Permethfin-Resistant Human Head Lice,Pediculus capitis, and Their Treatment

Kyong Stp Yoon, MS;jian-Rong Gao, PhD; Si Hyeock Lee, PiD; J. Marshall C!ark, PhD;Leon Brown, MS; David Taplin, PhD

Objective: To compare the pedicuticidai activity of Ovidelotion and its active ingredient, 0.5% malathion, with Nixand is active ingredient, 1% permethrin, in permethrin-resistant head lice.

Design: In vitro pediculicidaI product and active ingre-dient comparison. The presence ofknockdown resistance-t?pe mutations (T9291 and L932F) vas validated by DNAsequencing.

Setting: University of Massachusetts-Amherst; Univer-sit}, of Miami School of Medicine, Miami, Fla; Planta-tion and Homestead, Fla; and Mathis, Tex.

Other Participants: Lice were coilected in 3 geographi-cal regions within the United States and in Yamburara,Ecuador, from healthy but infested individuals.

Intervention: Within 3 to 6 hours of collection, lice-weregiven a blood meal, exposed to products or active ingre-dients, and observed at regular intervals.

Main Oufcome Measures: Percent mortality of liceat regular intervals after exposure to products or active

ingredients and presence of T929I and L932F muta-

tions.

Resu|s: South Florida lice exhibited a significantiysloxver mortality response to permethrin compared withsusceptible Ecuadorian lice. Ovide and malathion killedpermethrin-resisrant lice faster than Nix or permethrin.The presence of T929I and L932F in permethrin-resistant south Florida lice *was confirmed by DNAsequencing. The population of Texas lice from Mathiswas slightly resistant to permethrin and included 13%with resistant genotypes.

Conclusions: The presence of the T929I and L932F mu-tations was confirmed by DNA sequencing in lice col-lected from children in south Florida that were resistantto the pediculicidal effects of permethrin and the lead-ing permethrin-based head !ice product, Nix. Mala-thion resistance was not observed in this study. The dataalso show that Ovide killed these same permethrin-resistant head lice approximately 10 times faster than per-methrin or Nix.

Arch DermatoL 2003;139:994-1000

From the Department ofEntomology, University ofMassachusetts-Amherst(Mr Yoon and Drs Gao andClark); the Department ofAgricultural t3iotechnology,Seoul National University,Suwon, 5out,h Korea (Dr Lee);Martin & Cuhjati, San Antonio,Tex (Mr Brown); and theDepartment oJ Dermatoiogyand Cttaneots SurgecUniversity of Miami School ofMedicine, Miami, Fla(Dr Taplin). The authors havew relcvant_financial interest inhis article

EDICULOSIS, CAUSEDby the hu-man head touse (HL) Pedicu-lus capiis, is the most preva-lent parasitic infestation inchildren from the United

States. Infestations continue to be re-ported in the United States and in othercountries such as those of the United King-dom (UK),s-m Israel, -' the Czech Repub-lic,3 and Argentina despite the many over-

the-counter, self-treatment medicationsavailable today. In the United States, con-

sumer (nonprescription) treatment prod-ucts are almost exclusively- limited to thosethat contain the natural botanical active in-gredients, the pyrethfins (eg, R1D [Pfizer Inc,New York, NY], A-200 shampoo It-logilPharmaceutical Corp, Purchase, NY], andPronto [Del Laboratories, Farmingdale,NY]), or those that contain the synthetic py-

rethroid permethrin (eg, Nix [Pfizer/Warner-Lambert, Morrks Plais, NJJ), as theactive ingredients. These products have beenpreviously reported to be highly effective inthe treatnent of infestations,s

For editorial commentsee page 1061

It is well established in the pub-lished literature that resistance to per-metbrin exists in certain locations. In theUnited States, resistance to permethrin hasbeen found in lice from children in Brook-line and Carnbridge, Mass; Chicopee andHolyoke, Mass:e'; Boise, Idahol5; and Plan-ration, Fla.'- Although resistance to mala-thion has been reported in the UK, none

has been reported in the United States todate., 6

ARCH DERMATOL/VOL 139, AUG 2003 WWVq.ARCtIDERMATOE.COM994

Lee et al6 reported that US lice were resistant to per-methrin and also exhibited knockdown resistance (kdr)in behavioral assays. Knockdown resistance is associatedwith increased nerve insensitivitT and is similar to the kdrnoted for DDT, the pyrethrins, and the pyrethroids re-

ported originally in the housefly, Musca domesgica.>9 Twopoint mutations (T929t and L932F) have been found tobe associated with permethrin-resistant lice from Florida,Massachusetts, and Bristol, England, but not found in pe{methrin-stsceptible lice from Panama. 16 The T9291 mu-

tation functions as a kdr-ty-pe mutation in the diamond-back moth, Plutella x,jlostella.2 Recently, both mutationshave been identified in lice throughout the UK and sub-stantiate their importance in kdr.21

A recently reintroduced prescription treatment(1999), Ovide lotion (Medicis Pharmaceutical Corpora-tion, Phoenix, Ariz), is a pedicuticidal product that con-tains 0.5% pharmaceutical-grade malathion as the ac-

tive ingredient in a special base. Meinking et ala recentlyreported that Ovide has superior pediculicidal and ovi-cidal activity on both susceptible and permethrin-resistant lice compared with the teading permethrin- andlindane-based products. These investigators concludedthat Ovide killed permethrin-resistant lice and that theselice were not resistant to malathion.

The purpose of the present study was to confirmand extend the findings of Meinking et at.2' Standardmortality bioassays determined comparative mortalityprofiles for both the Nix and Ovide products and theiractive ingredients. In addition, DNA sequencing deter-mined whether the mutations associated with per-methrin resistance were present and what influencethey had on mortally,.

MEHODS

HUMAN HEAD LOUSE POPULATIONS

The Ecuador test population (EC-HL) was obtained from over

50 children in Yamburara, Ecuador (obtained by D.T.). Theyhad never been exposed to pesticides, including permethrin andmalathion, and were considered pediculicide susceptible. Amixed population of all stages and eggs were overnight ex-pressed to the University of Massachusetts-Amherst and placedinto a temporary colony fed on humans. The south Floridapopulation (SF-HL) was collected from over 30 infested chil-dren in Plantation and Homestead and are permethrin resis-

tant., The Texas population (TX-HL) was obtained from a

single heavily infested child (42 lice were obtained) fromMathis, Tex (obtained by L.B.). All collections and adminis-tration of informed consent forms were carried out using pro-tocols previously approved by the institutional reviexv boardof the University of Massachusetts-Amherst.

IN VITRO MORTALITY BIOASSAYS

After collections, the EC-HL were transported to the PesticideToxicology Laboratory, Department of Entomology, Univer-sity of Massachusetts-Amherst; the FL-HL to the Field Epide-miology Survey Team Laboratory; and the TX-HL o a tempo-rary field laboratory in Mathis. All lice were given a blood mealprior to initiation of mortality bioassays by feeding on the in-vestigators hand. Filter paper and contact bioassays were usedto determine lethality of 1% perrnethrin, 0.5% malathion, Nix

(ingredients: 1% permethrin, balsam Canada, cetyl alcohol, cit-ric acid, FDC yellow #6 fragrance, hydrolyzed animal pro-tein, hydroxTetI,ylcellulose, poiyoxyefhylene 10 cetyl ether, pro-pylene glycoi, stearalkonium chloride, water, isopropyi alcohoi,methylparaben, propylparaben) and Ovide shampoo (ingredi-ents: 0.5% malathion, terpineol, dipentene and pine needle oilin 78% isopropyl alcohol). Filter paper disks (Whatman No.I) were dipped for 10 seconds into 1% (vol/voi [1 part 10%permethrin in acetone to 9 parts acetone]) permethrin in ac-

etone, 0.5% (volvo1) malathion in acetone, or Ovide and weresubsequently air dried in a dark fume hood for hours. Diskswere likewise dipped into Nix for minute and dried in a darkfume hood for 24 hours owing to the slow drying characteris-tics of this product. Disks were also dipped into neet acetone,dried, and used as nontreatment controls.

The bioassay proce&are was conducted according to Lee

et al except that mixed developmenta! stages (first, second,and third instars and adults) were examined. Log time vs ]ogitmortafity regressions were performed (POLO PC; LeOra Soft-ware, Berkeley, Catif, i987) to determine lethal time 50% (LT0)values. To determine a susceptible or resistant phenotype fol-lowing 1% permetlqrin exposure, the lethaI tirne 95% (L%5) valueof the insecticide-susceptible EC-HL was used. Survival be-yond tie calculated 7.6 hour value of this susceptible strain wasused to assess perrnethrin resistance on a phenotypic basis. Theuse of timed bioassays at a single dose rather than multiple dosesat a single time point is becoming a standard procedure for theassessment of resistance, We have chosen the same concen-

tration that is used in the commercial products for both per-methrin and malathion because this is how lice are actually ex-

posed to the active ingredient (at a single concentration over a

set time interval). The speed in which permethrin and mala-thion Mils susceptible lice vs the resistant lice is therefore ger-mane in the delermination of resistance. Comparisons of themortality responses due to different test materials were madeusing the maximum log-likelihood ratio test, which tests thehypothesis of equality of slopes and intercepts of the logit re-

gressions (P=.05 [POLO PC]).

DETERMINATION OF THE T929IAND L932F MUTATIONS

To determine whether a correlation exists between increasedsurvivorship (due to resistance) and an increasing frequencyof kdr-type mutations (T929I and L932F) for permethrin re-

sistance, genomic DNA was extracted from individual SF-HLlice used in the mortality bioassays described previously, whichvere presepeed in 95% ethanol. Extractions were performed us-

ing DNAzol (Molecular Research Center, Cincinnati, Ohio) wi(h

a slight modification (addition of 1% polyacryl carrier) to banditsmali samples according to the manuiacturer's instruction. Ge-nomic DNA was also obtained from a mixed population of per-methrin-susceptble and -resistant lice from Mathis (TX-HL)and used as internal quality controls to assure that the DNAamplification and seo(uencing reactions vere successful and ca-

pable of identii,ing lice with and without mutations. T:a.o roundsofpotymerase chain reaction (PCR) were performed with nestedprimers (BLS'Gn/3SP1 for first PCR and 5SPIL/3SP5N for thesecond PCR; 'l'll to amplify a DNA fragment (approxi-mately 548 to 561 base pairs) containing the $4 to $6 regionsofdomain II ofpara-orthoiogous, voltage-sensitive sodium chan-nel a-subunit gent ofhead lice. The presence or absence of kdr-

type mutations (T929 or L932F) was determined by DNA se-

quencing (ABI 377XL; Applied Biosystems, Foster City Catif)at the Automated DNA Sequencing Facility, University of Mas-sachusetts-Amherst. Computer software, Gent Runner, Ver-sion 3.00 (Hastings Software, Bethesda, Md, 1994) was usedto analyze and manage sequencing daa.

ARCH DERMATOL/VOL 139, AUG 2003 WVW.ARCHDERMATOL.COM995

IN VITRO MORTAL|TY BIOASSAYS

Treatment of the EC-HL with either 0.5% malathion or

1% permethrin resulted in significantly different and sub-stantially reduced survival times compared with the ac-etone-treated EC-HL as judged by the maximum log-likelihood ratio test (X2= t74.8 [P<.001] and X[=312.9[P<.001], respectively) (Figure I A). Malathion re-

duced the LTs0 value 9.0-fold and permethrin reducedthe LTs0 value 5.2-fo]d compared with the acetone con-trol ('ab|e 2).

Yreatment of the SY-HL with 0.5% malathion, like-wise, resulted in significantly differen and substan-tially reduced suwival times compared with the acetone-treated SF-HL (X=I59.0 [P<.001]) (Figure 1B).Malathion reduced the LTs0 value 5.9-fold compared withno treatment (Table 2). Although treatment of the SF-HLwith 1% permethrin aIso resulted in a significantly dif-ferent response (X=59.9 [P<.001]) (Figure 1B), the LTovalue was not substantially reduced compared with theacetone reated SF-HL (1.3-fold, Table 2).

The SF-HL (Figure 1B) exhibited significantly slowermortality response to i% permethrin co-reputed with theEC-HL (Figure IA) (X[=218.5 [P<.001I). The mortal-ity resistance ratio (RR) based on the LT>0 values of theSF-HL vs the EC-HL was 3.1 (Table 2) and confirmedthat the SF-HL was resistant to permethrin.

Abbreviation: PCR, polymerase chain reaction.

The mortality response of the SF-HL (Figure 1B) to0.5% malathion, however, was not statistically differentfrom the EC-HL (Figure ]A) (X=2.7 [P=.26]). Al-though the LTo value of the SF-HL was slightly longerthan for the EC-HL, the magnitude ofdifference ,was small(RR= 1.2) and their confidence limits were overlapped(Tabte 2). These results confirm that the SF-HL and

,,6, eo

" 70a, 60

50J

0.5% Ma{ath{onAcetone Control

97-

95"

90-

80"

70-

60-50-40-30-{

//1'0 0 10'00

Log Time, rnin

Figure 1. Log time vs Iogit percent mortality regressions of human headtouse populations from Ecuador (EC-HL) (A) and south Florida (Piantationand Homestead [SF-HL]) (B) treated with 1% permethrin (EC-HL, n=45;SF-HL, n:30), 0.5% malathion (EC-HL, n=16; SF-HL, n=30), or acetone(EC-HL, =30; SF-HL, n=30) as soivent controi.

EC-HL SF-HL TX-HL

LT Louse Sample LT Louse Sample, LT Louse Sample.Treatments

t% Perrnethdn 1870.5% Matathort 107 (83q32] 16 129{ (79-197) 30 k A 1.2 NANix fPfJzer/Warner-Lambe, NA NA 551 (516-587) 22 N/. NA NA NA

Morris Pains.Ovide Medcis Parmaceut}ca Ng NA 57 {35-79} 30 NA NA }4A NA

Cor3oraion. Phoenix. Adz)Ovide vehicle NA NA 38 30-46 15 NA N NA NAContro 965 (93&993 30 748 {702-790 30 NA NA NA NA

Abbreviations: Ci, confidence interval; EC-HL, Ecuador test population; LTso, lethal time 50%; NA, not applicable; RR, mortality resistance ratio; SF-HL, southFlorida test population; TX-HL, Texas test population.RR LTo (SF-HL)/LT (EC-HL).I'RR LT.,o (TX-HL)/LTo (EO-HL).:}:Significantly different from EC-HL (maximum log-1 kelihood test, P<.05).Significantly not different from EO-HL (maximum iog-iiKelihood test, P>.05).Sig,qficantly not different from 1% permethrin treatment in SF-HL (maximum Iog-likeiihood test, P>.05).

ARCH DERMATOL/VOL 139, AUG 2003 W'WNV.ARCHDERMATOL.COM996

EC-HL share a similar level of susceptibility to mala-thion and that the SF-HL lice were not resistant to thispediculicide.

Comparatively, the mortality responses of the SF-HLto 0.5% malathion (Figure 1B) and Ovide (F|ga'e )were significantly faster than when treated with 1% per-methrin (Figure IB) (X= 141.9 [P<.001] and X[= 173.0[P<.001], respectively) or with Nix (Figure 2) (X= 1056[P<.001] and X= 140.6 [P<.00I], respectively). Thesefindings indicate that both 0.5% malathion and Ovide are

more efficient in killing permethrin-resistant lice in theSF-HL than 1% permethrin or Nix. The data also showthat Ovide kills permethvin-resistant lice from the SF-HLapproximately 10 times faster than 1% permethrin andNix (Table 2).

The mortality response of the SF-HL to Nix (Fig-ure 2) is statistically the same as 1% permethrin (FigureiB) (X2= 1.8 [P<.4]). Similarly, the magnitude of thedifference using the LTs0 values was small (LTs0 of 1%permethrin-treated SF-HL/LT0 of Nix-treated SF-HL= 1.0) and their confidence limits were overlapped(Table 2).

In the SF-HL, Ovide (Figure 2) exhibited a some-what faster mortality response (2.3-fold, Table 2) com-

pared with 0.5% malathion (Figure ]B) (X=61.1[P<.001]). Additionally, Ovide vehicle (Ovide withoutmalathion) also elicits lethality. These results suggest thatother ingredients (eg, isopropanol and terpenes) in Ovidemay have pediculicidal activity. These differences mayor may not be clinically meaningful but deserve furtherinvestigation.

LINKAGE OF PERMETHRIN-RESISTANTPHENOTYPES AND KDR-TYPE MUTATIONS

Two populations were used to establish a linkage be-tween increasing sarvivorship to 1% permethriu and in-creasing frequency of the 2 kdr-type mutations. TheTX-HL was one of the most permethrin-susceptible USpopulations assayed to date as judged by its LT:0 valuecompared with that of the SF-HL (2.2-fold more suscep-tiN< Table 2) and by its calculated RR vs the EC-HL(RR= 1.4; Table 2). Additionally, the overlap of its re-

gression line with that of the permethrin-resistant SF-HLpopulation only at the longer survival rimes xvould in-dicate a population that consists mostly of susceptibleindividuals with some highly resistant individua]s(1=|' a). The SF-HL is resistant to permetbrin(RR 3.1; Table 2) and its regression ]ine is not substan-tially different from that of its acetone control line (Fig-ure 1B), which indicates a uniformly high tolerance topermethrin.

Using the LT95 vaiue of 7.6 hours from the EC-HLreated with 1% permethrin (Figure 1A) to assess the sus-ceptible or resistant phenotype of individual lice (sur-viva1 beyond 7.6 hours indicated resistance), it vas de-termined that of the thirty-three 1%-permethrin-treated lice of the TX-HL, 15% (5) were resistant and 85%(28) were susceptible ('l'tle a). These same lice weregenotyped using DNA sequence data, with t sample pro-ducing unreadable sequence (TPYE7). Only 13% (4/32)of these ]ice possessed the homozygous kdr-type muta-

97(R) Nx

/

soj

20

/

10 10Log Tme, rain

I000

Figure 2. Log time vs iogit percent mortality regressions of human head lousepopulations from south Ftorida (Plantation and Homestead [SF-HL]) treatedwith Nix (PfizerA'arner-Lambert, Morris Plains, NJ) (n=22) or Ovide (MedicisPharmecutical Corporation, Phoenix, Adz) (n=30) compared with acetonecontrol (n=30).ili97] I'SF'HLi o

20-{ /olO4,

/,10 i00 10'00

Log Time, rain

Fig|ire 3. Log time vs Iogit percent mortalib, regressions of human headiouse popuiations from south Florida (Plantation and Homestead [SF-HL],n=30) and Mathis, Tex (TX-HL, n=33) treated with 1% permethrin.

tions, and 87% (28/32) of the lice had no mutations or

-were heterozygous for these mutations (Table 3). It shouldbe noted that kdr-type mutation has been widely deter-mined to be inherited as a recessive factor and that het-erozygous individuals are susceptible to DDT, the pyre-thrins, and the pyrethroids in a variety of insect species,z-'xOf the lice that possessed the homozygous kdr-De mu-tations, all died at times greater than 7.6 hours (11-20hours). Only 1 sample (TPYE4) had a mismatch be-tween its phenotype (resistant) and its genotype (het-erozygous susceptible). Using these criteria, we found a97% agreement (31/32) between the phenotypic and thegenotypic determinations.

In contrast, it was phenotypically assessed that ofthe thirty 1%-permethrin-treaed lice of the SF-HL (Fig-ure 1B), 87% (26) were resistant and 13% (+) were sus-

ceptible ('l|e ). Two samples, both of which werephenot;qically resistanL failed to produced readable

quence (Per27 and 30). GenotypicaIly, it was deter-mined that in the SF-HL 96% (27/28) were resistant and% (1/28) were susceptible (Table 4). Using these crite-ria, we found an 86% (24/28) agreement between the phe-

ARCH DERMATOL/VOL i39, AUG 2003 WWCARCHDERMATOL.COM99"/

TaNs 3, Comparative Data of the Phenotype and Genu|ype of the Human Head Louse Population From Mathis, Tex,Foowing 1% Permethrin Nodality Bioassay (Phenotype) and DNA Sequencino Analysis (Genotype)

Genotype'Louse Samp|e Phenotype

(R 33} Time Di. ($ or R) T29! Lg32F

TPYEt I8 R R RTPYE2-3 ti R q R

TPYE4 8 R H

TPYE5 6 S H HTPYE6 3 S M P

TPYE7 S Unreadable readable$TPYE8 5 S H

TPYE9 5 S S STPYEIO-11 S HTPYE12- 5 S STPYEI5-17 5 S HRYE!8 6 S H HTYE19-20 6 S S STPYE21-22 6 H HTPYE23-27 S SYOONI-3 S S

S .S S

SYOON4 3 SYO05 3YOON6 26

Abbreviations: H, neterozygote allele; R, resistant al!ele; S, susceptible allele.Determined by iethal time 95% vaue (7.6 hours) of 1% permethrin bioassay using the Ecuador test population. A louse that died in 7.6 hours or less was

susceptib e. A louse that died in more than 7.6 hours was resistant.1-Genotype was assessed by actual D,A sequencing with T929 and L932 being susceptible and 1929 and F932 being resistant genot/pes.SThe DI'A sequence cannot be determined owing to the high background in chromatogram.

Abbrev ations: NA, not avaiiaNe (sampie iost following b oassay); R, resistant allele; S, susceptible aiie]e.*Determined by iethal time 95% vaiue (7.6 hours) of I% permethdn bioassay using the Ecuador population. A louse that died in 7.6 hours or less was

susceptible. A louse that died in more than 7.6 hours was resistant.tGenotype was assessed by actua', DNA sequenc ng with T929 and L932 being susceptibie and 1929 and F932 being resistant genotypes.SThe DNA sequence cannot be determined owing to the high background in chromatogram.Pfizer,.%arner-Lambert, Morris Plails: NJ.

notypic and genotypic determinations. These results in-dicate a stron correlation between the presence of theT929I and L932F mutations and survivorship beyond 7.6hours in the l%-permethrin bioassay, which is indica-tive of kdr-wpe permethrin resistance.

DETERMINATION OF THE FREQUENCYOF KDR-TYPE MUTATIONS

Lice were randomly selected from the SF-HL used in themortality bioassays for Nix (Figure 2), 0.5% malathion

ARCH DERMATO[/VOL 139, AUG 2003 wxrW.ARCIIDERiMATOL.COiM98

(Figure 1B), and Ovide (Figure 2) and used for geno-typic analysis. The LTo vaiues determined from the logtime vs logit mortality regression lines for the 9 lice ran-domly selected from the SF-HL treated with Nix (Table4, bottom), the 5 lice treated with 0.5% malathion('l'=ble 5, top), and the 7 lice treated with Ovide (Table5, bottom) were statistically not different from the LTs0values of their respective parent populations (Table 2)due to overlapping 95% confidence limits (552 {519-586] vs 642 [578-7t3], 129 [111-149] vs 227 [131-383], and 57 [51-64] vs 72 [55-92] minutes, respectively).Thus, the 3 randomly selected subpopulations used forsequencing were representative of their respective bio-assayed parent populations.

Of the 9 randomly selected SF-HL lice treated withNix, 100% (9/9) were phenotypicatly and t00% (8/8) weregenotypically resistant, with 1 sample (Nixr) that wasunreadable (Table 4). These results clearly establish thepresence of kdr-type mutations and permethrin resis-tance in this subpopulation.

Of the 5 randomly selected SF-HL lice treated with0.5% malathion and the 7 lice treated xvith Ovide, 100%were genotypically resistant to permethrin (5/5 and 7/7,respectively) (Table 5). Hmvever, all were assessed to bephenotypically susceptible to 0.5% malathion and Ovideasjudged by the LT95 value of 0.5% malathion on the pe-diculicidal-susceptible EC-HL (5.1 hours, Figure 1A), andall died prior to the 7.6 hour value used to assess phe-notypic resistance to permethrin, indicating that thereis no cross-resistance to malathion in permethrin-resistant lice. Although based on limited numbers, theseresults show that, even in the presence of the kdr-typemutations and permethrin resistance, both 0.5% mala-thion and Ovide provided much faster kill times than 1%permethrin or Nix.

COMMENT

We continue to confirm the presence of permethrin-resistant lice on individuals with pediculosis in the UnitedStates. Overall, the SF-HL is resistant to permethrin dueto kdr-typ,e mutations, but 0.5% malathion and Ovide killthese permethrin-resistant head lice in a manner not sig-nificantl different from that elicited by the insecticide-susceptible EC-HL. We were also able to demonstrate thata prescription-only product, Ovide, was able to kill thepermethrin-resistant lice at a rate that was approxi-mately 10 times faster than that observed for Nix.

Our mortality bioassay data are in agreement withfindings from 3 other independent studies. 2,x5, Al-though the overall conclusions are the same, the in vitrokill times observed in the present study are longer thanthose observed for both studies by Meinking et al2, andanother conducted by Pollack et al owing to several dif-ferences between the in vitro mortality bioassay tech-niques used. In view of these differences, there is a dearneed for standardizing the way in which pediculicidal ef-fectiveness and resistance are evaluated in vitro. It mustalso be noted that the sloxver kill times for permethrinand Nix may not be clinically relevant as long as they alsokill all the lice, albeit more slowly, in practice. This co-nundrum, however, can only be resolved by a clinical

Table 5. Comparative Data of the Phenotype and Genotypeof the Human Head Loose Pop|ahon From South FloridaFollowing 0.5% Malathion and Ovide Mortality Bioassay(Phenotype) and DNA Sequencing Analysis (Genotype)

Abbreviations: R, resistant aJieie; S, susceptible allele.*Determined by lethal time 95% vatue (5.1 hours) of 0.5% malathion

bioassay using the Ecuador test population. A louse that ded in 5.1 hours orIess was susceptibie. A louse that died in more than 5.1 hours was resistant.

?Geno,,pe was assessed by actual DNA sequencing with T929 and L932being susceptibie and 1929 and F932 being resistant genotypes.

SMedicis Pharmaceutical Corporation, Phoenix, Ariz.

study that assesses the effectiveness of these products andthe survivability of permethrin-resistant lice. The pres-ent work provides the means to carW out such a study.

The combination of mortality bioassays and DNAsequence analysis confirms the original findings of Leeand coworkers6 that the presence of kdr-type pointmutations T929I and L932F is highly associated withpermethrin resistance in field-collected head lice. Thedata presented also confirm and extend the recent find-ings by Meinking et al that Ovide is able to killpermethrin-resistant lice. This finding was expectedbecause the 2 mutations associated with permethrinresistance in licd do not confer cross-resistance to thediffering pharmacological and pediculicidal action ofmalathion in Ovide. Additionally, Ovide is not availableexcept as a prescription product and has only been rein-troduced to the market since 1999, which has limitedits impact on louse populations. Since Ovide was ableto kill lice many times faster than Nix and otherpermethrin-based products, its pediculicidal and ovi-cidal activity make it an alternative treatment whenpermethrin resistance is a problem. Nevertheless,permethrin/matathion-dual resistant lice already existin the UK and are a serious problem. Thus, surveil-lance for malathion resistance should be implemented ifOvide becomes the treatment of choice. Finaliy, our invitro bioassay and genotyping data clearly indicate thatpermethrin-resistant head lice are established in theUnited States and warrant the need for additional clini-cal studies to ensure the continued efficacy of the cur-rently- available over-the-counter formulations that con-

tain permethrin.

Acceptedfor publication February 11, 2003.This study was supported by grant PHS 1 R01 AI45062-

01A1 from the National Institutes qfHeath National Insti-tute ofAllergy and I@ctious Diseases, Bethesda, Md, andby a grant-in-aid from Medicis Pharmaceutical Corpora-tion, PhoenLx, Ariz.

We sincerely thank Lice Soarce Services, Inc, and FieldEpidemioog,)' Super; Team membersfor their assistance inlouse collections and shipments.

ARCH DERMATOL/VOL 139, AUG 2003 \VWV.ARCHDERMATOL.COM

Corresponding author and reprints: J. Marshall Cloth,PhD, Dartment of Entomolo,e, University of Massactm-serfs, Amhe'st, MA 01003.

1. Gra= NG. Human Lice: Ti,eir Prevalenc& OontrolandRedstance Insect..'ades, WHO/OTO/WHOPS/97.8. Geneva, Switzer!and: World Health Organization. 1997:1-61.

2. Meinking TL, Serrano L, Hard B, etai. Conparatve in-vitro pedicul cidal efficacyof treatments in resistant head lice population in the United States. Arch Oer-matoL 2002;138:220-224.

3. Meinkin TL, Entzel P, Villar ME, Vicar M, emard G, Porceiain SL. Compara-{ve efficacy of treatment ":or PedcWus cap#is infestations: update 2000. ArchDermatoL 2001 ;137:287-292.

4. MeJnking TL. Infestations. Grr Probl DermatoL 1999;11 (3):73-120.5. Chosidow O= Scabies and pediculosis. LanceL 2000;355:8i9-826.6. Poorbaugh JH. Head lice infestation--treatment fai!ures with i% lindane (Kwell).

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he 15th tbero-Latin American Congress ofDer-matology will take place from October 21 through

October 25, 2003, in Benos Aires, Argentina. For moreinformation, contact he Secretariat atAv. Rivadavia 1977,11th floor A, C1033AAJ, Buenos Aires, Argentina; ax:(54 11) 4-953-1505; e-mail: xvcongresso@xvcitad.org orxvcongreso@fibertet.com.ar.

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