Human lice: Past, present and future control

Pesticide Biochemistry and Physiology 106 (2013) 162–171

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Human lice: Past, present and future control J. Marshall Clark a,⇑, K.S. Yoon a, S.H. Lee b, B.R. Pittendrigh c a

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea c Department of Entomology, University of Illinois, Urbana-Champaign, IL 61801, USA b

a r t i c l e

i n f o

Article history: Available online 30 March 2013 Keywords: Human head louse Pediculus humanus capitis Pyrethroid resistance kdr monitoring SISAR Non-invasive induction assay

a b s t r a c t Human lice represent one of the longest ectoparasitic relationships associated with mankind. Whereas the body louse vectors bacterial diseases that have killed millions, the head louse does not but represents an economic and social concern worldwide. A crisis exists in the control of lice due to the availability of only a few new pediculicides and the occurrence of resistance. In this review, we summarize data that validates the knockdown resistance mechanism (kdr) as the main cause of control failures to the pyrethrin/pyrethroid-based pediculicides and the molecular diagnostics used to determine kdr worldwide. New commercially-available pediculicide are discussed in terms of their use in sustainable resistance management approaches. Lastly, the optimization of dose, the timing of exposure, and the assessment of transcript levels during tolerance is used to identify detoxification genes that metabolize ivermectin as a proof of principle experiment, indicating that such an approach may allow proactive resistance management. Ó 2013 Elsevier Inc. All rights reserved.

Contents 1.

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Extent and concerns of pediculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Pediculosis and medical importance of human head and body lice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Past and current control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Status of resistance to commercial pediculicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Resistance management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutations in VSSC cause target site insensitivity and permethrin resistance and can be used in resistance monitoring . . . . . . . . . . . . . . . . . . 2.1. Three point mutations in the a-subunit gene of the VSSC cause knockdown resistance (kdr) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Monitoring the allele frequency of kdr by SISAR in field populations of head lice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New and novel acting pediculicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Dimeticone-based formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Ivermectin-based formulations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Spinosad-based formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of detoxification genes involved in insecticide tolerance as a proactive resistance monitoring approach . . . . . . . . . . . . . . . . . . 4.1. Optimization of the non-invasive induction assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Extent and concerns of pediculosis 1.1. Pediculosis and medical importance of human head and body lice Pediculosis is the infestation of humans by lice. Pediculosis by the human head louse, Pediculus humanus capitis (De Geer), is ⇑ Corresponding author. Address: Department of Veterinary and Animal Sciences, University of Massachusetts, N311B Morrill 1, Amherst, MA 01003, USA. E-mail address: [email protected] (J.M. Clark). 0048-3575/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2013.03.008

162 162 163 163 164 164 164 165 167 167 167 168 169 169 170

one of the most prevalent parasitic infestation of humans and louse outbreaks may constitute one of many opportunistic infestations associated with a depressed immune system (e.g., people with HIV) [1]. U.S. pediculicide sales are >$240 million/yr and infestation rates range from 6–12 million cases annually with 2.6 million households affected and 8% of all schoolchildren infested [2]. Overall cost of infestations is estimated at $1 billion annually but the long-term impact of days of lost learning by almost 1 in every 10 school-aged children due to the ‘‘No-Nit’’ policy and the lack of effective control options overshadows these cost estimates [3].

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Unlike head lice, the human body louse, Pediculus humanus humanus, poses a serious public health threat because they transmit several bacteria (Rickettsia prowazekii, Borrelia recurrentis, and Bartonella quintana) that cause human diseases (epidemic typhus, louse-borne relapsing fever, five-day relapsing fever and trench fever) and these diseases have killed millions [4]. Since the advent of antibiotics, outbreaks are sporadic but in 1986 more than 50,000 people were infected with R. prowazekii in Burundi [5]. However, the increasing frequency of antibiotic resistance is of particular concern. Thus, body lice represent a health risk during times of war, famine and social unrest, and still serve as an important vector of re-emerging diseases in developed countries. Although not a significant disease vector, head lice represent a major economic and social concern worldwide because infestations are often associated with school-aged children, who miss substantial school days (12–24 million days) [6]. Infestations often cause intense itching, which can injure skin allowing secondary infections and self-inoculation of bacteria including R. prowazekii, B. recurrentis, B. quintana [7] and methicillin-resistant Staphylococcus aureus (MRSA). Most people find lice intolerable and repeatedly and prophylactically apply pediculicides (insecticides) without realizing their harm and lethality. Misapplications affect children in particular due to their small size and higher sensitivity. With the recent economic downturn, and increases in the homeless populations in states such as California, there has been resurgence in body louse populations in the USA (Dr. Jane Koehler, M.D., Div. of Infectious Diseases, UCSF, personal observations). R. prowazekii is a category B bioterrorism agent [8] and only emphasizes the potential problems associated with increased body louse infestations in human populations and the concomitant increased risk of louse-borne diseases even within the U.S. There are two ways to combat pediculosis: (1) proactive prevention or (2) post-infestation treatment. Emphasis is increasingly on prevention (education) and physical removal (combing or shaving) because a crisis exists in the chemical management of pediculosis. The pediculicide arsenal is limited and shrinking. Thus, health care providers are spending an increasing and inordinate amount of time and resources dealing with infestations. Effective management information is lacking and few, if any, alternatives exist when standard treatments fail. 1.2. Past and current control In addition to shaving your head and a variety of physical means to remove lice, treatment of pediculosis has been usually and most effectively accomplished by the topical application of insecticides. As recently reviewed by Durand et al. [9], there are currently six major pediculicides commercially available for the treatment of head louse infestation including: the natural pyrethrin esters (pyrethrum), synthetic ‘‘pyrethroids’’ (permethrin,

phenothrin), organochloine (lindane), organophosphorous (malathion) and carbamate (carbaryl) insecticides (Table 1). Of these, the pyrethrins/pyrethroids dominate the over-the-counter market worldwide, followed by prescription only malathion-containing formulations, such as OvideÒ. The pyrethrins/pyrethroids share a common target site in the nervous system, the voltage-sensitive sodium channel (VSSC), and act as agonistic neuroexcitants by increasing sodium current, leading to nerve depolarization and hyperexcitation followed by neuromuscular paralysis and death. Malathion is a phosphorodithioate-type organophosphorous insecticide, which is an indirect nerve toxin that acts as a competitive irreversible inhibitor of acetylcholinesterase found associated with the cholinergic nervous system. When inhibited, acetylcholinesterase cannot efficiently hydrolyze the neurotransmitter, acetylcholine, allowing overstimulation of post-synaptic effector organs, including muscle, and eventual paralysis and death.

1.3. Status of resistance to commercial pediculicides Insecticide resistance threatens the success of all control programs but is particularly problematic in the control of human lice for several reasons: (1) they are obligate human blood feeders that are exposed to pediculicides at all stages; (2) they have short generational time and high fecundity; and (3) there are few pediculicidal products, the majority of which share common chemistry and elicit cross-resistance. Because of these issues, louse resistance to most commercial pediculicides has occurred and is increasing [9]. Both clinical and parasitological pyrethroid resistance to dphenothrin was first reported in France in 1994 [10] with additional reports of clinical control failures following: permethrin (2001) in the USA [11], phenothrin (2005) in the UK [12], and permethrin (2005) in the UK [13]. Also, parasitological resistance has been reported in the Czech Republic [14], the UK [12], Denmark [15], Israel [16], the USA [17], Argentina [18], Japan [19] and Australia [20]. Malathion resistance was first reported in France in 1995 [21], followed by the UK in 1999 [22], Australia in 2003 [20], and Denmark in 2006 [15]. The lack of extensive resistance in the USA is likely due to the use of the OvideÒ formulation, which also includes pediculicidal terpenes likely resulting in a mixture that has redundant killing action on multiple target sites [23]. Current control and resistance problems underscore the need to understand the molecular mechanisms of insecticide resistance in lice. The identification of resistance mechanisms and novel target sites may allow the development of resistance-breaking compounds and specific non-toxic synergists useful in novel control strategies.

Table 1 Traditional formulations of pediculicidal products.

a

Active ingredients

Brands

Product form

Dosage

OTC products Phenothrin (0.2–0.5%) Permethrin (1%) Pyrethrum (0.33% with 4% PBO)

Full marks, Herklin NF, Various generics Nix Rid, Clear, Pronto, Various Generics

Lotion, Mousse, Liquid, or Shampoo Crème rinse Shampoo or Mousse

1–2 Applications 1 Applicationa 2 Applications (7–10 days apart)

Rx products Malathion (0.5%) Lindane (1%) Carbaryl (0.5 or 1%) Permethrin (5%) Ivermectin (0.4%)

Ovide Various generics Carylderm Elimite IVOMEC

Alcoholic lotion Shampoo or lotion Liquid or lotion Cream Oral

1 1 1 1 1

A second application is required after 7 days if infestation persists.

Application Applicationa Application Application Application

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1.4. Resistance management Resistance management entails processes that reduce resistant allele frequencies, dominance, and fitness of the resistant genotypes [24]. Many non-chemical processes used to delay resistance in agricultural settings (e.g., natural enemies, insect disease, and host-plant resistance) are not applicable for human lice and limit the operational choices to a chemical management format coupled with nit removal. Insecticides are applied to manage resistance by moderation, saturation, and multiple attack strategies [25]. Low tolerance of infestations and the ‘‘No-Nit’’ policy eliminate most moderation approaches. Saturation schemes that involve high concentrations of insecticides are not appropriate when treating children. Thus, only multiple attacks (e.g., mixtures, mosaics, and rotations of pediculicides) are available strategies. The use of mosaics on a human head is impractical and rotations have not worked in the U.K. [26], leaving only mixtures as a viable application strategy. To this point, a mixture of a-terpineol, terpine-4-ol, and 0.5% malathion, found in the OvideÒ formulation (Medicis Corp., Scottsdale, AZ), was effective in controlling a malathionand permethrin-resistant head louse strain from the U.K. [23]. The successful application of insecticide mixtures, used in a resistance management format, shows that: (1) these approaches suppress resistance and (2) they are likely to be effective in field situations when used in conjunction with efficient monitoring. Thus as new pediculicides are introduced into the market place, it is imperative that we understand how lice may develop resistance or cross-resistance to these compounds. Such knowledge will have practical applications, in terms of recommending mixtures of compounds, where lice may develop very different forms of resistance to the two separate compounds. In summary, resistance to traditional pediculicides has developed, leading to clinical failures, so management strategies are necessary. However, these resistance management strategies need to be built on sound scientific knowledge before resistance evolves. It is also imperative that the molecular mechanisms mediating resistance be identified for the selective targeting of novel compounds and proper formulation of mixtures aimed at controlling these insects. The body louse genome project [27] has now provided the necessary core information with respect to novel target sites for improved louse control, the means to identify genes

I

responsible for resistance and to establish cross- and negative cross-resistance relationships, and the tools for effective and affordable monitoring.

2. Mutations in VSSC cause target site insensitivity and permethrin resistance and can be used in resistance monitoring 2.1. Three point mutations in the a-subunit gene of the VSSC cause knockdown resistance (kdr) Heightened public and governmental concerns have occurred because of increased incidents of head louse infestations among school children (http://www.cdc.gov/ncidod/dpd/parasites/lice/ default.htm; http://www.headlice.org). Lee et al. [28] first reported that head lice from Massachusetts and Florida were resistant to a pyrethroid, permethrin, and exhibited in vivo responses in behavioral bioassays that were consistent with kdr. kdr is a heritable trait associated with nerve insensitivity to DDT, the pyrethrins and the pyrethroids, which was first discovered in the house fly, Musca domestica [29]. Point mutations in these genes are functionally responsible for the kdr, kdr-like and super-kdr traits and nerve insensitivity to DDT, the pyrethrins and pyrethroids [30]. Three point mutations located in the domain IIS1-2 extracellular loop (M815I) and in the domain IIS5 transmembrane segment (T917I and L920F) of VSSC a-subunit (numbered according to the head louse amino acid sequence) have been identified in permethrin-resistant head lice [29,31] (Fig. 1). All three mutations were found en bloc as a haplotype in permethrin-resistant field populations of head louse. T917I, corresponding to T929I in the house fly, has been functionally validated as a kdr-type mutation in the diamondback moth, Plutella xylostella [32]. The other two mutations (M815I and L920F) were novel and their functional significance in pyrethroid resistance unproven. Heterologous expression of insect VSSC in Xenopus laevis oocytes has been utilized to determine the functional characteristics and pharmacological significance of kdr-associated point mutations [33]. Studies with the wildtype house fly Vssc1 channel coexpressed either with the Drosophila melanogaster tipE auxiliary subunit (Vssc1WT/tipE channels) or the house fly Vsscb auxiliary subunit (Vssc1WT/Vsscb channels) in oocytes and with mutated

II

III

IV

M815I outside S1 S2 S3 S4 S5

S6

S1 S2 S3 S4 S5

S1 S2 S3 S4 S5

S6

i id inside

S6

S1 S2 S3 S4 S5

S6

CO2-

H3N+

T917I

L920F

Fig. 1. Transmembrane topology of the voltage-sensitive sodium channel (VSSC) a-subunit showing the location of the three mutations responsible for knockdown resistance (kdr) in the human head louse. Modified with permission from Ref. [37]. Copyright 2009 Elsevier Inc.

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A.Wt Vssc1

B. MITILF Vssc1 0.2 µA

Control Permethrin (µM) 0.1 1 0.2 µA

50 ms

50 ms

10 100 200

Fig. 2. Comparative sodium current traces from the house fly VSSC variants with and without head louse mutations expressed in Xenopus oocytes before and after exposure to increasing concentration of permethrin. (A) Permethrin effects on the wild-type VSSC. (B) Permethrin effects on the triple mutant VSSC. Modified with permission from Ref. [35]. Copyright 2008 Elsevier.

Vssc1 variants carrying the kdr-associated mutations demonstrated that the single (L1014F) or double (L1014F and M918T) kdr-type mutations significantly reduced or abolished pyrethroid sensitivity [34]. Using this system, Yoon et al. [35] inserted the three mutations associated with pyrethroid resistance in the head louse (M815I, T917I and L920F) in all possible combinations into the corresponding positions of the house fly Vssc1WT sequence, expressed wildtype and specifically-mutated channels along with the house fly Vsscb auxiliary subunit in Xenopus oocytes, and employed the two electrode voltage-clamp technique to electrophysiological assess the impact of these mutations on the expression, gating properties and permethrin sensitivity of the expressed channels. Fig. 2A (left side) shows superimposed sodium current traces obtained from a single oocyte expressing Vssc1WT/Vsscb channels before and after exposure to increasing concentrations of permethrin ranging from 0.1 lM to 200 lM. As depicted, there is a dosedependent increase in the late current seen during inactivation and a prolongation of the tail current seen during deactivation. These effects are consistent with the action of permethrin on many VSSCs expressed in Xenopus oocytes. Fig. 2B (left side) shows the results of the same experiment except that the expressed Vssc1 now has the three amino acid replacements (M815I, T917I and L920F) seen in the permethrin-resistant head louse. Superimposed current traces obtained 5 min after permethrin treatments were indistinguishable from DMSO control traces recorded prior to permethrin treatments. Like all other channel variants containing the T929I mutation, the Vssc1MITILF/Vsscb channel was virtually insensitive to permethrin over the range of permethrin concentrations examined. These results confirm that the MITILF haplotype results in target site insensitivity of the VSSC and contributes to permethrin resistance in the head louse.

2.2. Monitoring the allele frequency of kdr by SISAR in field populations of head lice The kdr-type resistance in head louse populations appears to be widespread, but varies in intensity and is not yet uniform [36] Thus, the establishment of a proactive resistance management strategy is essential to maximize the time over which the pyrethrin- and pyrethroid-based pediculicides remain effective control agents. Detection of resistance when the frequency of resistant lice

is still low is crucial for implementing proper management decisions that can delay and reverse resistance development. Early resistance detection, however, is often difficult using conventional mortality bioassay-based monitoring methods, particularly when the resistance trait is recessive, as it is for kdr. Several genotyping techniques for the determination of kdr allele frequencies using genomic DNA extracted from individual head lice have been developed to overcome this limitation and to improve time and cost effectiveness [37]. One such technique, the serial invasive signal amplification reaction (SISAR), employs a fluorescence resonance energy transfer (FRET) detection format [38] (Fig. 3) and has been used for the high-throughput detection of the kdr mutations (M815I, T917I and L920F mutations) in field populations of head lice [36]. The SISAR method uses PCR-amplified DNA targets prepared from individual lice and has been found to be a very accurate and efficient technique in obtaining detailed information on the genotypes and kdr allele frequencies of human head louse populations. Each of the three SISAR kits produced SISAR ratios (three SISAR ratios/louse sample) that fell into the previously determined ranges and allowed the genotyping of individual lice at each mutation site collected globally [36]. The kdr allele frequencies at the three mutation sites were determined for head louse populations collected from 14 countries using all three SISAR kits Eq. (1) and a global kdr map constructed (Fig. 4).

kdr allele frequencyð%Þ ¼ ½number of kdr alleles in N lice at the three mutation sites =ðN lice  6Þ  100;where N ¼ number of lice in a population ð1Þ All seven North American head louse populations were collected from the USA and their diplotypes were determined by SISAR as follows, Pinon, Arizona (50% RRR, 30% HHH, 20% SSS); Ocklawaha, Florida (100% RRR); West Palm Beach, Florida (100% RRR); Asginaw, Michigan (96.6% RRR, 3.4% HHH); Tracy, Minnesota (100% RRR); Mathis, Texas (11% RRR, 52% HHH, 37% SSS) and San Antonio, Texas (100% RRR). The combined data resulted in 63% RRR, 22% HHH and 15% SSS. Based on this result, kdr allele frequencies at the three mutation sites in the U.S. populations were 65% (Pinon, Arizona), 100% (Ocklawaha, Florida), 100% (West Palm Beach, Florida), 98.3% (Saginaw, Michigan), 100% (Tracy, Minnesota) and 100% (San Antonio, Texas). The only U.S. population that

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Fig. 3. Schematic of serial invasive amplification reaction (SISAR) based on matched enzyme (Cleavase)-substrate reactions. Top is primary reaction. Bottom is secondary reaction. Reproduced with permission from Ref. [38]. Copyright 2004 Elsevier Inc.

Fig. 4. Global kdr allele frequency map. Each pie chart shows diplotype proportions of lice collected from a country. Proportions of lice with resistance diplotype (RRR) are presented in red, proportionswith susceptible diplotype (SSS) are represented in yellow and proportionswith heterozygous diplotype (HHH) are represented in orange. In the Egyption population, six diplotypes are illustrated in different colors (HSH in brown, RSR in green, HSR in dark-brown, HHH in orange, RHR in light-brown, RSH in dark-green). Reproduced with permission from Ref. [36]. Copyright 2010 Wiley. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

displayed kdr allele frequency less than 50% was Mathis, Texas (37%). The combined data from U.S. populations resulted in a 74% kdr allele frequency. In South America, five head louse populations were collected. Head louse populations from Buenos Aires, Argentina (n = 54), Comodoro Rivadavia, Argentina (n = 5) and Brazil (n = 12) had the diplotypes of 90.7% RRR, 5.6% HHH, 3.7% SSS; 60% RRR, 20% HHH, 20% SSS; and 50 %RRR, 25% HHH, 25% SSS, respectively. The Ecuador population (n = 8) had 100% SSS and the Uruguay population (n = 8) had 100% RRR. The calculated kdr allele frequencies were 93.5% (Buenos Aires, Argentina), 70% (Comodoro Rivadavia, Argentina), 62.5% (Brazil), 0% (Ecuador), and 100%

(Uruguay). The overall kdr allele frequency for South America was calculated to be 79.9%. The kdr allele frequencies of louse populations collected from European Union (EU) were also varied, based on head lice collected from the U.K. (n = 8), Denmark (n = 12), and the Czech Republic (n = 7), where the diplotypes were 100% RRR in the U.K., 75% RRR, 17% HHH, 8% SSS in Denmark, and 29% RRR, 14% HHH, and 57% SSS in Czech Republic. Based on their diplotypes, kdr allele frequencies were 100% (Bristol, U.K.), 83.5% (Uvelse, Denmark), and 36% (Czech Republic). The overall kdr allele frequency for the EU was calculated to be 75.9%. Only four head louse samples from Israel were analyzed in this study. No

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susceptible homozygote lice were identified. Diplotypes of the Israel lice were 75% RRR and 25% HHH, resulting in a 87.5% kdr allele frequency. All head lice collected from Seoul and Hong-Sung, South Korea (n = 36), Thailand (n = 33), and Papua New Guinea (n = 3) possessed SSS diplotypes, resulting in 0% kdr allele frequencies. Comparatively, all head lice (n = 28) collected from Australia possessed the RRR diplotype, resulting in a 100% kdr allele frequency. Interestingly, six different diplotypes were identified in lice (n = 20) from Kafr-el Sheikh Governorate, Egypt. Fifty percent of the analyzed lice were HSH, 30% were RSR, and the remaining 4 lice possessed diplotypes of HSR, RHR, RSH, and HHH, respectively. Based on these results, the calculated kdr allele frequency for the Egyptian population was 47.5%. Unlike individual lice from other populations, however, no lice had either the RRR or SSS diplotype. This population had a 70% kdr allele frequency at the M815I mutation site, a 5% kdr allele frequency at the T917I mutation site, and a 70% kdr allele frequency at the L920F mutation site. In summary, early detection of head louse resistance to pyrethroid insecticides is a crucial factor for long-term resistance management designed to effectively suppress or slow the spread of this resistant pest. As an alternative to conventional mortality bioassay-based monitoring, the SISAR kits have been developed to estimate the diplotypes and the kdr allele frequencies of field-collected head louse populations. This technique, which allows for the determination of zygosity at each mutation site, is particularly useful to understand the population dynamics of kdr during the early stage of resistance because heterozygous lice can be detected in a population. 3. New and novel acting pediculicides Insecticide resistance to currently-used pediculicides, including permethrin, synergized pyrethrins and malathion, has occurred worldwide, is increasing [36,39] and is certainly contributing to increased incidences of pediculosis. Given this scenario, a crisis currently exists in the effective control of human lice at a time when the prevalence of pediculosis is increasing. The search for new effective products with novel modes of action is therefore a critical need. Recently, a number of new topical pediculicidal products have been introduced to the marketplace (Table 2). They possess novel modes of action, show little cross-resistance to existing commercially-available pediculicides and appear safe and effective.

attracted a good amount of attention due to their low mammalian toxicity, novel modes of action that are not neurotoxic and the possibility that they will have a low potential for the development of resistance. To date, particularly in Europe, different dimeticone-based products are commercially available. Dimeticones are linear polydimethylsiloxanes (CH3SiO[SiO(CH3)2]nSi(CH3)2), where n is the number of repeating monomers [SiO(CH3)2] of varying chain length. The chain length substantially influences the molecular weight and the viscosity of the substance. Although dimeticones in general are known for their low surface tension, they can vary considerably in spreading characteristics. Of the different dimeticone-based products available, two products are better characterized scientifically in terms of their effectiveness and probable modes of action. HedrinÒ 4% lotion (Thornton & Ross Ltd, Huddersfield, UK) is a 4% dimeticone lotion in 96% (w/w) decamethylcyclopentasiloxane (cyclomethicone D5). Head lice treated with this product are rapidly immobilized but small movements in their extremities over several hours indicate that death is delayed. Scanning electron microscopy coupled with X-ray microanalysis revealed that HedrinÒ 4% lotion was deposited in the spiracles, in some cases blocking the opening completely, and penetrated into the outer aspects of the tracheae [40]. Given the slow onset of mortality, asphyxia was discounted as a mode of action. The inability of the louse to excrete the excess water acquired during blood feeding by transpiration via the spiracles has been suggested as a mode of action with death occurring by either prolonged immobilization or by the rupture of organs such as the gut. The second dimeticone-based anti-louse product (NYDAÒ, G. Pohl-Boskamp GmbH & Co. Hohenlockstedt, Germany) contains a mixture of two dimeticones, one of low viscosity and the other of higher viscosity, at a final total concentration of dimeticones of 92% (w/w). Medium-chain length triglycerides, jojoba wax and two fragrances make up the remaining constituents. Because of its superior spreading characteristics, NYDAÒ rapidly enters the tracheal system, filling even the smallest branches [41]. Within one min of treatment with NYDAÒ, lice do not show any major vital signs. This effect appears to be due to an interruption in the oxygen supply leading to suffocation. Additionally, NYDAÒ has been shown to be an effective ovicide [42].

3.2. Ivermectin-based formulations 3.1. Dimeticone-based formulations Because of increasing instances of resistance, particularly to the neurotoxic insecticides currently used as pediculicides, and the increasing scrutiny of the use of such products on children, there has been a trend, primarily in Europe, for the development of physical means to control head lice. Of these types of products, dimeticone-based anti-louse products (silicone oils) have

Ivermectin is a macrocyclic lactone produced fermentatively by Streptomyces avermitilis and is a widely-used oral anthelmintic agent for both humans and companion animals. It has a unique mode of action by reducing motility and feeding in treated nematodes [43]. In addition to muscles used in motility, ivermectin also acts to paralyze the muscles associated with the pharyngeal pump, inhibiting the pumping action needed for feeding and attachment

Table 2 New formulations of pediculicidal products.

a b

Active ingredients

Brands

Product form

Dosage

OTC products Dimeticones (92%) Dimeticones (4%) Dimeticones (95%)

NYDA Hedrin Linicin lotion 15 min

Topical Spray or lotion Lotion or spray gel Lotion

1 Applicationb 2 Applications 2 Applications

Rx products Spinosad (0.9%) Ivermectin (0.5%)

Natroba SKlice

Topical suspension Lotion

1 Applicationa 1 Application

A second application is required after 7 days if infestation persists. In some countries 2 applications, according to national guidelines.

J.M. Clark et al. / Pesticide Biochemistry and Physiology 106 (2013) 162–171

Spinosad is a macrocyclic lactone insecticide produced fermentatively by a soil actinomycete bacterium, Saccharopolyspora spinosa. It has two active ingredients, spinosyn A and spinosyn D, in a 5–1 ratio. Spinosad is a neurotoxic agonist at the nicotinic acetylcholine receptor of the cholinergic nervous system where it selectively modifies the non-desensitizing aspect of the current flowing through this ligand-gated channel, causing prolonged excitability and paralysis [51]. Spinosad has been commercially formulated as a 0.9% viscous topical suspension for the treatment of head louse infestation (NatrobaÒ, ParaPRO, LLC, Carmel, IN).

Contact

m

500

Clade 3287

Clade 4

* 58 *

50 30

• CYP6CJ1 6

• CYP9AG2

*

* 10

**

0

3050

Clade 3 398

Clade 4

358

*

* 92

*

*

26

22

*

4BS2

6CJ1 9AG1 9AG2

*

4BU1 4BU2 4BV1 4G38 4G39

*

6CF1

350 100 50 25 0

m

301A1

1000

*

Immersion

3500

P450 Fig. 5. Relative increases in the transcript levels of cytochrome P450 monooxygenase (P450) genes determined by qPCR following 4 h post ivermectin (IVM) treatments of female lice, where normalized fold-increase = normalized basal transcript level X [relative fold-increase of transcript level following IVM treatment (106 M for direct contact (upper panel); 109 M IVM in ethanol for immersion (lower panel)) – 1]. Asterisks (⁄) indicate a significant increase in transcript levels over respective controls using Student’s t-test (P < 0.05). Solid bars are highly expressed (normalized basal level transcription (NBLT > 220), hatched bars are modestly expressed 30 < NBLT < 220) and open bars are weakly expressed (NBLT < 30) genes. Modified with permission from Ref. [54]. Copyright 2011 The Royal Entomological Society.

150

111 Contact

*

B

C

G

100 75 50 40

• ABCC4

35

*

• ABCG10

17

*

20 0

F

69

*94 *

*

• ABCG12

173

235

* Immersion

B

185

C

F

156G164 89

** *

135 44

*

PhABCF1

*

PhABCG2

*

*

PhABCC4 PhABCC5

0 **

PhABCB6

50

25

PhABCG10

38

PhABCG7 PhABCG9 PhABCG12

85

PhABCB4 PhABCB5

3.3. Spinosad-based formulation

1000

Normalized fold-increase

[44]. The concentration of ivermectin needed to cause paralysis of the pharyngeal pump is 10- to 100-fold lower than the concentration needed to cause mortality [45]. Ivermectin has been shown to increase chloride permeability in insect [46] and nematode [47] neurons and muscle membranes through binding to glutamate-gated chloride ion channels. These channels are highly expressed in the neuromuscular system of the pharyngeal pump in the mouthparts of the free living nematode, Caenorhabditis elegans, which has been shown to be highly sensitive to ivermectin. It is believed that during de-worming with ivermectin, nematode parasites are killed by ivermectin acting on glutamate-gated chloride channels in the cells of the neuromuscular system. Ivermectin increases Cl influx, which hyperpolarizes the cells, leading to paralysis of the mouthparts. This action causes the worms to detach from the mammalian gut and be excreted. A similar mode of action in head lice, however, has not been directly characterized. Recently, successive oral ivermectin treatments have been used to treat hard-to-control head louse infestations [48]. Ivermectin also has been formulated as a less invasive topically-applied pediculicide that possesses the ability to kill permethrin-resistant head lice [49]. The need for successive treatments in the oral ivermectin studies indicates that one treatment, while effective against feeding lice in situ, needs to be supplemented by a second systemic treatment to kill nymphs that emerge from eggs present at the time of the initial treatment. This implies an absence of an ovicidal effect of oral ivermectin. A 0.5% ivermectin topical cream formulation (SkliceÒ, Sanofi Pasteur Inc., Swiftwater, PA) was determined to kill permethrinresistant head lice [49] but was not directly ovicidal to treated eggs of head lice, as hatchability was not decreased [50]. Nevertheless, the percent of hatched lice from treated eggs that took a blood meal significantly decreased (80–95%) compared to lice that hatched from untreated eggs and all treated lice died within 48 h of hatching, including those that fed. Dilutions of ivermectin formulation of 0.15 and 0.2 lg/ml, which were topically applied to 0–8 days old eggs, were not lethal to lice at 24 h post-eclosion. However, 9 and 16% less lice fed when hatched from these treated eggs, respectively. Total [3H] inulin ingested by untreated 1st instars significantly increased over a 48 h feeding interval but was significantly less in instars that hatched from eggs receiving the 0.15 (36% less) and 0.2 (55% less) lg/ml ivermectin treatments compared to placebo. The reduced feeding that occurred following the 0.15 and 0.2 lg/ml ivermectin treatments occurred in the absence of mortality and suggests a unique mode of action of ivermectin on feeding that is separate from the mode of action of ivermectin leading to mortality. Failure of hatched instars to take a blood meal following egg treatments with formulated ivermectin is likely responsible for its action as a post-eclosion nymphicide.

Normalized fold-increase

168

ABC transporter Fig. 6. Relative increases in the transcript levels of ABC transporter genes determined by qPCR following and 4 h post ivermectin (IVM) treatments of female lice, where normalized fold-increase = normalized basal transcript level X [relative fold-increase of transcript level following IVM treatment (106 M for contact (upper panel); 109 M IVM for immersion (lower panel)) – 1]. Asterisks (⁄) indicate a significant increase in transcript levels over respective controls using Student’s ttest (P < 0.05). Solid bars are highly expressed (normalized basal level transcription (NBLT > 220), hatched bars are modestly expressed 30 < NBLT < 220) and open bars are weakly expressed (NBLT < 30) genes. The letters B, C, G, and F designate ABC transporter subfamilies. Modified with permission from Ref. [54]. Copyright 2011 The Royal Entomological Society.

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J.M. Clark et al. / Pesticide Biochemistry and Physiology 106 (2013) 162–171

A

CYP9AG2 Opa

1.00

*

0.75

1.25

ABCC4 relative transcript level

CYP9AG2 relative transcript level

1.25

0.50

0.25

0.00

0.75

0.50

Logit % Mortality

*

0.25

0.00

( • ) ABCC4 dsRNA injected

*

80

Injection

N

Buffer

19

LT50 (95% CL), min

CYP9AG2 dsRNA 19 600

D

90

5 % Ivermectin

400

48 hr after dsRNA injection

95

( • ) CYP9AG2 dsRNA injected* ( ° ) Buffer only injected

200

ABCC4 pQE-30

1.00

48 hr after dsRNA injection

C

B

800

646 (606-697) 525 (483-574) * 1000

Log Time (min)

70 60 50 40 30

( ° ) Water only injected

5 % Ivermectin

20 10 7 200

Injection

N

LT50 (95% CL), min

Water

15

615 (563-668)

ABCC4 dsRNA 15

400

600

479 (434-527)*

800

1000

Log Time (min)

Fig. 7. Relative transcript levels (panel A and B) and mortality responses (panel C and D) of body louse females to a lethal contact amount of ivermectin (5% IVM) following injection of dsRNA targeting either louse CYP9AG2 or ABCC4. Lice were also injected with either dsRNA of the odd-paired gene, opa, (GeneBank accession # S78339) for P450 silencing or with dsRNA of the Escherichia coli plasmid, pQE30, for ABC transporter silencing as sham injected controls. Asterisks (⁄) in panels A and B indicate that CYP9AG2 and ABCC4 dsRNA significantly suppress the levels of CYP9AG2 and ABCC4 transcripts, respectively (Student’s t-test, P < 0.05). In panel C, the bioassay was started 48 h after CYP9AG2 dsRNA injection. In panel D, the bioassay was started 12 h after ABCC4 dsRNA injection. Asterisks (⁄) in panels C and D indicate that the mortality responses of lice injected with dsRNAs were significantly different from their respective controls (buffer or water only injected, maximum log-likelihood ratio test, P < 0.05). Reproduced with permission from Ref. [54]. Copyright 2011 The Royal Entomological Society.

4. Determination of detoxification genes involved in insecticide tolerance as a proactive resistance monitoring approach 4.1. Optimization of the non-invasive induction assay Identifying insect detoxification genes, based on induced transcript profiles, has been repeatedly suggested as a means of identifying the major metabolic pathways involved in insecticide resistance [52], and initial attempts using transcriptional profiling following insecticide induction in a susceptible strain of D. melanogaster did identify a number of detoxification genes [53]. However, only a limited number of the genes induced appeared to be involved in insecticide metabolism. To investigate whether detoxification genes can be selectively induced by insecticides, and thereby identify those genes involved in the actual metabolism of the insecticide, the induction scheme should be optimized by including: (1) an assessment of gene transcript levels at a time of peak gene induction that results in insect tolerance; (2) insecticide doses that do not result in physiological stress that can mask the identification of primary detoxification genes due to a large number of genes of secondary importance

being co-induced; and (3) application of insecticides in a non-invasive manner, such as contact exposure without solvent carriers. Insecticide induction that leads to tolerance also needs to be fast enough to protect the insect from the rapid onset of toxicity and temporary so the fitness cost associated with the long-term overexpression of detoxification gene products is minimal. For these reasons, a non-invasive induction assay (brief exposure to sublethal levels of insecticide administered in a low stress fashion with a rapid assessment of transcript levels that overlaps with tolerance) was envisaged to optimize the identification of inducible detoxification genes that produce tolerance via metabolism. It is likely that some of the induced genes will result in resistance once inheritable mutations causing constitutive over-expression, more sensitive induction or structural alteration occur. Because resistance monitoring is an absolute requirement for any sustainable vector control program, there is a critical need to efficiently identify detoxification genes that metabolize insecticides during the process of induced tolerance prior to resistance evolving. Some of these genes will certainly be involved in phenotypic resistance that will evolve after pesticide selection and can then be used proactively to monitor for metabolic resistance. This

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approach is particularly relevant with the advent of pesticides that possess ‘‘green chemistries’’, such as ivermectin, and prone to rapid detoxification by xenobiotic metabolism. The optimization of dose, the timing of exposure, and the assessment of transcript levels during tolerance was used to identify detoxification genes that metabolize ivermectin as a proof of principal experiment. Transcriptional profiling results, using our ‘‘optimized’’ non-invasive induction assay [short exposure intervals (2–5 h) to sub-lethal amounts of insecticides (
[11] R.B. Hipolito, F.G. Mallorca, Z.O. Zuniga-Macaraig, P.C. Apolinario, J. WheelerSherman, Head lice infestation: single drug versus combination therapy with one percent permethrin and trimethoprim/sulfamethoxazole, Pediatrics 107 (3) (2001) E30. [12] I.F. Burgess, C.M. Brown, S. Peock, J. Kaufman, Head lice resistant to pyrethroid insecticides in Britain, BMJ 311 (1995) 752. [13] N. Hill, G. Moor, M.M. Cameron, A. Butlin, S. Preston, M.S. Williamson, et al., Single blind, randomised, comparative study of the Bug Buster kit and over the counter pediculicide treatments against head lice in the United Kingdom, BMJ 331 (2005) 384–387. [14] V. Rupes, J. Moravec, J. Chmela, J. Ledvinka, J. Zelenkova, A resistance of head lice (Pediculus capitis) to permethrin in Czech Republic, Cent. Eur. J. Public Health 3 (1995) 30–32. [15] M. Kristensen, M. Knorr, A.M. Rasmussen, J.B. Jespersen, Survey of permethrin and malathion resistance in human head lice populations from Denmark, J. Med. Entomol. 43 (2006) 533–538. [16] K.Y. Mumcuoglu, J. Hemingway, J. Miller, I. Ioffe-Uspensky, S. Klaus, F. BenIshai, et al., Permethrin resistance in the head louse Pediculus capitis from Israel, Med. Vet. Entomol. 9 (1995) 427–432. [17] T.L. Meinking, P. Entzel, M.E. Villar, M. Vicaria, G.A. Lemard, S.L. Porcelain, Comparative efficacy of treatments for pediculosis capitis infestations: update 2000, Arch. Dermatol. 137 (2001) 287–292. [18] M.I. Picollo, C.V. Vassena, A.A. Casadio, J. Massimo, E.N. Zerba, Laboratory studies of susceptibility and resistance to insecticides in Pediculus capitis (Anoplura; Pediculidae), J. Med. Entomol. 35 (1998) 814–817. [19] T. Tomita, N. Yaguchi, M. Mihara, M. Takahashi, N. Agui, S. Kasai, Molecular analysis of a para sodium channel gene from pyrethroid-resistant head lice, Pediculus humanus capitis (Anoplura: Pediculidae), J. Med. Entomol. 40 (2003) 468–474. [20] J.A. Hunter, S.C. 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