Detection of permethrin resistance and phylogenetic clustering of turkish head lice (Pediculus humanus capitis; De Geer, 1767 populations

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Detection of Permethrin Resistance and Phylogenetic Clustering of Turkish Head Lice (Pediculus humanus capitis; De Geer 1767 Populations ˘ ¸ e Atıcı , S¸emsi Nur Karabela , Mehmet Karakus¸ , Tugc ˘ Orhan Baylan , Mehmet Emin Limoncu , úIbrahim Cuneyt Balcıoglu ¨ PII: DOI: Reference:

S0001-706X(20)30003-6 https://doi.org/10.1016/j.actatropica.2020.105362 ACTROP 105362

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Acta Tropica

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2 January 2020 27 January 2020 27 January 2020

˘ ¸ e Atıcı , S¸emsi Nur Karabela , Orhan Baylan , Please cite this article as: Mehmet Karakus¸ , Tugc ˘ , Detection of Permethrin Resistance and PhyloMehmet Emin Limoncu , úIbrahim Cuneyt Balcıoglu ¨ genetic Clustering of Turkish Head Lice (Pediculus humanus capitis; De Geer 1767 Populations, Acta Tropica (2020), doi: https://doi.org/10.1016/j.actatropica.2020.105362

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Highlights  First molecular detection of permethrin resistance in field collected Pediculus humanus capitis specimens  We detected high resistance allele frequency (RAF) in three mutation sites T917I L920F, and M815I.  Phylogenetic analysis of head lice specimens revealed the presence of overlapping Clade A and Clade B in the present study.

Detection of Permethrin Resistance and Phylogenetic Clustering of Turkish Head Lice (Pediculus humanus capitis; De Geer 1767 Populations

Mehmet Karakuş1*, Tuğçe Atıcı2, Şemsi Nur Karabela3, Orhan Baylan1, Mehmet Emin Limoncu4, İbrahim Cüneyt Balcıoğlu5

1

Department of Medical Microbiology, Hamidiye Faculty of Medicine, University of Health

Sciences, İstanbul, Turkey 2 3

Department of Parasitology, Faculty of Medicine, Ege University, İ mir, Turkey Department of Infectious Diseases and Clinical Microbiology, Bakırköy Dr. Sadi Konuk

Training and Research Hospital, University of Health Sciences, İstanbul, Turkey 4 5

Vocational School of Health Services, Manisa Celal Bayar University Manisa, Turkey. Department of Parasitology, Faculty of Medicine, Manisa Celal Bayar University, Manisa,

Turkey

*Corresponding Author e-mail: [email protected]

Abstract Head lice infestation caused by Pediculus humanus capitis De Geer, 1767 is one of the most common public health problems. The relationship between humans and head lice dates back millions of years ago that differentiated into different phylogenetic clades. Treatment of head lice infestation usually based on insecticide-based products, which promotes the resistance in the head lice populations. In the present study, we aimed to screen the presence of permethrin resistance among collected P. h. capitis specimens in Turkey. Three mutation sites (T917I, L920F, and M815I) were screened using real-time PCR and resistance was identified by melt analysis. Of the studied specimens, resistance allele frequency (RAF) was found 0.98 for T917I, 0.99 for L920F, and 1.00 for M815I. The phylogenetic study revealed that Clade A and Clade B are present and overlap in Turkey. The present study is first to screen the resistance among Turkish head lice specimens. To not stimulate the pyrethroids resistance in head lice populations, early detection of resistance is crucial and will help the health professionals to choose suitable formula in the treatment. We suggest that the resistance status needs to be screened in randomly selected populations before any treatment application is given.

Keywords: Pediculus humanıs capitis, Head lice, Resistance, Pyrethroids, Turkey

Introduction Pediculosis, an infestation caused by Pediculus humanus capitis De Geer, 1767 is one of the most important health problems in school-age children for almost all countries. It is estimated that blood-sucking lice were coevolved with primates around 25 million years ago and P. h. capitis is differentiated during the last 2 million years of this coevolution (Reed et al., 2004, Amanzougaghene et al., 2016). The prevalence of the infestation is generally high in vulnerable groups, especially school-age children and refugees. Pediculus h. capitis is an obligate ectoparasite that can only survive on the human scalp and the first stage nymph starts feeding soon after it hatches from the egg. Since P. h. capitis have an incomplete metamorphosis, three different life stages (egg, nymph, and adults) are observed. It usually takes 20 days to develop eggs but this period might change according to weather conditions and single adult females may lay 150 eggs during their lifetime (Boutellis et al., 2014). Besides the intense irritation and discomfort among the infested people, P. h. capitis infestation also causes social ostracism, anxiety, and absence at school in school-age children. Also, several studies reported that P. h. capitis may serve as a vector for several bacterial agents like Borrelia spp., Bartonella and Acinetobacter spp. (Bonilla et al., 2009, Angelakis et al., 2011, Bouvresse et al., 2011). The head lice infestation is usually associated with low personal or general hygiene but high infestation rates were reported in both developed and developing countries. The estimated infestation in the USA is between 6-12 million per year and between 1%-20% in European countries (CDC, 2016, Feldmeier, 2012). Another survey study revealed that 36.4% of all households had head lice infestation experience in Norway (Rukke et al., 2011). Apart from country and culture, girls are more frequently infested (2-12 fold higher) comparing to boys (Kokturk et al., 2003, Hunter and Barker, 2003). Pediculosis is widespread in Turkey and according to the previous studies, the prevalence is changing between 0.54%-29.4%. Also, pediculosis among school-age children in Turkey is related to the socioeconomic status of the family (Karakus et al., 2014). The treatment of pediculosis is usually based on the topical application of insecticide-based products. Because the infestation is widespread worldwide, the extensive use of these products is inevitable. Treatment is usually done by using over the counter products (OTC), which may contains different types of pyrethroids. As a result of this, increasing resistance in different head

lice populations is reported worldwide (Picollo et al., 1998, Downs et al., 2002, Hunter and Barker 2003, Kristensen 2005). Pyrethroid resistance leads to disruption of the treatment and the emergence of chronic infestations, which approximately costs a billion $ per year only in the USA (Yoon et al., 2014). It’s been known for a long time that OTC products are not effective in the control of head lice in Turkey. Several studies were performed to evaluate the effectiveness of these products and results showed that most of them were failed to control the head lice even in in-vitro experiments (Balcıoğlu et al. 2015). Most of these products contain the same or similar active ingredient (sumithrin, permethrin, lindane, pyrethrin, and phenothrin). All these products are easy to access and no need any prescription to buy and apply to the infested person in Turkey. The pyrethroid targets the site of voltage-sensitive sodium channels (VSSC), which results in muscle paralysis in the head lice and followed by death. The excessive use of pyrethroids causes the point mutations within the subunit of the VSSC and those mutations functionally affect the nerve insensitivity in the head lice (Williamson et al., 1993, Dong and Scott 1994). Recently, identification of these mutation sites was performed and three sites (M815I, T917I, and L920F) in the VSSC gene were identified (Lee et al., 2000). The mutation resulting in the change in the amino acid substation of the para-orthologous voltage-sensitive sodium channel. Several methods were evaluated to determine these single nucleotide mutations (SNP) using genomic DNA extracted from P. h. capitis specimens (Lee et al. 2010). The relationship between humans and head lice dates back to millions of years ago and just like their human hosts, P. h. capitis can be differentiated into different clades (Mumcuoglu and Zias, 1988, Araujo et al., 2000, Reed et al., 2004) Distinct clade structure were identified using the mitochondrial DNA (mtDNA) data and three clades (Clade A, Clade B, and Clade C) were identified. Further studies identified more clades (Clade D, Clade E and Clade F) and subclades (Boutellis et al., 2013, Drali et al., 2015, Amanzougaghene et al., 2019). The identified clade structures are grouped according to their geographical distribution. The most specific clade is Clade C consist of only P. h. capitis populations of Nepal and Ethiopia, while Clade A comprises the head and body lice from all over the world. Clade B only consists of populations from Europe, Australia, and the Americas (Amanzougaghene et al., 2019). Yet there is no study performed in Turkey to identify the clade structure of Turkish P. h. capitis populations. In the present study, we aimed to screen the permethrin resistance among collected P. h. capitis specimens from a primary school located in Manisa City, The Aegean region of Turkey. Also,

phylogenetic clade analyses were performed to understand the clade distribution of Turkish P. h. capitis populations.

Material and Methods Sample Collection Pediculus h. capitis specimens used in this study were collected from a village primary school (Manisa city, Turkey, Lat: 38844083’N, Long: 27278344’E) using special plastic and metal combs as used in a recent study (Balcıoglu et al. 2015). Head lice collection protocol was approved by the Directorate of the school and Manisa Celal Bayar University, Faculty of Medicine, Ethics Committee under the number of 2015-107. The written informed consent form was provided by a parent of each child. Collected P. h. capitis specimens were transferred to the laboratory and preserved in 95% EtOH until the molecular experiments were done. DNA extraction and molecular experiments Head lice specimens were transferred to ZR Bashing BeadTM (Zymo Research Corp., Irvine, CA, USA) tubes individually. Each specimen was homogenized using Magna Lyzer (Roche Molecular Diagnostics, Mannheim, Germany) at 7000 g for 90 s and DNA extraction was made using the commercial kit (Qiagen, Hilden Germany) by following the instructions of the manufacturer. A real-time PCR protocol targeting the three mutation sites (M815I, T917I, and L920F) of head louse sodium channel α-subunit gene was performed using the gDNA of each head lice (Drali et al., 2012). Fluorescence Resonance Energy Transfer (FRET) probe technology was used for each target with specific primers using LightCycler® FastStart DNA Master HybProbe master mix (Roche Life Science, Germany). Real-time PCR reactions were performed on thin-walled (50 l) stripe tubes. The reaction conditions were similar for both T917I and L920F markers, while annealing was optimized for M815I. The reaction starts with 90°C hold for 10 min., followed by 45 cycles containing 95°C for 5 sec., 51°C (64°C for M815I) for 10 sec. and 72°C for 15 sec. and last hold step for 30 sec. at 95°C. Melt analysis was started from 40°C and finalized at 85°C. Gain optimization was done before melt analysis for all tubes to obtain high quality melting peaks. Each specimen was named as resistant (RR), susceptible (SS), and heterozygous (RS) according to the melting temperature obtained by this protocol. Each target was amplified individually and melting analysis was performed. Due to transitions causing replacement in the specific region, the T m difference between RR and SS specimens was

7-8 °C. Homozygote and heterozygote alleles were identified according to the T m and peak values as reported previously (Drali et al., 2012). Because the probe attachment was specific to the mutated region, high Tm was specific to wild type allele, lower Tm was specific to homozygous allele and the double peak was identified as a heterozygous allele. Haplotypes and allele frequency were calculated using the formula [(number of resistant homozygotes x 2) + (number of heterozygotes)]/[(number of resistant homozygotes + number of heterozygotes + number of susceptible homozygotes) x 2] and the resistance level was evaluated on a scale within 0 to 1.00, where 1.00 indicated the highest resistance. Phylogenetic studies To reveal the genetic diversity of the head lice found in Turkey, all were involved in the mitochondrial clade study. The primer pair CytbF/CytbR is used to amplify the Cytochrome b (Cytb) region by conventional PCR. Genomic DNA of the P. h. capitis specimens were used as a template and previously reported conditions were applied (Light et al., 2008). Amplified products were visualized on agarose electrophoresis and sequence analysis was done commercially (Letgen Biotechnology, Izmir) with the primers used in the amplification. Sequence data analyzed on Geneious R8 Software and phylogenetic analysis was performed using the database of previously deposited cytb data in NCBI Genbank (Kearse et al., 2012). The phylogenetic tree was constructed with the Tamura-Nei genetic distance model (Neighbor-Joining) using previously deposited sequence data (Supplementary Table 1).

Results Real-time PCR application Hundred-and-fifty adult head lice were collected and used for the present study. All subjects involved in the present study were in the adult stage. Before real-time applications, standard peaks for SS, RR, and RS were created using wild type, resistant and heterozygote DNA samples obtained commercially from Letgen Biotechnology, Izmir, Turkey. A real-time PCR technique revealed the mutations in the VSSC gene and both homozygote and heterozygote peaks were obtained in the present study (Figure 1). Melting peaks obtained from real-time PCR were analyzed for each point mutation site and were determined as RR (homozygote resistant), RS (heterozygote) and SS (homozygote susceptible) according to the specific Tm. For the marker M815I, RR was noted in 150 (100%) specimens while no RS peak was recorded. None of the studied specimens was found SS for this marker. Obtained peaks were evaluated for marker T917I and RR was recorded in 146 (95.6 %) specimens and six specimens were found RS (4.4 %). Lastly, melt peaks for marker L920F were analyzed and RR was recorded in 149 (99.3 %) specimens, while no RS recorded in the studied specimens. According to those findings, resistance allele frequency (RAF) was calculated for each mutation points and was found 1.00 for M815I, 0.98 for T917I, and 0.99 for L920F (mean= 0.99) (Table 1). Specimen-based results can be found on supplementary data (Supplementary Table 1). Cluster Analysis of Cytb gene (Clade determination) A phylogenetic tree was constructed using Cytb gene sequence data obtained in the present study by comparing it with previously submitted data to GenBank. Neighbor-joining (NJ) phylogram showed that the analyzed specimens were belonging to two different clades (Clade A and Clade B). To maximize the figure quality, only two specimens (identical sequence data were excluded from the phylogenetical graph) from each clade are represented in the phylogenetic cladogram figure (Figure 2). The majority of the analyzed specimens were belonging to Clade B (n=123/150, 82%), while the rest were belonging to Clade A (n=27/150, 18%). All sequence data obtained in the present study were deposited to GenBank (MN337632-MN337781). The GenBank accession codes obtained in the present study and reference sequences used in the phylogenetic analyses to construct a cladogram can be found in data (Supplementary Table 1).

Discussion Head lice infestation is one of the major public health problems among school-age children and the damage it costs to the economy is greater than it is estimated. More than 100 research study on head lice infestation was performed in Turkey between 1982-2019 and all were conducted in primary schools (Karakus et al. 2014, Ö kan et al. 2015, Acıö and Ö türk, 2018). Infestation rate differs between 0.54%-34.1% and compared to boys, girls always showed a higher infestation rate in previous studies (Kokturk et al., 2003, Hunter and Barker, 2003). As reported in a recent study, the socio-economic level difference was one of the major factors that affect the infestation rate among school-age children (Değerli et al. 2013). The major limitation of previous studies performed in Turkey was to not give proper information to families of infested children, which leads to re-infestation even if successful treatment is implemented. Also, lack of the school nurse system in Turkey, which will be the first to notice the head lice infestation in primary schools, makes it harder to control the head lice infestation in primary schools. The use of mitochondrial genes provides suitable data in the classification of head lice due to the lack of recombination throughout the historical period. Head lice found in Turkey were speculated to belong to the Clade A according to the proposed migration route (Light et al. 2008). Interestingly, two clades (Clade A and Clade B) were determined in a single sampling location in the present study. The majority (n=123, 82%) of the studied head lice specimens were found to be classified in Clade B, while 23 specimens (18%) were belonging to Clade A. According to the proposed human migration route, Clade B was reported to be a New world Clade found in North and Central America, Europe, and Australia and reported to be strongly present in Algeria (Boutellis et al. 2015). Both Clade A and Clade B consist of specimens from Asia, Europe, and Central America, which makes them overlap in some certain timeline. Sympatric lifestyle was reported in several studies suggesting that the head lice belonging to different clades can live and interbreed in the same individual. The sympatric presence of Clade A and Clade B was reported by researchers in recent studies (Boutellis et al. 2013, 2015, Xiong et al (2014). The same results were obtained in the present study and the existence of overlapping Clades (Clade A and Clade B) were shown for the first time in Turkey. Unfortunately, we could not identify whether those different clade subjects were collected from the same individual or not. However, the presence of both clades in the same village school strongly supports the idea that Clade A and B can live and

interbreed in the same individual. More studies are needed to identify the presence of different haplotypes in Turkey. A high number of resistant specimens were reported in the present study. We believe that the fact that there is no obstacle to access pyrethroid-based products is one of the major reasons for such a developed resistance in Turkey. There are several studies performed in Turkey questioning the effectiveness of those OTC products and the treatment rate was found 96% for sumithrin (Budak et al. 1996), 84.7% for permethrin (Yazar et al. 2002), 75.5% for d-phenothrin (Tanyuksel et al. 2003), 35% for pyrethrin + piperonyl butoxide (Noyan and Demir, 2006), and 67.5% for lindane (Budak et al. 1996). Interestingly only one study reported that they successfully treated the head lice infestation using hair shampoo containing sumithrin (Eroglu et al. 2016). Since there is no data published in Turkey about the molecular surveillance of the resistance in head lice specimens, we failed to compare our results and understand the resistance trend in different head lice populations of Turkey. With the help of a real-time PCR protocol described recently by Drali et al. (2012), it was faster and easier to determine the permethrin resistance in each head lice specimen. This method allows us to determine the resistance status on an individual base, which is perfectly suitable for the epidemiological studies (Drali et al. 2012). Heterozygous alleles found in the present study recorded as RS for each specific allele and all (n=6; 4.4%) were found in the T917I site in the present study, as it was in a recent study (Toloza et al. 2014). The presence of heterozygous allele only in the T917I site supports the idea that marker M815I and L920F are the first sites to mutate under permethrin exposure. This finding is also suggested in several studies and mutation site T917I was found to have a lower prevalence comparing to M815I and L920F (Hodgdon et al. 2010, Drali et al. 2012). It is considered that mutated M815I and L920F sites are not the main cause of permethrin resistance, but responsible for the reduced sensitivity to permethrin. In the present study, we found the same trend in mutation sites as it was reported previously (Yoon et al. 2008, Drali et al. 2012). A high prevalence of pyrethroid resistance was reported to be one of the most important problems encountered in the control of pediculosis (Drali et al. 2012). Three markers were analyzed using the Hardy-Weinberg equilibrium model and the observed frequency for each marker was very high compared to previous studies performed in Europe and The Americas. According to the

published data, mean RAF was ranging between 0.57 and 0.93 in France, 0.44 in Wales UK, and 0.95 in Denmark, 0.35-0.96 in Guadeloupe, and 0.99 in The USA (Kristensen et al. 2006, Durand et al. 2007 and 2011, Gellatly et al. 2016). In the present study, only six specimens were found to be RS and all were T917I. Observed high RAF values can be explained by the extensive use of insecticide based anti-head lice products in the study area. Interestingly, RAF values can be dramatically changed even in the same city but in a different primary school. As it is reported in recent studies, changing rates varied between 0.32 to 0.83 in Guadeloupe and 1.00 to 0.78.5 in Argentina (Ponce-Garcia et al. 2017; Toloza et al.2014). Resistance to anti-head lice products were demonstrated in several countries, including Canada, Britain, Korea, Japan, USA, and Israel (Marcoux et al. 2010, Mienking et al. 2002, Burgess et al. 1995, Mumcuoğlu et al. 1995, Picollo et al. 1998, Hemingway et al. 1999, Clark 2009). Since no study performed to screen permethrin resistance in head lice populations of Turkey, high mutation rates were reported for the first time in the present study.

Conclusion The present study is performed to screen the permethrin resistance in head lice population of Turkey. The results obtained from this study showed high resistance allele frequency among the specimens. To not stimulate the pyrethroids resistance in head lice populations, early detection of resistance is crucial and will help the health professionals to choose suitable formula in the treatment. We suggest that the resistance status needs to be screened in randomly selected populations before any treatment application is given. Also, phylogenetic analysis was performed and overlapping clades were shown in the present study. Author Contribution M.K. designed and performed the experiments, analyzed the data, wrote the manuscript. T.A. performed several molecular experiments. C.B. and MEL. designed the fieldwork. S.N.K. and O.B. critically read the article. Conflict of interest statement On behalf of all authors, the corresponding author states that there is no conflict of interest. Acknowledgments We thank Drs. Suha Kenan Arserim, Seray Töz and Yusuf Özbel for their valuable helps. This study is supported by the Scientific Research Project Fund of the University of Health Sciences under project number 2018/067.

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Figure 1: RT PCR graph showing positive controls and head lice specimens (RR: Resistant, RS: Heterozygous, SS: Susceptible)

Figure 2: Cladogram of P. capitis specimens constructed using Neighbor-Joining method (NJ) with Tamura and Nei model (previously submitted sequence data is shown with accession codes) Bootstrap values (1000 number of replicates support threshold 55%) are shown above the branches.

Sampling Location

Sampling Size

Marker

Number of RR

Number of Number of Frequen RS SS cy

M815I 150 0 0 1.00 (ATG>ATT) T917I Manisa, Turkey 150 144 6 0 0.98 (ACA/ATA) L920F 149 0 1 0.99 (CTT/TTT) Table 1: Resistance allele frequency (RAF) of the studied head lice specimens. RR: Resistant, RS: Heterozygous, SS: Susceptible

Supplementary Table 1: Detailed results of studied markers (M815I, T917I, and L920F) and sequence data of all specimens