A family cluster of schistosomiasis acquired in Solenzara River, Corsica (France) - Solenzara River is clearly a transmission site for schistosomiasis in Corsica.

We report a patient with urogenital schistosomiasis and three cases of subclinical infection within one family acquired from Solenzara River, Corsica, in 2019. Our cases confirm that transmission of schistosomiasis in Corsica is ongoing and has been extended from the Cavu River to the Solenzara River. Solenzara River is clearly a transmission site for schistosomiasis in Corsica. Public health efforts are recommended to uncover and prevent further cases.

Oxamniquine resistance alleles are widespread in Old World Schistosoma mansoni and predate drug deployment.

Do mutations required for adaptation occur de novo, or are they segregating within populations as standing genetic variation? This question is key to understanding adaptive change in nature, and has important practical consequences for the evolution of drug resistance. We provide evidence that alleles conferring resistance to oxamniquine (OXA), an antischistosomal drug, are widespread in natural parasite populations under minimal drug pressure and predate OXA deployment. OXA has been used since the 1970s to treat Schistosoma mansoni infections in the New World where S. mansoni established during the slave trade. Recessive loss-of-function mutations within a parasite sulfotransferase (SmSULT-OR) underlie resistance, and several verified resistance mutations, including a deletion (p.E142del), have been identified in the New World. Here we investigate sequence variation in SmSULT-OR in S. mansoni from the Old World, where OXA has seen minimal usage. We sequenced exomes of 204 S. mansoni parasites from West Africa, East Africa and the Middle East, and scored variants in SmSULT-OR and flanking regions. We identified 39 non-synonymous SNPs, 4 deletions, 1 duplication and 1 premature stop codon in the SmSULT-OR coding sequence, including one confirmed resistance deletion (p.E142del). We expressed recombinant proteins and used an in vitro OXA activation assay to functionally validate the OXA-resistance phenotype for four predicted OXA-resistance mutations. Three aspects of the data are of particular interest: (i) segregating OXA-resistance alleles are widespread in Old World populations (4.29-14.91% frequency), despite minimal OXA usage, (ii) two OXA-resistance mutations (p.W120R, p.N171IfsX28) are particularly common (>5%) in East African and Middle-Eastern populations, (iii) the p.E142del allele has identical flanking SNPs in both West Africa and Puerto Rico, suggesting that parasites bearing this allele colonized the New World during the slave trade and therefore predate OXA deployment. We conclude that standing variation for OXA resistance is widespread in S. mansoni.

Mastomys natalensis (Smith, 1834) as a natural host for Schistosoma haematobium (Bilharz, 1852) Weinland, 1858 x Schistosoma bovis Sonsino, 1876 introgressive hybrids.

Cercarial emission of schistosomes is a determinant in the transmission to the definitive host and constitutes a good marker to identify which definitive host is responsible for transmission, mainly in introgressive hybridization situations. Our goal was to test the hypothesis that micro-mammals play a role in Schistosoma haematobium, S. bovis, and/or S. haematobium x S. bovis transmission. Small mammal sampling was conducted in seven semi-lacustrine villages of southern Benin. Among the 62 animals trapped, 50 individuals were investigated for Schistosoma adults and eggs: 37 Rattus rattus, 3 Rattus norvegicus, 9 Mastomys natalensis, and 1 Crocidura olivieri. Schistosoma adults were found in four R. rattus and two M. natalensis, with a local prevalence reaching 80% and 50%, respectively. Two cercarial chronotypes were found from Bulinus globosus experimentally infected with miracidia extracted from naturally infected M. natalensis: a late diurnal and nocturnal chronotype, and an early diurnal, late diurnal, and nocturnal chronotype. The cytochrome C oxidase subunit I mtDNA gene of the collected schistosomes (adults, miracidia, and cercariae) belonged to the S. bovis clade. Eleven internal transcribed spacer rDNA profiles were found; four belonged to S. bovis and seven to S. haematobium x S. bovis. These molecular results together with the observed multi-peak chronotypes add M. natalensis as a new host implicated in S. haematobium x S. bovis transmission. We discuss the origin of the new chronotypes which have become more complex with the appearance of several peaks in a 24-h day. We also discuss how the new populations of offspring may optimize intra-host ecological niche, host spectrum, and transmission time period.

Cattle as natural host for Schistosoma haematobium (Bilharz, 1852) Weinland, 1858 x Schistosoma bovis Sonsino, 1876 interactions, with new cercarial emergence and genetic patterns.

Schistosomiasis remains a parasitic infection which poses serious public health consequences around the world, particularly on the African continent where cases of introgression/hybridization between human and cattle schistosomiasis are being discovered on a more frequent basis in humans, specifically between Schistosoma haematobium and S. bovis. The aim of this paper is to analyze the occurrence of S. bovis in cattle and its relationship with S. haematobium in an area where cattle and humans share the same site in Benin (West Africa). We used the chronobiology of cercarial emergence as an ecological parameter and both molecular biology (COI mtDNA and ITS rDNA) of the larvae and morphology of the eggs as taxonomic parameters. The results showed a chronobiological polymorphism in the cercarial emergence rhythm. They showed for the first time the presence of S. bovis in Benin, the presence of introgressive hybridization between S. bovis and S. haematobium in domestic cattle, and the presence of atypical chronobiological patterns in schistosomes from cattle, with typical S. haematobium shedding pattern, double-peak patterns, and nocturnal patterns. Our results showed that the chronobiological life-history trait is useful for the detection of new hosts and also may reveal the possible presence of introgressive hybridization in schistosomes. Our results, for the first time, place cattle as reservoir host for S. haematobium and S. bovis x S. haematobium. The consequences of these results on the epidemiology of the disease, the transmission to humans, and the control of the disease are very important.

Host-parasite life-histories of the diurnal vs. nocturnal chronotypes of Schistosoma mansoni: adaptive significance.

OBJECTIVES: To optimise host-to-host transmission, digenean trematodes (parasites) synchronise their cercarial emission patterns with the aquatic activities of their vertebrate hosts. Schistosoma mansoni has two different circadian chronotypes: a diurnal shedding pattern with a mean peak occurring at 11:00 h, and a nocturnal shedding pattern with a mean peak occurring at 20:00 h. We analysed the life-history variations between these two chronotypes at the levels of the parasite and its hosts. METHODS: For each chronotype, we quantified three life-history traits related to the parasite (prepatent period, infection rate and cercarial production) and analysed the morphometry and the morphology of the parasite eggs; we also quantified three life-history traits related to the snail intermediate host (shell diameter, fecundity and survival rate) and one life-history trait related to the experimental definitive host (survival rate). A phylogeny based on the mitochondrial cytochrome-oxidase gene was made on samples of both chronotypes. RESULTS: Life-history analysis revealed significant variations between the two chronotypes. Life-history traits were optimal for both the parasite and the snail host for the diurnal chronotype compared to the nocturnal one. The new chronotype behaved like an allopatric population towards its snail host. Phylogenetic analysis supports the hypothesis of a lateral transfer of S. mansoni from humans to Rattus rattus. These results were interpreted in terms of an ongoing sympatric speciation. CONCLUSION: The nocturnal chronotype of S. mansoni showed non-adapted life-history traits in its relation with the snail intermediate host Biomphalaria pfeifferi. The emergence of this new phenotype is probably linked to divergent natural selection.

OBJECTIFS: Afin d'optimiser la transmission d'hôte à hôte, les trématodes digènes (parasites) synchronisent leurs schémas d'émission cercarienne avec les activités aquatiques de leurs hôtes vertébrés. Schistosoma mansoni a deux chronotypes circadiens différents: un schéma de libérations diurnes avec un pic moyen survenant à 11h00 et un schéma nocturne avec un pic moyen à 20h00. Nous avons analysé les variations de l'histoire de vie entre ces deux chronotypes aux niveaux du parasite et de ses hôtes. MÉTHODES: Pour chaque chronotype, nous avons quantifié trois traits d'histoire de vie liés au parasite (période prépatente, taux d'infection et production cercarienne) et avons analysé la morphométrie et la morphologie des œufs du parasite; nous avons également quantifié trois traits d'histoire de vie liés à l'hôte intermédiaire escargot (diamètre de la coquille, fécondité et taux de survie) et un trait d'histoire de vie lié à l'hôte définitif expérimental (taux de survie). Une phylogénie basée sur le gène mitochondrial de la cytochrome oxydase a été réalisée sur des échantillons des deux chronotypes. RÉSULTATS: L'analyse de l'histoire de vie a révélé des variations significatives entre les deux chronotypes. Les traits d'histoire de vie étaient optimaux à la fois pour le parasite et pour l'hôte escargot pour le chronotype diurne par rapport au chronotype nocturne. Le nouveau chronotype se comportait comme une population allopatrique vis-à-vis de son hôte escargot. L'analyse phylogénétique soutient l'hypothèse d'un transfert latéral de S. mansoni de l'homme à Rattus rattus. Ces résultats ont été interprétés en termes de spéciation sympatrique en cours. CONCLUSION: Le chronotype nocturne de S. mansoni montre des traits d'histoire de vie non adaptés dans sa relation avec l'hôte intermédiaire escargot, Biomphalaria pfeifferi. L'émergence de ce nouveau phénotype est probablement liée à une sélection naturelle divergente.

Acute anuria after a family vacation to Corsica/France.

A 12-year-old male patient suffered hematuria. Histopathology of a biopsy showed granulomata suspicious for schistosomiasis. The patient had never travelled outside Europe during his entire lifetime. He had taken frequent bathes in various rivers during his last family holidays 5 months earlier in Corsica. Microfiltration of urine revealed viable ova of Schistosoma haematobium with alterated size and shape. Ultrasonography showed a large focal echopoor mass attached to the bladder roof. Four days after antihelminthic therapy, the patient suffered inferior abdominal pain and acute anuria. Ultrasound revealed an approximately 5-cm mass in the bladder lumen suspicious for a large blood clot. After taking non-invasive measures such as drinking high amounts of fluid and treating the lower abdomen with a warm water bag and massage, the clot was excreted with urine and symptoms subsided. The further course was uneventful until 11 months later when hematuria recurred. This time, parasitological urine examination confirmed non-viable schistosome ova. Hematuria was likely due to erosion of the bladder mucosa by calcified non-viable ova.

Genetic diversity, multiplicity of infection and population structure of Schistosoma mansoni isolates from human hosts in Ethiopia.

BACKGROUND: Human intestinal schistosomiasis caused by Schistosoma mansoni and urinary schistosomiasis caused by Schistosoma haematobium are endemic in Ethiopia. Although schistosomes look morphologically uniform, there is variation in infectivity, egg productivity and virulence due to variation in their genetic make. Knowing the genetic diversity and population structure of S. mansoni isolates will enable to understand and consider the possible variability in terms of infectivity, egg productivity and virulence. METHODS: Between 2010 and 2011, genetic diversity and population structure of Schistosoma mansoni isolates from four endemic areas of Ethiopia was assessed using previously published 11 polymorphic microsatellite loci. Miracidia were hatched from eggs of S. mansoni collected from stools of human subjects residing in Kemissie, Wondo Genet, Ziway and Sille-Elgo villages. DNA was extracted from single miracidium and PCR was run following standard protocol. Allelic polymorphism and population genetic structure was analyzed using different software. RESULT: At a population level (i.e. different villages), the mean number of alleles per locus, allelic richness, expected heterozygosity in Hardy-Weinberg equilibrium and pairwise F ST values ranged from 8.5 to 11.5, 3.46-20.8, 0.66-0.73 and 3.57-13.63%, respectively. All analyzes on population genetic structure reveals strong genetic structuration corresponding to the four sampled villages. At infrapopulation level (i.e. different hosts) the mean number of alleles per locus, allelic richness, expected heterozygosity in Hardy-Weinberg equilibrium and F IS values ranged from 3.09 to 7.55, 1-1.96, 0.59-0.73 and 0.1763-0.4989, respectively. Mean estimated genetically unique adult worm pairs within hosts ranged from 66 to 92% revealing the occurrence of infection of a single host with genetically unique multiple S. mansoni strains. The data also indicated the occurrence of genetic variation within inter- and intra-hosts. CONCLUSION: High level of genetic diversity and significant population differentiation characterized the S. mansoni isolates of Ethiopia. These results are quite different from previous studies demonstrating that it is difficult to generalize schistosome transmission patterns because epidemiological situation tends to vary. These are important factors to be considered in relation with morbidity, drug resistance or vaccine development.

Introgressive hybridizations of Schistosoma haematobium by Schistosoma bovis at the origin of the first case report of schistosomiasis in Corsica (France, Europe).

This study concerns the first urinary schistosomiasis case observed in Corsica (France, Europe) occurring in a 12-year-old German boy. The aim was to identify the relationship between this Schistosoma haematobium infection and other schistosomes of the Schistosoma group with terminal-spined ova. Morphological and molecular analyses were conducted on the ova. The results showed that the schistosome responsible for the emergence of schistosomiasis in Corsica was due to S. haematobium introgressed by genes from S. bovis.

Genetic diversity, fixation and differentiation of the freshwater snail Biomphalaria pfeifferi (Gastropoda, Planorbidae) in arid lands.

The freshwater snail Biomphalaria pfeifferi is the main intermediate host of human intestinal Bilharziasis. It is widely distributed in Africa, Madagascar and middle-eastern countries, and its habitat includes wetlands, and arid to semi-arid areas. Based on analysis of 18 microsatellites, we investigated reference allelic variation among 30 populations of B. pfeifferi from three drainage basins in Dhofar, Oman (the eastern limit of its distribution). This is an arid to semi-arid region, with a 9,000-year history of very low rainfall, but is subject to unpredictable and destructive flash floods. In this context we showed that genetic fixation was very high compared to genetic differentiation which was moderate and, that, relative to B. pfeifferi populations from wetlands, the populations in Dhofar show evidence of lower levels of genetic diversity, a higher degree of genetic fixation, a quasi-absence of migration, and a higher level of genetic drift. Despite the extreme conditions in the Dhofar habitat of this species, it is able to survive because of its very high self-fertilization (approaching 100 %) and fecundity rates.

Snail intermediate host/Schistosoma haematobium relationships from three transmission sites in Benin (West Africa).

The relationships between three strains of Schistosoma haematobium (Doh, Sô-Tchanhoué and Toho-Todougba; from Benin, West Africa) and their snail hosts were assessed by measurement of several life-history traits, including the infection rate; pre-patent period; cercarial production of each parasite strain; and growth, fecundity and survival of the host snails. Adaptations to its local snail host was found for the Toho-Todougba strain and included a short pre-patent period, a long patent period and production of more cercariae in its local snail host. In contrast, the life-history traits of the Doh and Sô-Tchanhoué strains indicated non-local adaptations, as some sympatric host-parasite combinations were not compatible, the highest infection rates occurred in the allopatric snail Bulinus wrighti, and the duration of cercarial production was short because of the high level of mortality of the snails. Furthermore, snail reproduction ceased following infection by each of the three parasite strains, and the life-history traits were not influenced by the miracidial dose.

A new chronotype of Schistosoma mansoni: adaptive significance.

OBJECTIVES: To optimise host-to-host transmission, digenean trematodes (parasites) synchronize their cercarial emission patterns with the aquatic activities of their vertebrate hosts. Schistosoma mansoni has a strictly diurnal shedding pattern involving two circadian chronotypes: an early shedding pattern with a mean peak occurring at 11:00 h and a late pattern with a mean peak occurring at 16:00 h. We analysed the cercarial emergence pattern of three schistosome populations from Oman where S. mansoni is resurgent. METHODS: For each schistosome population, the cercarial emergence pattern was assessed hourly over several days. Because we identified a new chronotype hitherto unknown in S. mansoni, we undertook taxonomic characterisation based on egg morphology and mitochondrial DNA sequence (COX1). RESULTS: Taxonomic characterisation revealed that the three schistosome populations belong to the species S. mansoni. Hence, this is the first report of this species exhibiting a nocturnal chronotype, with the mean peak occurring at 20:00 h. We interpreted the new chronotype as being the result of a lateral transfer of S. mansoni from humans to Rattus rattus. CONCLUSION: The cercarial emergence pattern of S. mansoni from Oman is circadian, exhibiting either a diurnal or a nocturnal phenotype.

Natural Interactions between S. haematobium and S. guineensis in the Republic of Benin.

Schistosomiasis is a parasitic disease which affects millions of people around the world, particularly in Africa. In this continent, different species are able to interbreed, like Schistosoma haematobium and Schistosoma guineensis, two schistosome species infecting humans. The Republic of Benin is known to harbor S. haematobium, but its geographical situation in between Nigeria, Mali, and Burkina Faso, where S. guineensis was found, raises the question about the possible presence of S. haematobium/S. guineensis hybrids in this country. We conducted morphological analyses on schistosome eggs and molecular analyses on schistosome larvae (high resolution melting (HRM) analysis and gene sequencing) in order to detect any natural interaction between these two species of schistosomes. The morphological results showed the presence of three egg morphotypes (S. haematobium, S. guineensis, and intermediate). Three genotypes were detected by ITS2 rDNA HRM analysis: S. haematobium, S. guineensis, and hybrid, and their percentages confirmed the results of the morphological analysis. However, sequencing of the CO1 mtDNA gene showed that all the samples from Benin belonged to S. haematobium. Our results provide the first evidence of introgression of S. guineensis genes in S. haematobium in Benin.

Automated ChIPmentation procedure on limited biological material of the human blood fluke Schistosoma mansoni.

In living cells, the genetic information stored in the DNA sequence is always associated with chromosomal and extra-chromosomal epigenetic information. Chromatin is formed by the DNA and associated proteins, in particular histones. Covalent histone modifications are important bearers of epigenetic information and as such have been increasingly studied since about the year 2000. One of the principal techniques to gather information about the association between DNA and modified histones is chromatin immunoprecipitation (ChIP), also combined with massive sequencing (ChIP-Seq). Automated ChIPmentation procedure is a convenient alternative to native chromatin immunoprecipitation (N-ChIP). It is now routinely used for ChIP-Seq in many model species, using in general roughly 10 6 cells per experiment. Such high cell numbers are sometimes difficult to produce. Using the human parasite Schistosoma mansoni, whose production requires sacrificing animals and should therefore be kept to a minimum, we show here that automated ChIPmentation is suitable for limited biological material. We define the operational limit as ≥20,000 Schistosoma cells. We also present a streamlined protocol for the preparation of ChIP input libraries.

Genetic analysis of praziquantel response in schistosome parasites implicates a transient receptor potential channel.

Mass drug administration with praziquantel (PZQ) monotherapy is considered the mainstay for control and elimination of the parasites causing schistosomiasis in humans. This drug shows imperfect cure rates in the field, and parasites showing reduced PZQ response can be selected in the laboratory, but the extent of resistance in Schistosoma mansoni populations is unknown. We examined the genetic basis of the variation in response in a PZQ-selected S. mansoni population (SmLE-PZQ-R) in which 35% of the parasitic worms survive high-dose PZQ (73 micrograms per milliliter) treatment. We used genome-wide association to map loci underlying PZQ response and identified a transient receptor potential (Sm.TRPMPZQ) channel (Smp_246790) within the major chromosome 3 peak that is activated by nanomolar concentrations of PZQ. The PZQ response showed recessive inheritance and marker-assisted selection of parasites at a single Sm.TRPMPZQ SNP that produced populations of PZQ-enriched resistant (PZQ-ER) and PZQ-enriched sensitive (PZQ-ES) parasites, exhibiting >377-fold difference in PZQ response. The PZQ-ER parasites survived treatment in rodents at higher frequencies compared with PZQ-ES, and resistant parasites exhibited 2.25-fold lower expression of Sm.TRPMPZQ relative to sensitive parasites. Specific chemical blockers of Sm.TRPMPZQ enhanced PZQ resistance, whereas Sm.TRPMPZQ activators increased sensitivity. We surveyed Sm.TRPMPZQ sequence variations in 259 parasites from different global sites and identified one nonsense mutation that resulted in a truncated protein with no PZQ binding site. Our results demonstrate that Sm.TRPMPZQ underlies variation in PZQ responses in S. mansoni and provides an approach for monitoring emerging PZQ-resistant alleles in schistosome elimination programs.

Transplantation of schistosome sporocysts between host snails: A video guide.

Schistosomiasis is an important parasitic disease, touching roughly 200 million people worldwide. The causative agents are different Schistosoma species. Schistosomes have a complex life cycle, with a freshwater snail as intermediate host. After infection, sporocysts develop inside the snail host and give rise to human dwelling larvae. We present here a detailed step-by-step video instruction in English, French, Spanish and Portuguese that shows how these sporocysts can be manipulated and transferred from one snail to another. This procedure provides a technical basis for different types of ex vivo modifications, such as those used in functional genomics studies.

Case Report: Hemianopia: From Suspected Glioblastoma to the Diagnosis of Ectopic Schistosomiasis Haematobium Infection in a Traveler Returning from the Republic of the Congo.

Schistosomiasis due to Schistosoma haematobium is a widespread disease usually affecting the urinary tract associated with hematuria and kidney disorders. Neurological damage is rarely reported and symptoms are nonspecific and may suggest brain tumors such as glioma. We describe the first double ectopic haematobium schistosomiasis case involving the brain and intestine.

Baseline Mapping of Schistosomiasis and Soil Transmitted Helminthiasis in the Northern and Eastern Health Regions of Gabon, Central Africa: Recommendations for Preventive Chemotherapy.

In order to follow the Preventive Chemotherapy (PC) for the transmission control as recommended by WHO, Gabon initiated in 2014 the mapping of Schistosomiasis and Soil Transmitted Helminthiasis (STH). Here, we report the results of the Northern and Eastern health regions, representing a third of the land area and 12% of its total population. All nine departments of the two regions were surveyed and from each, five schools were examined with 50 schoolchildren per school. The parasitological examinations were realized using the filtration method for urine and the Kato-Katz technique for stool samples. Overall 2245 schoolchildren (1116 girls and 1129 boys), mean aged 11.28 ± 0.04 years, were examined. Combined schistosomiasis and STH affected 1270 (56.6%) with variation between regions, departments, and schools. For schistosomiasis, prevalence were 1.7% across the two regions, with no significant difference (p > 0.05) between the Northern (1.5%) and the Eastern (1.9%). Schistosomiasis is mainly caused by Schistosoma haematobium with the exception of one respective case of S. mansoni and S. guineensis. STH are more common than schistosomiasis, with an overall prevalence of 56.1% significantly different between the Northern (58.1%) and Eastern (53.6%) regions (p = 0.034). Trichuris trichiura is the most abundant infection with a prevalence of 43.7% followed by Ascaris lumbricoides 35.6% and hookworms 1.4%. According to these results, an appropriate PC strategy is given. In particular, because of the low efficacy of a single recommended drug on T. trichiura and hookworms, it is important to include two drugs for the treatment of STH in Gabon, due to the high prevalence and intensities of Trichuris infections.

Outbreak of urogenital schistosomiasis in Corsica (France): an epidemiological case study.

BACKGROUND: Schistosomiasis is a snail-borne parasitic disease endemic in several tropical and subtropical countries. However, in the summer of 2013, an unexpected outbreak of urogenital schistosomiasis occurred in Corsica, with more than 120 local people or tourists infected. We used a multidisciplinary approach to investigate the epidemiology of urogenital schistosomiasis in Corsica, aiming to elucidate the origin of the outbreak. METHODS: We did parasitological and malacological surveys at nine potential sites of infection. With the snails found, we carried out snail-parasite compatibility experiments by exposing snails to schistosome larvae recovered from the urine of a locally infected Corsican patient. Genetic analysis of both mitochondrial (cox1) and nuclear (internal transcribed spacer) DNA data from the Schistosoma eggs or miracidia recovered from the infected patients was conducted to elucidate the epidemiology of this outbreak. FINDINGS: We identified two main infection foci along the Cavu River, with many Bulinus truncatus snails found in both locations. Of the 3544 snails recovered across all sites, none were naturally infected, but laboratory-based experimental infections confirmed their compatibility with the schistosomes isolated from patients. Molecular characterisation of 73 eggs or miracidia isolated from 12 patients showed infection with Schistosoma haematobium, S haematobium-Schistosoma bovis hybrids, and S bovis. Further sequence data analysis also showed that the Corsican schistosomes were closely related to those from Senegal in west Africa. INTERPRETATION: The freshwater swimming pools of the Cavu River harbour many B truncatus snails, which are capable of transmitting S haematobium-group schistosomes. Our molecular data suggest that the parasites were imported into Corsica by individuals infected in west Africa, specifically Senegal. Hybridisation between S haematobium and the cattle schistosome S bovis had a putative role in this outbreak, showing how easily and rapidly urogenital schistosomiasis can be introduced and spread into novel areas where Bulinus snails are endemic, and how hybridisation could increase the colonisation potential of schistosomes. Furthermore our results show the potential risk of schistosomiasis outbreaks in other European areas, warranting close monitoring and surveillance of all potential transmission foci. FUNDING: WHO, ANSES, RICET, and the Ministry of Health and Consumption.