Molecular Characterization and Genetic Diversity of Clade E-Human Head Lice from Guinea

Aix Marseille Univ, IRD, APHM, MEPHI, IHU-Méditerranée Infection, Marseille, France. Aix Marseille Univ, IRD, APHM, VITROME, IHU-Méditerranée Infection, Marseille, France. 3 Faculté des Sciences, Université M'Hamed Bougara Boumerdes, Boumerdès, Algérie. 4 Institut de Recherche en Biologie Appliquée de Guinée (IRBAG), Kindia, Guinée 5 Centre de Formation et de Recherche en Santé Rurale de Mafèrinyah B.P. 2649, Conakry, Guinée. 6 Faculté de Médecine, Pharmacie et Odontostomatologie, Université Gamal Abdel Nasser de Conakry, Guinée. 7 Maladies Infectieuses Et Vecteurs : Écologie, Génétique, Evolution Et Contrôle, MIVEGEC UMR 224, Univ Montpellier, IRD, CNRS, Montpellier, France

It is therefore obvious that it is important to prevent potential epidemics related to these ectoparasites.
Robust phylogenetic studies of human lice based on mitochondrial DNA, mainly cytochrome b [cytb] and cytochrome oxidase subunit 1 [cox1] genes, have inferred Pediculus humanus into six divergent mitochondrial clades (haplogroups): A, D, C, E, B and F, each with distinct geographical distribution [5,14,[31][32][33]. Human lice also present an intra-clade diversity in addition to their inter-clade diversity, which is illustrated by several distinct haplotypes for each haplogroup [21,31,34]. Unlike body lice, which only belong to clades A and D, head lice encompass all the genetic diverse clades [18]. Clade A has a global continental distribution and it is the most prevalent [31,34], while clade D is restricted to sub-Saharan African countries, and has so far been reported in the Democratic Republic of Congo (DRC), the Republic of Congo (Congo-Brazzaville), Ethiopia and Zimbabwe [14,21,31]. Clade C has been identified mainly in 29]. Clade B is found in a high diversification in America, it has been reported in Western Europe, Australia, North Algeria, South Africa, Saudi Arabia, and is also present among the remains of head lice from the Roman period dating back to about 2000 years [31,[34][35][36][37].
Recently, a novel clade F, the sister group of clade B, was described in French Guiana, in head lice recovered from the Wayampi community living in a remote Trois-Sauts village. This clade was also found in Argentina and Mexico [33]. All these data confirm important facts about the evolutionary history of lice, as well as the ancestors of their human hosts since their migration out of Africa [38].
Despite their clade diversification and ecological niches, The Pediculus humanus ecotypes are morphologically and biologically almost similar [3,5]. Previous genetic studies targeting intergenic spacers, using a highly polymorphic markers were not able to differentiate between body and head lice [38,39]. Moreover, a study based on the comparison of head and body lice transcriptomes, reported that the two ecotypes had a single 752-base pair (bp) difference in the Phum_PHUM540560 gene, with differential expression that encodes a hypothetical 69-amino acids protein of unknown function [40]. The PHUM540560 gene and 13 others were thought to be missing in head louse. However, a study conducted by Drali and collaborators, showed that the head louse also harbors this gene, but with a rearranged sequence compared to body louse.
The variation of the Phum_PHUM540560 gene within the two ecotypes allowed the design and development of a novel molecular tool based on multiplex real-time PCR assays, in order to differentiate the Clade A body and head lice [41].
In Guinea, West Africa, human lice infestation is very frequent but never investigated. In this study, we aimed to identify for the first time in Guinea, the genetic diversity status of head lice collected from two sites: rural (Maférinyah village) and urban (Kindia city), the Phum_PHUM540560 gene polymorphisms, as well as to assess the occurrence of bacterial pathogens in these lice.

Lice collection and DNA extraction
In December 2018, head lice collection was carried out at Medical Centers in two areas: Maférinyah (9.5466° N, 13.2866° W) and Kindia (10.0407° N, 12.8630° W) from Guinea in West Africa (Fig.1). All individuals in medical centers with a head lice infestation were asked to perform a complete self-examination for the presence of head and body lice. The medical center personnel obtained verbal consent from the participants and authorization from the head of the medical center to supervise the collection process.
A total of 155 head lice were obtained from 49 individuals (Mean age: 11 [2,62] In order to decontaminate the external surface and avoid bacterial contamination, each louse specimen was washed and decontaminated as previously described [42]. Dried louse specimen was cut in half lengthwise, the first half was frozen at −20 °C for subsequent studies. Total DNA was extracted from the remaining half using a DNA extraction kit, QIAamp Tissue Kit (Qiagen, Courtaboeuf, France) in the EZ1 apparatus following the manufacturer's instructions.  In this study, all lice specimens were collected from the scalp region of each individual; however, we were curious to further study our lice ecotypes. Therefore, all lice samples were analyzed by multiplex real-time PCR, targeting a portion of the PHUM540560 gene. This assay was developed to discriminate between body lice and head lice belonging to clade A [41], which has always been known as a worldwide clade that encompass the two ecotypes [31,34]. This assay had never been used to study lice belonging to other clades. We used a clade A head and body louse as positive controls.

Louse ecotype investigation by conventional PCR assays and sequencing
To analyze the sequences of the PHUM540560 gene of the Guinean human lice, 40 specimens were randomly selected. Additionally, clade A-human lice were randomly selected from our lice collection, including 7 specimens of Orlando strain from our rabbit rearing colony and 11 Algerian body lice collected from homeless people [45]. These samples were then subjected to standard PCR and sequencing of the target PHUM540560 gene. The sequences obtained were aligned with the specific PHUM540560 sequences of the clade A-body and head lice, described thorough the previous study [41]. Alignment was performed using the BioEdit v 7.0.5.3 software (available online: http://en.bio-soft.net/ format/BioEdit.html), in order to reveal the rearranged sequences of the head lice PHUM540560 gene collected in Guinea compared with those of the clade A-human lice.

Identification of pathogen's DNA by qPCR assays
In order to screen louse-borne pathogens, a qPCR was performed for all head lice samples targeting the presence of various pathogenic bacteria: Rickettsia spp., Borrelia spp., B. quintana, Y. pestis, C. burnetii, Anaplasma spp. and Acinetobacter spp., using specific primers and probes, as previously reported (Table1).
Initially, we pooled our DNA's template into 8 samples of 10μl/specimen for each pool; each sample was in a proportion of 1 Log of its initial concentration. Modifications of the qPCR cycles threshold (Ct) from 40 Ct to 45 Ct were performed to ensure that no potentially positive samples were missing from each pool. Thereafter, each louse from each positive pool with a Ct ≤ 38, was tested individually. The qPCR analysis was performed as described above for the cytb, a positive control of the targeted DNA and a negative control of master mixtures were included in each PCR run. Samples positive for Acinetobacter spp. were subjected to qPCR specific for Acinetobacter baumannii, targeting the OmpA/MotB gene, as previously reported (Table 1).

Identification of pathogen's DNA by conventional PCR and sequencing
In order to identify the species of Acinetobacter, positive samples from qPCR were subjected to a standard PCR targeting a portion of the rpoB gene (zone1) using the primers and conditions previously described (Table 1). Successful amplification was confirmed by electrophoresis via agarose gel, amplicons were prepared and sequenced using similar methods as described above. Due to a low specificity of above-mentioned primers, a new specific PCR system was designed in this study in order to amplify only and specifically the DNA of

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This study included 155 head lice specimens collected from 49 individuals, (96% female 4 and 4% male), from two regions of Guinea, Maférinyah and Kindia. First, All P. h. capitis 5 specimens were subjected to a duplex qPCR to determine their clade. The results of the 6 amplification curve revealed that our samples were positive for the clade C-E. Standard PCR and 7 sequencing showed that all our samples belonged to the haplogroup E. For the phylogenetic 8 analysis, we were able to generate 141 sequences from the 155 samples analyzed, due to the low 9 DNA concentration. The generated Guinean sequences were then aligned and combined with all 10 sequences available for cytb haplotypes and were then used to construct a maximum-likelihood 11 (ML) tree (Fig. 2)    Besides, all Pediculus humanus lice were tested by multiplex qPCR targeting the 31 PHUM540560 gene to investigate their ecotype; this method was used previously to distinguish 32 between head and body lice belonging to clade A. All our specimens were collected by the 33 patients from their scalp hair, and belong to clade E. Using this method, all the 155 Guinean head  Table 2. Details of PHUM540560 sequences alignments are listed in Figure 2. 58 59  Acinetobacter species including: 2/7 (28.6%) E69 and E39 head lice matching with 89 Acinetobacter nosocomialis, 2/7 (28.6%) E39 and E48 head lice with Acinetobacter variabilis, 90 1/7 (14.2%) E69 head louse with Acinetobacter towneri and finally, 1/7 (14.2%) E48 specimen 91 with Acinetobacter haemolyticus. The remaining generated sequence 1/7 (14.3%), shared a lower 92 similarity (<94%, coverage 100%) with two Acinetobacter species: A. johnsonii and A.
E48 and E39. The remaining seven of the 14 sequences (50%) also had similarities with 99 Acinetobacter spp. However, the sequences were of poor quality, which is assumed to be due to 100 the co-infection of several Acinetobacter species. The phylogenetic tree of all Acinetobacter 101 species identified in this study is presented in Fig.4. 102 In our study, none of the DNA A. baumannii samples tested positive for carbapenem's-103 resistant encoding genes ( bla OXA-21 , bla OXA-24 , bla  . 104 105 Figure 5. Phylogenetic tree highlighting the position of the Acinetobacter species 106 identified in the head lice collected from Guinea compared to Acinetobacter spp. available in the 107 GenBank database. Phylogenetic inferences were conducted in MEGA 7 using the maximum 108 likelihood method based on the Kimura 2-parameter model for nucleotide sequences. Statistical 109 support for internal branches of the tree was evaluated by bootstrapping with 1000 replicates. 110 There was a total of 7 positions in the final dataset. 111

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To the best of our knowledge, the present study is the first to investigate both the 113 phylogeny and associated pathogens of head lice collected in Guinea. A total of 155 head lice 114 were collected from 47/49 (95.9) females and 2/49 (4.1%) males living in two different rural and 115 urban areas, Maférinyah, a village, and the city of Kindia, Guinea, West Africa. A genetic study 116 using the qPCR duplex first showed that all the lice samples analyzed belonged to the 117 mitochondrial clade C or E. Standard PCR and sequencing revealed that all the head lice 118 belonged to the haplogroup E. The qPCR duplex method is not discriminative enough for the 119 screening of African human lice, which mainly belong to haplogroups C and E. In the future, this 120 method should be optimized by a design that also includes a specific clade E monoplex, to obtain 121 a better identification of African human lice, as well as to establish an optimal qPCR duplex for 122 the discrimination of all human lice belonging to all existing haplogroups, including the recently 123 described clade F. The phylogenetic study showed the existence of 8 haplotypes including 6 124 novels described for the first time in this study. The presence of clade E in both rural and urban 125 communities in Guinean lice is not surprising, as it confirms the high prevalence of the "African 126 endemic" clade E, as previously reported so far [18,26,28,29]. The most prevalent haplotype 127 reported in our head lice is the E39 obtained with 68.1%, followed by the haplotype E48 with more specifically the E62 haplotype in Central Africa, in Congo, suggesting that Congonians are 135 in direct contact with West African populations or travelers arriving form West African countries 136 [28]. More recently, this clade has also been found in head lice collected from individuals in 137 Gabon, belonging to haplotype E46 [29], already reported among lice collected in Mali [18]. 138 These results suggest that the significant migratory exchange between Gabon and the Republic 139 of the Congo can be the source for the clade E expansion [29]. In addition, among the 141 head 140 lice cytb sequences analyzed, 4 lice with two different haplotypes, E39 and E70, were collected 141 from the same 34-year-old woman. In addition, four haplotypes were also identified within the 142 same person infested with 11 lice, in Kindia, belonging to haplotypes E39, E48, E71 and E73.  necessary to study a larger portion of the Phum_PHUM540560 gene. These investigations will 169 allow a better understanding and will probably lead us to design a more efficient molecular tool, 170 which will be able to discriminate between the two ecotypes. At this point, we can affirm the fact 171 that the morphological, biological and genetic characteristics of P. humanus species are almost 172 similar and remain obscure. However, body and head lice are extremely different in their 173 ecological niches, which remain, until now, the main criterion for distinguishing between these 174 two ecotypes. 175 In recent decades, the paradigm that P.h.humanus was the only vector of dangerous 176 diseases has been challenged [8]. Indeed, many studies have reported the presence of several 177 pathogenic agents in head lice specimens collected worldwide [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]; thus, demonstrating that 178 the potential pathogen-vector-competence of the head louse is not yet understood [8]. In this head lice infesting 11 individuals. Findings from previous studies reported a worldwide spread 181 of several Acinetobacter species, including A. baumannii, A. junii, A. ursingii, A. johnsonii, A. 182 schindleri, A. lwoffii, A. nosocomialis, A. towneri, A. variabilis, A. radioresistens, A. 183 calcoaceticus, A. soli, A. pittii  A. haemolyticus, because of their multiple resistance to many common antibiotics [58]. 206 In recent decades, Acinetobacter bacteria have shown high ability to develop resistance to 207 almost all major classes of antibiotics. So far, the incidence of carbapenem resistance in A. 208 baumannii has continued to increase worldwide [54]. In human lice, A. baumannii isolates were 209 remarkably susceptible to carbapenems [55]. Indeed, a study performed on head lice collected in 210 Senegal reported that 21.4% of the positive A. baumannii-head lice harbored a bla OXA-23 211 carbapenem resistant encoding gene [56]. A precedent study reported for the first time the 212 presence of bla OXA-23 gene in positive A. baumannii human head lice in Senegal [54]. None of 213 our positive-A. baumannii head lice were positive to the carbapenem's-resistant encoding genes 214 ( bla OXA-21 , bla OXA-24 , bla  . Further studies are needed to investigate the association between 215 Acinetobacter infections and human lice, the compatibility of Acinetobacter strains present in 216 lice and those responsible for human infections, as well as investigate the carbapenem's-resistant 217 in A. baumannii strain present in human lice. 218

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Herein, to the best of our knowledge, we report here the first molecular data on the genetic 220 diversity and associated pathogens of head lice collected in Guinea. Overall, polygenetic analysis 221 reveal that all our specimens belonged to haplogroup E within 8 haplotypes, with 6 novels 222 described for the first time in this study. Genetic study of the PHUM540560 Gene 223 polymorphisms profile revealed that the majority of our Guinean head lice exhibit a clade A-224 body lice PHUM540560 gene polymorphism profile, showing the importance of conducting a 225 more in-depth genetic study of the PHUM540560 gene, targeting human lice belonging to the six 226 divergent mitochondrial clades to better understand this paradigm. None of the pathogenic 227 bacteria tested were detected in our P. h. capitis samples, except for Acinetobacter spp. for 228 which we were able to identify several species, including A. baumannii, A. nosocomialis, A. 229 variabilis, A. towneri, a potential new specie "Candidatus Acinetobacter P.h capitis Guinea" 230 and, for the first time in head lice, A. haemolyticus. Further studies are needed to study the 231 genetic diversity of Guinean head lice and to evaluate their role as a potential vector for their 232 associated bacteria. 233