First Determination of Pyrethroid Knockdown Resistance Alleles in Human Head Lice (Phthiraptera: Pediculidae) From Chile

Abstract

The infestation with the human ectoparasite, Pediculus humanus capitis (De Geer), is a common public health problem affecting schoolchildren worldwide. In Chile, the main active ingredients present in the over-the-counter pediculicides contain pyrethroids. Despite the extended use of these products, there is no evidence of the insecticide resistance status of the head lice geographically located in Chile. The most extended resistant mechanism of pyrethroids consists of the target site insensitivity (Kdr) determined by the presence of mutations linked to insecticide-binding sites in the voltage-sensitive sodium channel. T917I is recognized as the main mutation in head lice, and detection is considered to be a biomarker of resistance. The goal of the present study was to detect the presence and distribution of T917I mutation in five geographic locations of Chile. All five geographically selected louse populations had a frequency of pyrethroid resistance genes that ranged from 36 to 77%, and 94.9% of the collected head lice had one or two T917I mutant alleles. Moreover, the frequency of the aggregate resistant alleles was 50.5%. This is the first evidence that head lice in Chile had the mutations commonly associated with the resistance to pyrethroids. Moreover, the overrepresentation of heterozygotes in the studied populations suggests that head lice in Chile are currently under active selective pressure.

Pediculus humanus capitis, head lice, kdr, resistance, pediculosis

The human head louse, Pediculus humanus capitis (De Geer), is an obligate ectoparasite responsible for Pediculosis capitis. This infestation of the human hair and scalp causes symptoms that include itching, irritability, and secondary bacterial infections that may result from scratching (Burgess 2004). Head lice are cosmopolitan blood-sucking insects with a worldwide prevalence (Falagas et al. 2008). The main route of transmission is by head-to-head contact (Heukelbach 2010), mainly on school-aged children and is heavily influenced by either social or behavioral transmission factors (Mumcuoglu et al. 2009, Toloza et al. 2009). Although head lice is not considered a significant disease vector, several studies reported that they can carry Bartonella quintana (the agent of trench fever) and Acinetobacter baumannii (an emerging hospital-acquired bacterial infestation; Bonilla et al. 2009, Angelakis et al. 2011, Bouvresse et al. 2011, Eremeeva et al. 2017).

Head lice control relies on health education, early diagnosis, and postinfestation treatments. This includes a wide variety of options such as combing (dry or wet) and the use of pediculicides (Burgess 2004, Mumcuoglu et al. 2009).

Chemical pediculicides are the first-line treatment by national institutions and consist of over-the-counter (OTC) products and those available under medical prescription. Of these, OTC topical products are still the method of choice with estimated pediculicide sales in the United States at >USD 200 million/yr (Clark 2009). Pyrethroid formulations are still the most widely used in the current pediculicide market worldwide, with permethrin and d-phenothrin as the most active ingredients included in the products (Heukelbach 2010). Pyrethroids have been considered excellent insecticides due to their low mammalian toxicity and relatively safe environmental residuality. However, resistance to this class of insecticide has been well documented in human head lice populations from several countries, including the United Kingdom, France, Japan, Israel, Israel, United States, Argentina, and Russia (Thomas et al. 2006, Durand et al. 2007, Kasai et al. 2009, Lindh and Mumcuoglu 2012, Clark et al. 2013, Toloza et al. 2014, Eremeeva et al. 2017). Use of pyrethroids (or DDT) can lead to the evolution of target site insensitivity due to single-nucleotide polymorphisms in the voltage-sensitive sodium channel (VSSC) gene of insects. When these channels function correctly, the nerve impulse is transmitted properly. On the contrary, when this process is altered, a number of neurotoxic symptoms such as incoordination, tremors, paralysis, and death occur. Knockdown resistance (Kdr) is associated with mutations in the sodium channel gene (Hemingway and Ranson 2000). Three point mutations M815I, T917I, and L920F, all located in domain II, are responsible for resistance worldwide (Heukelbach 2010). Moreover, all these mutations were determined to coexist en bloc as a resistant haplotype (Lee et al. 2003). Importantly, it was demonstrated that the mutation T917I either alone or in combination abolished permethrin sensitivity. Also, this mutation may be similar to the super kdr trait identified in other insects, a genetic trait by which permethrin sensitivity is almost completed abolished. Thus, this point mutation can be used as a molecular biomarker for resistance detection (SupYoon et al. 2008).

In Chile, pediculosis is a common public health problem with an overall infestation rate in schoolchildren of 29% determined several years ago (Devera 2012). Moreover, prevalence may or may not vary in relation to geographic area. For instance, studies performed in elementary schools of Santiago de Chile and Valparaíso (central region), and Antofagasta and Arica (northern region) found a prevalence of 22, 47, 25, and 40%, respectively (Hernández et al. 1981, Neira et al. 1986, Sagua et al. 1997, Paola Gazmuri et al. 2014). There are several OTC pediculicides approved in Chile whose main active ingredients consist of permethrin, deltamethrin, crotamiton, and Quassia amara (Instituto de Salud Pública de Chile 2017). Despite the numerous authorized products, the insecticide resistance status of head lice from Chile has not been evaluated (Kuncar et al. 2004, Avello et al. 2016). The purpose of this study was to investigate the presence and occurrence of the T917I sodium channel mutation in head lice collected from five selected geographic regions of Chile.

Materials and Methods

Head Lice Collection

Head lice were collected from heads of infested children attending the Center for Pediculosis Control Liceteam, Inc., Santiago, Chile. The hair of each child was first combed with a regular hairbrush followed by combing the entire scalp with a fine-toothed louse comb for a period of 5–10 min. All the combings were performed by healthcare-trained professionals. Insects were then picked-up by entomological forceps and placed in individual vials containing 95% ethyl alcohol. Nymphs, adult female, and male lice were classified under a stereoscopic zoom microscope (Nikon SMZ 10). This was approved by the protocol for lice collection and identification of the Centro de Investigaciones de Plagas e Insecticidas (CIPEIN-UNIDEF, Buenos Aires, Argentina; # BA20061995ARG, June 1995). Once in the laboratory, head lice were stored at −20°C until studied. In total, 99 human head lice from two to five children were collected along different locations of Chile (Table 1; Fig. 1).

Table 1.

Frequency of Kdr alleles in Chilean head lice populations

Population	No. of head lice obtained (no. of infested subjects)	Genotypea			Resistance allele frequency (%)	H-Wb (χ2)	Fisd
		S/S	R/S	R/R
Vallenar	11 (2)	0	5 (45.5)	6 (54,5)	77.3	0.95	−0.25
Talca	24 (2)	1 (4.1)	23 (95.9)	0	47.9	20.31c	−0.91c
Las Condes	11 (2)	3 (27.3)	8 (66.7)	0	36.4	3.59	−0.53
Lo Barnechea	19 (4)	1 (5.3)	18 (91.7)	0	47.3	15.39c	−0.83c
Vitacura	34 (5)	0	34 (100)	0	50	34c	−1c
Total	99 (15)	5 (7)	88 (88.8)	6 (6.1)	50.5	59.91c	−0.849c

^aS and R are abbreviations for the susceptible and resistant alleles. Between brackets are the percentages of each genotype proportion.

^bPopulations were tested for the Hardy–Weinberg equilibrium by the χ² (P < 0.05; df = 1; χ² = 3.84).

^cValues that are statistically significant at P < 0.05. Significance level indicates rejection of the null hypothesis F_is = 0 at P < 0.05.

^dF_is values > 0 indicate heterozygote deficiency, whereas F_is values < 0 indicate heterozygote excess.

Fig. 1.

Geographic location of the head louse populations from Chile.

Genomic DNA Isolation and Detection of the Kdr Mutation

Genomic DNA was extracted from individual lice (third nymphal stage or adults) following the methodology of Ascunce et al. (2013). Briefly, each louse was cut in half, deposited into 1.5-ml Eppendorf centrifuge tubes that contain cell lysis solution and proteinase K, and ground using a plastic pestle. Then, DNA was extracted from each louse using Wizard genomic DNA purification kit (PROMEGA, Madison, WI) following manufacturer protocol to insect tissues. We used a NanoDrop 1000 spectrophotometer (Thermo Scientific, ON, Canada) to estimate the amount of DNA in each sample. Finally, a dilution from the original concentration to approximately 5–10 ng/µl was made.

The PCR protocol was used to amplify a 332-bp fragment of VSSC gene containing a site affected by C/T mutation and corresponding T917I amino acid substitution on each human head louse genomic DNA fragments (Durand et al. 2007). Reactions were carried out in a 25-µl reaction volume containing 12.5 µl of MasterMix (PROMEGA), 0.25 µM of each primer 5′-AAATCGTGGCCAACGTTAAA-3′ (sense) and 5′-TGAATCCATTCACCGCATCC-3′ (antisense), 2 µl of DNA template, and 8.5 µl of pure water (Toloza et al. 2014). The PCR conditions were as follows: 10 min at 94°C followed by 40 cycles of 94°C for 30 s, 56°C for 30s, 65°C for 1 min, and a final extension step at 65°C for 10 min. Then, 10 µl of each PCR amplicon was digested with the SspI restriction enzyme 10U (Thermo Scientific) to detect the C/T mutation. Finally, this fragment was analyzed by 2% agarose gel electrophoresis and visualized by ethidium bromide under UV light.

Statistical Analysis of Genotype Frequencies

The estimation of genotypic frequencies was calculated by dividing the number of lice of each genotype (RR, RS, SS) by the total number of analyzed human head lice. Then, genotype frequencies were compared with Hardy–Weinberg expectations using the program Genepop (v. 4.2), Option 1 (Hardy–Weinberg exact tests), Suboption 3 (probability test; Rousset 2008). This software (Genepop) was also used to estimate Wright’s inbreeding coefficient (F_is) using the method of Weir and Cockerham (1984) and departure of genotype frequencies from Hardy–Weinberg proportions. These values were used to test for heterozygote deficiency and excess (Genepop Option 1, Suboptions 1 and 2, respectively) using the U test as described in Raymond and Rousset (1995).

Results

It was possible to determine the Kdr T917I mutation in all of the 99 human head lice studied from several localities of Chile (Table 1). The presence of one or two fragments after the digestion with the SspI restriction enzyme was a diagnostic biomarker for the detection of mutations related to pyrethroid resistance in head lice from Chile. Thus, three possible amplified fragment with restriction profiles corresponding to resistant or susceptible genotypes were found: a homozygous susceptible head louse genotype was identified by a fragment of 332 bp; a heterozygous head louse had three fragments of 332, 261, and 71 bp; and a genotype assigned to a homozygous resistant louse could be identified by two bands of 261 and 71 bp. Globally, the frequency of the T917I mutation was 0.505 in the five louse populations from different sites of Chile. All the populations had a frequency of Kdr-like genes that ranged from 36 to 77%. Five of these lice (7%) were homozygous susceptible, 88 (88.8%) were heterozygotes, and 6 (8.4%) were homozygous resistant. Thus, 94.9% of the collected Chilean head lice had one or two T917I mutant alleles. The only population that harbored homozygous resistant individuals was the Vallenar population (n = 11 head lice from two kids) with a resistant allele frequency of 77.3%. In addition, this same geographic population had no homozygous susceptible lice. Notably, the population of Vitacura had only heterozygous head lice.

The Hardy–Weinberg equilibrium test showed that the distribution of the genotypes in three of the five analyzed populations varied significantly from the other two. In addition, these populations (Talca, Lo Barnechea, and Vitacura) had an inbreeding coefficient (F_is) < 0, indicating an excess of heterozygotes (Table 1).

Discussion

Pediculosis persists most everywhere in the human population being the most common parasitic infestation of humans (Gratz 1997). Reinfestation is a serious problem with both social and economic issues that effective pediculicides can only partial solve. Generally, concerns associated with lice and schoolchildren may affect both normal school day activities as well as parent’s financial status by missing work to care for children not allowed in school. Because most people find infestations with lice disgusting and an ensuing stressful situation, it has lead to drastic methods for louse control (Mumcuoglu et al. 2009). Today, chemical insecticides are still recommended as the first option to treat head lice infestations. Of these, permethrin is the most widely used neurotoxic ingredient contained in pediculicides. Thus, the overuse of this compound (and another pyrethroids such as deltamethrin) has led to the emergence and, in some cases, widespread resistance in many parts of the world (Heukelbach 2010). Resistance to insecticides may have severe consequences for lice control by adversely affecting treatment outcome. Such failure leads to an increased chronic infestation requiring additional, as well as, episodic treatments. Such repeated treatment episodes generate undue costs and disturbance and increase insecticide selection pressure.

This is the first study that analyzed the pyrethroid resistance status of head lice across several regions of Chile. The T917I haplotype in head lice associated with pyrethroid resistance was present in all of the studied populations. The replacement of the C → T substitution of the single-nucleotide coding for the T917I mutation results in a cutting site for the restriction endonuclease SspI (Kristensen 2005). The frequency of the Kdr biomarker was at 0.505, with a high proportion of heterozygotes at 0.44. Notably, these values are in accordance with those found in head lice collected in schoolchildren of Wales (Thomas et al. 2006). Those authors found a global frequency of Kdr T929I mutation at 0.43, with a heterozygote proportion of 0.38. However, the frequency of Kdr-like alleles is extremely variable due to their geographic origin (Hodgdon et al. 2010). Those authors performed a world Kdr map that included United States, European Union, Asia, Oceania, Africa, and South America with a resistance allele frequency of 74, 75.9, 29.1, 100, 47.5, and 79.9%, respectively. Although head lice had a Kdr allele at the T917I mutation, site related to pyrethroid resistance that is prevalent in many locations globally does not appear to be uniform geographically due to differential selective pressure.

Prior to this report, the reported prevalence in Chile of head lice varied from 17 to 47% (Devera 2012, Paola Gazmuri et al. 2014) and did not trigger much attention despite being above the epidemiological value considered to be of epidemic importance (>5%; Clore 1998). Until 2011, the available OTC pediculicides consisted of pyrethroids and lindane, and each of them with equivalent estimated market sales (Moreno 2011). Since the banning of lindane by the Chilean Public Health Institute (ISP) in March 2012, pyrethroids functioned as the only available pediculicide (Valenzuela Bravo 2012). Currently, permethrin and deltamethrin remain the most commonly used products to treat head louse infestations, covering 75% (21/28) of the approved products available on the market (Instituto de Salud Pública de Chile 2017). Because of this, Chilean head lice remain exposed to pyrethroid-based pediculicides at an unduly high selective pressure.

Notably, distribution of Kdr genotypes significantly departed from Hardy–Weinberg proportions in three of the five population examined in this study. Thus, deficiency of either susceptible (5/99) or resistant (6/99) homozygous, and the overrepresentation of heterozygotes (88/99) suggests that the studied populations are consistent with head lice in Chile being currently under active selective pressure. Similarly, a study performed in schools in Wales showed that of the 316 analyzed lice, 17.4% were homozygous susceptible, 5.4% were homozygous resistant, and 77.2% were heterozygotes (Thomas et al. 2006). On the contrary, several authors reported that head lice collected in France, Argentina, United States, and Russia had a high frequency of Kdr-resistant homozygotes reflecting that they were nearly fixed frequency (Durand et al. 2007, 2011; Toloza et al. 2014; Gellatly et al. 2016; Eremeeva et al. 2017).

Beyond our findings of excess heterozygotes, Thomas et al. (2006) proposed that because few individuals were homozygous resistant, there may be a substantial fitness cost related to this genotype. In other words, those individuals carrying only one copy of the mutation have more probability to survive under a selective environment than those individuals that have two copies of the mutation. Platt et al. (2015) revealed that the occurrence of a target site resistance mechanism affected the mating competitiveness of male Anopheles gambiae mosquitoes. They found that heterozygote male mosquitoes with the Kdr mutation L1014F were statistically more likely to mate than homozygote-resistant males suggesting that this mutation had a detrimental effect on the An. gambiae males. In other words, this suggests that there was a fitness cost associated with possessing double alleles of the L1014F mutation rather than having just one allele. The reduced mating success of RR-Kdr males might be explained by the important impact that this change on the VSSC can induce on the neural network, possibly affecting many physiological traits such as mobility, perception of the stimuli or the olfactory system (Rivero et al. 2010). Further studies are warranted to test the potential heterozygote advantage for head lice in Chile.

Another possible alternative related to an excess of heterozygotes in the studied populations (the simplest scenario) is that Kdr-like alleles have no or little effect on the fitness of resistant head lice. Hence, this leads to speculation that the return to the susceptible state might be slow, and the presence of resistant individuals could persist within the studied populations (Yu 2014). The occurrence of resistance to permethrin via Kdr balancing mutations has not been associated with any fitness disadvantage among head lice from the United States (Takano-Lee et al. 2003), possibly owing to the occurrence of two balancing mutations that exist in association with Kdr mutations (SupYoon et al. 2008). In practice, this means that resistant lice to permethrin can compete equally with susceptible individuals persisting in the population without ongoing insecticide selection pressure (Grayson et al. 2017).

In middle- and long-term resistance managements designed to detect and suppress insecticide resistance, detection of Kdr genes at an early stage is of vital importance. Early resistance detection by traditional toxicological bioassay-based monitoring methods is typically recommended but is difficult to apply in practice. This is the case of the toxicological studies performed in head lice populations because collecting a large number of living individual lice is often impractical and difficult (Heukelbach 2010). To avoid these limitations, several genotyping techniques and detection platforms were developed (Kristensen 2005, Durand et al. 2007, Kwon et al. 2008, Clark 2009). Thus, they can be readily employed for routine resistance monitoring of head louse populations (Heukelbach 2010). Based on the evidence that permethrin resistance mediated by Kdr trait is well established in head lice populations worldwide, differential control actions for resistance management could be successfully implemented. In those regions where allele frequency is fixed or nearly fixed, pyrethroid-based pediculicide use should be discontinued as soon as feasible and replaced by alternative products with a different mode of action. On the contrary, in regions where the allele frequency is low or near zero, formulations containing pyrethroids might continue to be used cautiously and in conjunction with a resistance-monitoring program (Clark 2009). This strategy should extend the effective life span of this valuable group of pediculicides. In regions with intermediate allele frequency, monitoring the susceptibility status of the exposed populations, together with the promotion of preventive measures to avoid pediculosis incidence, is of vital importance. This latter case is the current situation for the head lice populations of Chile. These recommendations are designed to avoid overexposure of children to unnecessary pyrethroids or to potentially dangerous alternative nonapproved remedies that may lead to poor outcome, as well as acute and chronic intoxication episodes (Mumcuoglu et al. 2009). Further research should focus on the determination of standardized toxicological bioassays together with epidemiological studies to determine the frequency and magnitude of the exposure to current pediculicide treatments in Chile. The findings in this work serve to correlate these parameters with the frequency of Kdr-like alleles and to inform a more appropriate approach to the management of pediculosis in Chile and even in other regions around the world.

Acknowledgments

We thank Monica Martinez Acuña for the coordination of the collection of head lice. We also thank two anonymous reviewers for their extremely helpful comments. A.T. and C.R.A. are members of the CONICET Research category. This research was supported by the CONICET Award PIP-0198CO to A.C.T. and Agencia Nacional de Promoción Científica y Tecnológica (Argentina) (PICT) 2015-1023 to G.R.A.