_REF] Saenz | Clonal population expansion in an outbreak of Plasmodium falciparum on the northwest coast of Ecuador[END_REF] or in Bolivia in 2010 [START_REF] Viana | Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia[END_REF]. The situation can be very different within a country. From 2008-2010, 53.6% of the parasites lacked the pfhrp2 gene in southeast Colombia (Amazonas) versus 0% in the northwest (Antiquia) [START_REF] Dorado | Genetic characterisation of Plasmodium falciparum isolates with deletion of the pfhrp2 and/or pfhrp3 genes in Colombia: The Amazon Region, a challenge for malaria diagnosis and control[END_REF]. In 2012, 31.6% of the P. falciparum parasites collected in Acre, located on the western edge of the Amazon region of Brazil and bordering Peru, lacked the pfhrp2 gene while none of the samples collected were deleted in Para, which is located in the eastern end of the Brazilian Amazon basin in northern Brazil [START_REF] Viana | Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia[END_REF]. In 2011, a French traveller imported malaria from Brazil and presented a false-negative PfHRP2-based RDT with a parasite density of 3.2%, resulting from combined deletions of pfhrp2 and pfhrp3 [START_REF] Houzé | Combined deletions of pfhrp2 and pfhrp3 genes result in Plasmodium falciparum malaria false-negative rapid diagnostic test[END_REF]. Similar evidence of the emergence of false negative PfHRP2-based RDTs, due to parasites lacking pfhrp2 gene was followed more recently in Africa [START_REF] Wurtz | Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests[END_REF][START_REF] Parr | Pfhrp2deleted Plasmodium falciparum parasites in the Democratic Republic of the Congo: a national cross-sectional survey[END_REF][START_REF] Menegon | Identification of Plasmodium falciparum isolates lacking histidine-rich protein 2 and 3 in Eritrea[END_REF][START_REF] Amoah | Plasmodium falciparum histidine-rich protein-2 diversity and the implications for PfHRP 2-based malaria rapid diagnostic tests in Ghana[END_REF][START_REF] Beshir | Plasmodium falciparum parasites with histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in two regions of Kenya[END_REF][START_REF] Koita | False-negative rapid diagnostic tests for malaria and deletion of the histidine-rich repeat region of the hrp2 gene[END_REF][START_REF] Gupta | Molecular surveillance of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum isolates from Mozambique[END_REF][START_REF] Kozycki | False-negative malaria rapid diagnostic tests in Rwanda: impact of Plasmodium falciparum isolates lacking hrp2 and declining malaria transmission[END_REF]. In Africa, and more particularly in the Democratic Republic of the Congo in 2013-2014, the prevalence of parasites lacking the pfhrp2 gene differed within the country with a high prevalence of pfhrp2 gene deletions observed in Sankuru, Nord-Kivu, Sud-Kivu and Kinshasa (from 14.8 to 21.9%) compared to other regions in the Democratic Republic of the Congo, such as Bas-Uele, Haut-Katanga, Kasai or Equateur (from 0.2 to 2.8%) [START_REF] Parr | Pfhrp2deleted Plasmodium falciparum parasites in the Democratic Republic of the Congo: a national cross-sectional survey[END_REF]. These data show the difficulty to monitor the emergence and the spread parasites lacking the pfhrp2 gene. The global proportion of parasites lacking the pfhrp2 gene was 6.4% in the Democratic Republic of the Congo. High proportions of pfhrp2 gene deletion were reported in 2015 in some areas of Ghana (36%) and Rwanda (23%) [START_REF] Amoah | Plasmodium falciparum histidine-rich protein-2 diversity and the implications for PfHRP 2-based malaria rapid diagnostic tests in Ghana[END_REF][START_REF] Kozycki | False-negative malaria rapid diagnostic tests in Rwanda: impact of Plasmodium falciparum isolates lacking hrp2 and declining malaria transmission[END_REF]. A retrospective study in Mali showed that 2% of P. falciparum parasites lacked the pfhrp2 gene in 1996 [START_REF] Koita | False-negative rapid diagnostic tests for malaria and deletion of the histidine-rich repeat region of the hrp2 gene[END_REF]. Isolates lacking the pfhrp2 gene were identified in Senegal from 2009-2011 [START_REF] Wurtz | Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests[END_REF], in Eritrea from 2013-2014 [START_REF] Menegon | Identification of Plasmodium falciparum isolates lacking histidine-rich protein 2 and 3 in Eritrea[END_REF], in Kenya in 2014 [START_REF] Beshir | Plasmodium falciparum parasites with histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in two regions of Kenya[END_REF] and in Mozambique [START_REF] Gupta | Molecular surveillance of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum isolates from Mozambique[END_REF].

Concerning Asia, a low proportion of parasites lacking the pfhrp2 gene were reported in India [START_REF] Bharti | Sequence variation in Plasmodium falciparum histidine-rich proteins 2 and 3 in Indian isolates: implications for malaria rapid diagnostic test performance[END_REF][START_REF] Bharti | Prevalence of pfhrp2 and/or pfhrp3 gene deletion in Plasmodium falciparum population in eight highly endemic states in India[END_REF][START_REF] Kumar | Genetic deletion of HRP2 and HRP3 in Indian Plasmodium falciparum population and false negative malaria rapid diagnostic test[END_REF] and at the border between China, Myanmar and Thailand [START_REF] Li | Genetic diversity of Plasmodium falciparum histidine-rich protein 2 in the China-Manmar border area[END_REF]. A PfHRP2-based RDT negative sample with pfhrp2 gene-deleted parasites was reported in Bangladesh in 2017 [START_REF] Nima | Case report: A case of Plasmodium falciparum hrp2 and hrp3 gene mutation in Bangladesh[END_REF].

Implications for clinical management

P. falciparum parasites lacking pfhrp2 and pfhrp3 genes are an important source of falsenegative cases using PfHRP2-based RDTs and, more particularly, in samples with low parasite densities. Parasites that lack the pfhrp2 gene cannot express the PfHRP2 protein and therefore cannot be detected by PfHRP2-based RDTs. High proportions of pfhrp2-deleted isolates were reported in some areas of South America (Peru, Brazil, Colombia) and in Africa (Ghana, Rwanda, Democratic Republic of Congo). Those countries are major endemic regions of P. falciparum malaria, and therefore, the clinical consequences must be considered.

Emergence of parasites lacking pfhrp2 gene poses a major threat to malaria control programs because P. falciparum infected patients are not diagnosed and properly treated. High proportions of pfhrp2-deleted parasites will lead to a high rate of false-negative PfHRP2based RDTs, particularly in areas with low malaria transmission and will lead to a high proportion of P. falciparum infected patients who will not be diagnosed and properly treated, especially in Africa where the diagnosis is mainly based on RDTs (177 million of RDTs used in sub-Saharan Africa in 2016). The common hypothesis is that parasites lacking pfhrp2 gene will emerge in a low-transmission area and spread then to a high-transmission area. First, in low-transmission area, each infection is more likely to result in a higher parasite load because of the lower levels of clinical immunity. Second, because immunity is less developed in low transmission areas, parasites lacking pfhrp2 gene are more likely to survive the host immune response and subsequently to be spread. Third, individuals tend to be infected concurrently by fewer genetically distinct parasites, so parasites lacking pfhrp2 gene face less competition within the host and an increased probability of transmission success. We can expect a selection and a spread of the pfhrp2 deletion in sub-Saharan Africa, and more particularly in Ghana, Rwanda and Democratic Republic of Congo, where high proportions of pfhrp2deleted isolates have been reported and where malaria diagnosis is mainly based on RDTs.

False negative RDT results due to parasites lacking pfhrp2 and pfhrp3 genes also have implications for non-endemic areas. Untreated, approximately 10% of uncomplicated imported malaria will progress to severe malaria [START_REF] Calderato | High prevalence of malaria in a non-endemic setting: comparison of diagnostic tool and patient outcome during a four-year survey (2013-2017)[END_REF].

By modelling the prevalence of parasites lacking PfHRP2 and the treatment coverage estimates, Watson et al could predict the spread of the pfhrp2 deletion in sub-Saharan Africa and regions in which surveillance should be prioritized [START_REF] Watson | Modelling the drivers of the spread of Plasmodium falciparum hrp2 gene deletions in subsaharan Africa[END_REF]. The model showed that low malaria prevalence and high rates of people treated by antimalarial drugs resulted in the greatest selection of parasites lacking the pfhrp2 gene. The selection pressure favouring parasites lacking the pfhrp2 gene was predicted to be weaker in the presence of the pfhrp3 gene. Using all these data, Watson et al created a map of 160 locations in Africa most at risk of rapid selection of parasites lacking the pfhrp2 gene, and more particularly in Angola, Cameroon, Gabon, Kenya, Mali, Mauritania, Republic of Central Africa, Senegal Sudan or Tanzania. These maps could be used by public health authorities to determine where they should more closely monitor the emergence and spread of parasites lacking the pfhrp2 gene.

Additionally, a recent study using an agent-based stochastic simulation model of P. falciparum transmission predicted that the use of PfHRP2-based RDTs in populations where P. falciparum parasites that lacked the pfhrp2 gene are introduced was sufficient to select parasites with the pfhrp2 gene deletion. These parasites were therefore undetectable by PfHRP2-based RDTs [START_REF] Gatton | Implications of parasites lacking Plasmodium falciparum histidine-rich protein 2 on malaria morbidity and control when rapid diagnostic tests are used for diagnosis[END_REF]. Parasites lacking the pfhrp2 gene were transmitted in more than 80% of cases compared with 30% of parasites with a normal pfhrp2 gene when detection of P. falciparum depended solely on PfHRP2-based RDTs. The only use of PfHRP2-based RDTs leads to select selects individuals with parasites lacking pfhrp2 gene as parasites reservoir.

Additionally, misdiagnosis due to the non-detection of parasites lacking the pfhrp2 gene leads to delayed treatment resulting in a higher load of parasites lacking the pfhrp2 gene and a longer period for mosquitoes feeding, leading a better transmission of parasites with this deletion. In Eritrea, in March 2016, 80.8% (21/26) and 92.3% (24/26) of the patients infected by P. falciparum at Ghindae Hospital were infected with parasites lacking the pfhrp2 and pfhrp3 genes, respectively [START_REF] Berhane | Major threat to malaria control programs by Plasmodium falciparum lacking histidine-rich protein 2, Eritrea[END_REF]. All the samples were confirmed by microscopy and PCR. The spread of pfhrp2-negative parasites was probably due to selection by use of HRP2-based RDTs. The conditions were ideal in Eritrea for selection of parasites lacking the pfhrp2 gene: very good compliance with RDT before treatment since 2006, low malaria prevalence and high treatment adherence. The introduction and spread of parasites lacking the PfHRP2 protein will increase the mutant parasite prevalence, transmission, morbidity, and ultimately malaria mortality.

Due to the potential impact at the clinical, public health and economic levels, recommendations are being developed to monitor the presence of pfhrp2 gene deletions in P.

falciparum [START_REF] Cheng | Plasmodium falciparum parasites lacking histidine-rich protein 2 and 3: a review and recommendations for accurate reporting[END_REF]. The WHO has formulated guidelines [START_REF]False-negative RDT results and implications of new reports of Plasmodium falciparum histidine-rich protein 2/3 gene deletions[END_REF]. PfHRP2 deletion must be investigated when a patient sample gives negative results by at least two quality-assured HRP2-based RDTs; this sample must be assessed microscopically for P. falciparum by two qualified microscopists. When parasites lacking the pfhrp2 gene have been confirmed, surveillance of febrile patients and other people around the index case must be carried out to determine the prevalence of the pfhrp2 gene deletion in P. falciparum in the specific region and country. In countries where the prevalence of pfhrp2-deleted parasites is low, many isolates are required to properly assess the proportion of parasites lacking the PfHRP2 protein.

There are still many regions where the evaluation of the presence of pfhrp2 gene deletions in P. falciparum has not been yet investigated.

It is imperative to use three-band RDTs that incorporate both pfHPR2 and pLDH antigens over a two-band RDT (HRP2 alone) to minimize misdiagnosis [START_REF] Hawkes | Use of a three-band HRP2/pLDH combination rapid diagnosis test increases diagnostic specificity for falciparum malaria in Ugandan children[END_REF] or to abandon the use of PfHRP2-based RDTs.

Further studies are needed to identify and develop new protein targets for RDTs. The P. falciparum insulin-degrading enzyme homologue (PfIDEh) could be a suitable candidate to replace PfHRP2 by using immuno-polymerase chain reaction detection [START_REF] Mu | High-sensitivity assays for Plasmodium falciparum infection by immuno-polymerase chain reaction detection of PfIDEh and PfLDH antigens[END_REF]. This assay could detect less than one parasite/µl. It would be desirable to develop immunochromatographic flow device for detection of this new protein in practical field diagnostic. Detection of HRP2 at sub-picogram levels was effective using an antigen capture-based immunoassay through a bead-based system [START_REF] Rogier | Bead-based immunoassay allows sub-picogram detection of histidine-rich protein 2 from Plasmodium falciparum and estimates reliability of malaria rapid diagnostic tests[END_REF]. This bead-based immunoassay could be developed for other antigens, such as PfLDH.

Transparency declaration

The authors declare that they have no competing interests.

  . The number of AHHAHHAAD (type 2) x AHHAAD (type 7) repeats for PfHRP2 were determined. PfHRP2 sequences were classified into four groups as a function of the number of type 2 x type 7 repeats. Isolates were classified as follows: very sensitive for the detection of PfHRP2 protein (group A) if the number of type 2 × type 7 repeats was ≥ 100; sensitive (group B) if the number of type 2 × type 7 repeats ranged from 50 to 100; borderline (group I) if the number of type 2 × type 7 repeats ranged from 43 to 50; and non-sensitive (group C) if the number of type 2 × type 7 repeats was < 43. The variation in the number and combination of repeats within PfHRP2 affected the sensitivity of PfHRP2based RDTs [17]. This classification would predict the performance of PfHRP2-based RDTs at low parasite density (below 250 parasites/µl). The low number of type 2 × type 7 repeats in group C and potentially in group I changes the conformation of the epitopes enough so that they are no longer recognized by by antibodies used in PfHRP2-based RDTs.

  region of Peru near Iquitos between 2003 and 2007 lacked the pfhrp2 and pfhrp3 genes, respectively. The proportion of parasites lacking pfhrp2 increased from 20.7% in 1998-2001 to 40.6% in 2003-2005 in the Amazon region of Peru [35]. During a P. falciparum outbreak on the northern Pacific coast of Peru in 2010-2012, 100% of the 54 analysed samples lacked the pfhrp2 gene

Figure 1 .

 1 Figure 1. Geographical distribution of the prevalence of P. falciparum parasites lacking the

Figure 2 .

 2 Figure 2. Geographical distribution of the prevalence of P. falciparum parasites lacking the

Table 1 .

 1 Prevalence of the Plasmodium falciparum histine-rich protein 2 (phhrp2) and pfhrp3 genes in P. falciparum isolates in the world

	Country	Region	Year	deletion Isolates with pfhrp2	Isolates with pfhrp3 deletion	Reference
				Total	Deletion	Deletion	Total	Deletion	Deletion
				no	no	%	no	no	%
	South America
	Bolivia	Beni	2010	25	1	4	25	17	68	[30]
	Brazil	Acre	2012	79	25	31,6	79	30	38	[30]
	Brazil	Rondonia	2010-2011	60	2	3,3	60	11	18,3	[30]
	Brazil	Para	2011-2012	59	0	0	59	30	50,9	[30]
	Colombia	Antioquia, Amazonas, Guaviare,	2003-2010	253	15	5,9	253	106	41,9	[27]
		Choco, Narino
	Colombia	Cordoba (22%), Narino (39%),	2008-2009	100	18	18	100	52	52	[31]
		Valle del Cauca (39%)
	Colombia	Antioquia, Amazonas, Guaviare,	2011-2012	112	0	0	112	51	45,5	[27]
		Choco, Narino
	Ecuador	Esmaraldas	2012-2013	32	1	3,1	32	1	3,1	[32]
	French Guiana	Cayenne (59%), Saul (19%), St	2009	221	0	0	221	10	4,5	[26]
		Laurent du Maroni (12%)
	Guyana	Cuyuni-Mazaruni (55%), Potao-	2010	97	40	41	97	1	1	[33]
		Siparuni (34%)

Funding

The study was supported by the Délégation Générale pour l'Armement, grant no PDH-2-NRBC-4-B-4104.