Permethrin and malathion resistance in head lice:

Results of ex vivo and molecular assays

Sophie Bouvresse, MD,a Zohra Berdjane, PhD,b R
emy Durand, Pharm D, PhD,b Julie Bouscaillou, MD,c
Arezki Izri, MD,b and Olivier Chosidow, MD, PhDa
Paris, France

Background: Treatment of head lice infestation relies on the application of topical insecticides. Overuse of
these products has led to the emergence of resistance to pyrethroids and malathion worldwide. Permethrin
resistance in head lice is mostly conferred by the knockdown resistance (kdr) trait.

Objective: To evaluate the occurrence of permethrin- and malathion-resistant head lice in Paris.

Methods: A prospective survey was conducted in 74 elementary schools. Live lice collected on
schoolchildren were randomly selected and submitted to ex vivo bioassays or underwent individual
DNA extraction. A fragment of kdr-like gene was amplified and compared with wild-type sequences.

Results: Live head lice were detected in 574 children. Ex vivo assays showed no surviving lice after a
1-hour contact with malathion while most lice died after a 1-hour exposure to permethrin and piperonyl
butoxide (85.7%, 95% confidence interval [CI]: 83.9-87.5). Among the 670 lice with workable DNA
sequences, 661 lice (98.7%, 95% CI 97.7-99.3) had homozygous kdr mutations.

Limitations: The findings of this large-scale survey of the occurrence of insecticide-resistant head lice
indicated a major insecticide pressure in the study population, but it was not sufficient to draw conclusions
about other populations. The presence of T917I-L920F mutations in kdr gene may not correlate with
treatment failure in prospective studies.

Conclusion: The high occurrence of kdr mutant allele suggests that insecticide resistance was already
strongly established in the studied population. This finding must be interpreted with caution as it may not
be predictive of treatment failure. ( J Am Acad Dermatol 2012;67:1143-50.)

Key words: head lice; malathion; Paris; permethrin; Pediculus humanus capitis; resistance.

INTRODUCTION
Abbreviations used:
Head lice infestation due to Pediculus humanus
capitis is the most prevalent human ectoparasitic CI: confidence interval
DASES: Direction de lAction Sociale,
disease worldwide. It is particularly frequent among de lEnfance et de la Sant

e
children 3 to 11 years of age and may cause skin kdr: knockdown resistance
irritation, superinfection from scratching, social stig- PB: piperonyl butoxide
VGSC: voltage-gated sodium channel
matization, and psychological distress.1 Treatment
relies widely on the use of topical insecticides, that is,

From Universit
e Paris-Est Cr
eteil Val de Marne and Assistance Accepted for publication April 8, 2012.
Publique-H^opitaux de Paris, Department of Dermatology, Henri Reprints not available from the authors.
Mondor Hospitala; Department of Parasitology and Mycology, Correspondence to: R
emy Durand, Pharm D, PhD, Department of
Assistance Publique-H^opitaux de Paris, Avicenne Hospitalb; and Parasitology and Mycology, H^ opital Avicenne, 125, rue de
Epidemiology of Emerging Infectious Diseases, Institut Pasteur.c Stalingrad, 93009 Bobigny Cedex, France. E-mail: remy.
Funding sources: French Society of Dermatology and the Mairie de durand@avc.aphp.fr.
Paris. Published online May 24, 2012.
Disclosure: Professor Chosidow reports receiving consulting fees 0190-9622/$36.00
from Laboratoire Pierre Fabre and lecture fees from Pohl 2012 by the American Academy of Dermatology, Inc.
Boskamp; Dr Izri reports receiving consulting fees from doi:10.1016/j.jaad.2012.04.011
Laboratoire Johnson & Johnson, Pierre Fabre, and Omega
Pharma. No relationships were declared by the other authors.

1143
1144 Bouvresse et al J AM ACAD DERMATOL
DECEMBER 2012

malathion or pyrethrins/pyrethroids, recommended The importance of the phenomenon of resistance

as first-line treatment by national health authorities
such as those in France.2 Insecticidal pressure in-
duced by these treatments has led to the emergence
and spreading of resistance to pyrethroids and, to a
lesser extent, to malathion in many parts of the world.
Resistance to insecticides may induce treatment fail-
ures and result in chronic
infestations requiring addi-
tional episodic treatments. CAPSULE SUMMARY
This process generates eco-
Permethrin resistance in head lice is
d
to pyrethroids and malathion is not exactly known.
Previous studies including a few schoolchildren
demonstrated a high frequency of the resistant kdr
trait (0.93) in an urban area of France, Bobigny, in
2007.19 Data about resistance to malathion in France
are lacking or anecdotal.
We present herein the re-
sults of a large-scale obser-
vational study performed to
evaluate the occurrence of

nomic costs, possible toxic- head lice resistance to pyre-

mostly conferred by the kdr trait.
ity, anxiety, inconvenience, throids and malathion in the
Pyrethroid- and malathion-resistant head
and a further increase in elementary schools of Paris.
lice are described worldwide.
drug pressure.
Pyrethroids, including Malathion efficacy appeared preserved
d

D-phenothrin and permeth- in head lice obtained from elementary METHODS

rin, have been registered as schoolchildren in Paris. The relatively Design of the study
pediculicides since the 1970s good efficacy of pyrethroids contrasted This prospective observa-
and have been widely with the high occurrence of the kdr tional study was conducted
available as over-the-counter mutant allele. from January 2008 to June
products since the 1980s. 2009. According to Direction
The high occurrence of kdr mutant allele
d
de lAction Sociale, de
Clinical and parasitologic re- suggests an already strongly established
sistance to pyrethroids was lEnfance et de la Sant

e
insecticide resistance in Paris, but this (DASES) (the Paris City
first reported in France in a may not correlate with treatment failure
randomized controlled trial3 Health Organisation), there
in prospective studies. are 345 public elementary
and followed by additional
reports from Europe (Czech schools in Paris in which
4 5
Republic, United Kingdom, and Denmark ), the 6 83,830 children are enrolled.
7 8
Middle East (Israel ), North (United States ), and At the start of each academic year, a letter was sent to
9
South (Argentina ) America, Asia (Japan ), and 10 every elementary school director explaining the pur-
11
Australia. Early publications indicated that permeth- pose of our work and its practical course. The list of
rin resistance in head lice was mostly conferred by the directors was provided by the Paris City Health
12
knockdown resistance (kdr) trait. Three point mu- Organisation. The letter was accompanied by written
tations (M815I-T917I-L920F) in the voltage-gated forms to be distributed to every parent with a child
sodium channel (VGSC) a-subunit gene associated enrolled in an elementary class at the school. Parents
with permethrin-resistant phenotypes were sug- were informed about the study and they could indicate
gested to be responsible for kdr-type resistance. their refusal to participate.
Sequence analyses of cloned cDNA fragments and During the first part of the study (January 2008 to
genomic DNA fragments from individual louse sam- January 2009), directors were asked to notify head
ples confirmed that all the mutations existed en bloc lice infestation cases to DASES and only schools that
as a resistant haplotype. Further experiments using notified head lice infestation were included in the
site-directed mutagenesis at the corresponding amino study. During the second part of the study (January
acid sequence positions of the house fly para- 2009-June 2009), other schools were randomly se-
orthologous VGSC a-subunit (Vssc1WT ) gene and lected to ensure an equal distribution among the 20
heterologous co-expression with the sodium channel districts of Paris (Fig 1).
auxiliary subunit of house fly (Vsscb) in Xenopus A total of 74 schools were included. Of these 74
13
oocytes showed that the T917I mutation was the schools, 42 (57%) were included on notification of
principal cause of permethrin resistance in head lice head lice infestation during the first part of the study
via a kdr-type nerve insensitivity mechanism. and 32 (43%) were included randomly during the
Resistance to malathion was reported in France 14 second part of the study.
but seemed to be more common in the United
Kingdom and Denmark.15,16 Alterations of the Ethics statement
organophosphate-targeted acetylcholinesterase have The protocol was reviewed and approved by the
been suggested to be involved in resistance.15,17,18 Comit
e de Protection des Personnes (institutional
J AM ACAD DERMATOL
VOLUME 67, NUMBER 6
Fig 1. Map of Paris showing the location of schools where
schildren with head lice were identified. Blue circle:
Schools included upon notification during first part of
the study. Red circle: Schools included after random
selection during the second part of the study.
review board) of the ethic committee CPP- Ile-de-
France VI. The study protocol was registered at the
CNIL (French Data Protection Agency) (1223375v0).
The agreement of DASES and the authorization of
the regional academy offices (Academic Inspection
of Paris) were obtained for the study.
Study sites and patients
For each selected school, a trained clinical inves-
tigator examined all children (except for those with-
out parental consent to participate) in their
classroom. Children harboring lice or nits on visual
screening examination of the scalp and hair were
reconvened between classes and were subjected to
dry-combing of the head with a fine-toothed comb
onto a white paper sheet as previously described.20
Hair was combed with two different lice-combs: one
with larger plastic teeth (KSL consulting) was used to
untangle hair to facilitate the use of a finer comb with
metallic teeth (Innomed). The combing procedure
confirmed active head lice infestation (ie, presence
of live lice) and facilitated the removal of as many lice
as possible from each child.
For infested children, a semiquantitative evalua-
tion of the infestation was performed (children were
categorized as harboring less than 5 lice, 5 to 20 lice,
or more than 20 lice) and basic morphologic (char-
acteristics of hair such as length, color, and texture)
and sociodemographic (birth date, gender status)
data were collected. The children were asked if they
had head lice (during the preceding week) and if
they had treatment for lice during the week prior to
the study. Written information in a sealed envelope
was given to each parasitized child, providing advice
Bouvresse et al 1145
for treatment. This information was based on the
French Health Service recommendations published
in 2003.2
Sampling of head lice
Collected lice were kept in Petri dishes on tap
wateremoistened Whatman filter paper to prevent
desiccation and sent directly to the laboratory. One
Petri dish was used for each child and each dish was
anonymously identified with a code for school, the
sample number, and the date of specimen collection.
For each child, only live and non-injured lice were
selected and distributed randomly for different tests
depending of the number of lice per head:
d For fewer than 10 lice per head, all lice were used
for ex vivo testing with malathion.
d For 10 to 20 lice per head, lice were randomly
divided in two groups. One group was used for
ex vivo bioassays with malathion and the other
was tested with saline as a control group.
d For more than 20 lice per head, 20 lice were
randomly selected and divided into two groups.
One group was used for ex vivo bioassays with
malathion and the other was tested with saline as a
control group. Surplus specimens were tested for
permethrin sensitivity with an ex vivo bioassay.
Ex vivo pediculocidal assay
A standardized method for ex vivo assessment of
efficacy of pediculicidal products was used as pre-
viously described.3 The average time between col-
lection of samples and the beginning of the bioassay
was about 3 hours. Briefly, live and non-injured lice
were placed onto impregnated Whatman filter paper
in 8.5-cm petri dishes. The filter paper was impreg-
nated with 1 mL of a lotion containing the studied
compound. For malathion, a 1% malathion lotion
(Prioderm) was used; for permethrin a 0.3% per-
methrin lotion synergized with 1% PB (Pyreflor), and
saline water was used as the control group. The lice
were examined for activity under a binocular loupe
after 15, 30, 45, and 60 minutes.
Predefined criteria for evaluation of survival of
lice were based on activity, ataxic signs, as well as gut
and leg movements. Lice were judged as dead if
there were no vital signs or minor vital signs present
(merely internal gut movements, movements of
antennae, minimal leg movements with or without
stimulation by a forceps). Lice showing major vital
signs such as inability to walk in a progressive
fashion or no righting reflex when rolled onto the
back were judged as moribund. Because of differ-
ences in product odor, complete blinding was im-
possible. After pediculicidal bioassays, lice were
frozen at e808C.
1146 Bouvresse et al
Molecular assays of resistance to pyrethroids
A few lice (at least one per parasitized child) were
randomly selected to undergo molecular assays.
Each selected louse was placed individually in a
sterile microcentrifuge tube, incubated at 658C for 15
minutes and crushed by using Tissue Lyser U
(Qiagen, Courtaboeuf, France) for 8 minutes at the
frequency of 30 times per second. The sample was
homogenized in 180 L of ATL and 20 L of protein-
ase K, vortexed for 20 seconds and then incubated preceding week (66%, 95% CI: 62-70). More than half
for 3 hours at 568C. Further steps were done accord-
ing to the manufactures instructions of QIAamp
DNA Mini Kit (Qiagen, Courtaboeuf, France).
The amplification of a 560-bp portion of the voltage
sensitive sodium channel-subunit gene (kdr-like gene)
encompassing the 917 and 920 codons was performed
on 3 L of DNA solution under the following condi-
tions: in a 25-L reaction mixture containing 0.3 M of
each primer: kdrzF 59-GGTCGAACTGTTGGAGCTTT-
39 and kdrzR 59 ACCTGAAATACAAATGTAGG-39
(SIGMA, France), 200 M dNTPs, buffer (50mMKCl,
10mM Tris-HCl, pH8.3, and 1mM MgCl2), and 2.5 units
of Thermus aquaticus DNA polymerase (AmpliTaq
Gold; PerkinElmer Life, and Analytical Sciences,
Boston, MA). The sample was incubated for 10 minutes
at 958C followed by 40 reaction cycles (948C 3 40
seconds; 548C 3 45 seconds, and 728C 3 40 seconds).
After 40 cycles, primer extension was continued for 10
minutes at 728C. Polymerase chain reaction (PCR)
products purification and DNA sequencing were per-
formed by the Qiagen Laboratory (Qiagen,
Courtaboeuf, Germany). Electropherograms were vi-
sualized and analyzed with the AutoAssembler pro-
gram (Applied Biosystems). The obtained sequences
were compared with wild-type sequences (GenBank
accession numbers DQ062568.1, DQ062567.1). The
presence of single mutations T917I and L920F was
confirmed by reading both forward and reverse strands.
Statistical analysis
Data were entered using Excel spreadsheets and
checked for entry-related errors. Binomial confi-
dence interval rates were calculated using Stata
software version 8.2 (Stata Corporation, College
Station, TX). The significance of the difference of
relative frequencies was compared by applying
Fishers exact test. To calculate the confidence inter-
val of global pediculosis prevalence, a ratio estima-
tion approach (based on an adjustment of the
standard Pearson chi-square statistic) was applied
to account for clustering.
RESULTS
Epidemiologic findings
The population of the included schools represented
16,915 schoolchildren. Children without parental
J AM ACAD DERMATOL
DECEMBER 2012
consent to participate were excluded and, eventually,
14,436 children were visually screened in the 74
schools. Live head lice were detected in 574 children.
Prevalence of head lice infestation was found to be
3.98% (95% confidence interval [CI] 3.45-4.50) (Fig 2).
The main demographic characteristics of the in-
fested children are shown in Table I. The mean age
was 8.9 years. They were mainly girls (70%, 95% CI: 66-
74), and most reported head lice infestation in the
of the children (54 %, 95% CI: 48-60) mentioned the
use of an anti-lice treatment in the past week. The
mean parasitic carriage was 13 head lice per head with
a high variability between children. Twenty-six per-
cent (95% CI: 22-30) were heavily infested with more
than 20 lice per head. A total of 7400 head lice were
collected.
Pediculocidal bioassays
Ex vivo assays were carried out on 3797 head lice
to assess resistance to malathion: no lice survived
after 1 hour of contact with malathion. Ex vivo assays
with permethrin synergized with piperonyl butoxide
(PB) were carried out on 1531 head lice: After a
1-hour exposure, 39 lice (2.5%, 95% CI: 1.7-3.4) were
alive, 180 (11.8%, 95% CI: 10.1-13.4) were moribund
and 1312 (85.7%, 95% CI: 83.9-87.5) were dead. At
shorter exposure times, fewer head lice were dead,
particularly for the permethrin plus PB formulation
(Table II). In comparison, of the 2021 lice tested with
saline solution, 1962 (97%, 95% CI: 96.3-97.8) sur-
vived after a 1-hour exposure.
Molecular markers of resistance to
pyrethroids
Of the 7400 collected lice, 864 lice were used for
molecular studies as at least one louse per infested
child was selected. Readable sequences were ob-
tained for 670 lice from 445 children distributed in 67
schools. Among these, 661 lice (98.7%, 95% CI: 97.7-
99.3) had homozygous kdr mutations at each of the
two sites examined (T917I and L920F). Only 9 lice
distributed in 7 districts were found as homozygous
susceptible. To assess genetic diversity in heavily
parasitized scalps, 10 children were randomly se-
lected from those harboring more than 20 lice and 10
randomly selected lice per head were analyzed.
Molecular results were interpretable in 77 cases
and all the studied lice had homozygous kdr muta-
tions at each of the two sites examined.
DISCUSSION
To our knowledge, this is the largest epidemio-
logic population-based survey studying occurrence
of insecticide-resistant head lice. The sampling
J AM ACAD DERMATOL Bouvresse et al 1147
VOLUME 67, NUMBER 6

Fig 2. Results of screening for head lice infestation in elementary schoolchildren.

Table I. Characteristics of children infested by head lice in Parisian elementary schools

No. of lice per head
No. of infested No. of collected Mean no. of lice
District children No. of girls (%) Mean age lice \5 5 to 20 [20 per head
1 19 10 (52.6) 8.44 209 11 7 1 11
2 4 4 (100) NA 15 2 2 0 4
3 22 17 (77.2) 8.71 224 10 8 4 10
4 17 12 (70.6) 9.33 161 4 9 4 9
5 16 8 (50) 9.35 183 3 8 5 11
6 21 18 (85.7) 8.88 269 11 7 3 13
7 11 6 (54.5) 8.65 154 3 4 4 14
8 13 10 (76.9) 9.08 172 4 2 7 13
9 17 11 (64.7) 9.16 228 6 4 7 13
10 42 23 (54.8) 8.64 518 17 15 10 12
11 48 30 (62.5) 8.79 772 12 25 11 16
12 30 23 (76.7) 9.3 386 11 12 7 13
13 70 49 (70) 9.2 1158 20 27 23 17
14 16 13 (81.2) 8.94 173 3 9 4 11
15 44 32 (72.7) 8.29 481 16 17 11 11
16 23 15 (65.2) 8.5 248 7 14 2 11
17 38 28 (73.7) 8.84 450 13 15 10 12
18 60 39 (65) 8.95 899 15 28 17 15
19 23 19 (82.5) 9.17 309 5 12 6 13
20 40 35 (87.5) 9.15 395 15 12 13 10
Total 574 402 (70) 7404 188 237 149 13

NA, Not available.

method was chosen to ensure an equal distribution absence of a significant difference in head lice
of the selected schools among different districts of prevalence and refusal to participate between the
Paris. The validity of this method is assessed by the institutions included upon notification of infestation
1148 Bouvresse et al J AM ACAD DERMATOL
DECEMBER 2012

Table II. Results of ex-vivo assays showing survival have easy access to pyrethroid-based compounds
(as percentage) of head lice at 4 times after have higher levels of kdr mutant allele.24 Our study
exposure to malathion (n = 3797) or permethrin demonstrated that 98.7% of the tested head lice had
(n = 1531) formulations homozygous kdr mutations. The overrepresentation
Live head Moribund Dead head
of homozygous kdr mutations probably reflects an
Compound lice (%) head lice (%) lice (%) increased insecticide selection pressure because of
Malathion lotion 1% overuse of these products. The frequency of the kdr
15 min 3.1 9.6 87.3 mutant allele, and the almost complete lack of heter-
30 min 0.8 5.5 93.7 ozygous genotypes suggested that the diffusion of
45 min 0.2 3.3 96.5 the kdr mutant allele was not a recent phenomenon
60 min 0 2.1 97.9 and that the resistance was already strongly estab-
Permethrin lotion 0.3% 1 piperonyl butoxide 1% lished in the studied population.
15 min 5.6 19.5 74.9 The high frequency of the kdr mutant allele was
30 min 3.9 18.4 77.7 comparable to those found in South Florida (97%),25
45 min 3.8 15 81.2
in Denmark (95%),16 in Germany,26 or recently in
60 min 2.5 11.8 85.7
France in a smaller series (93%).22 Our results con-
Controls (isotonic sodium chloride solution) (n = 2021)
15 min 98.7 1.1 0.1 firmed that resistance to pyrethroids has reached a very
30 min 98.5 1.4 0.1 high frequency in schoolchildren in the Paris region.
45 min 97.2 1.9 0.9 The purpose of the present study was not to
60 min 97 2 1 evaluate the correlation between ex vivo bioassays
and molecular typing as the association of single-
nucleotide polymorphisms in the kdr gene and
and the schools randomly selected during the second permethrin resistance in head lice has been docu-
inclusion period. The reported confidence interval mented in several limited series.6,16,21,24,27 We used
could seem inappropriate considering the purposive 0.3% permethrin synergized with 1% PB for ex vivo
sampling method, but it was estimated with a ratio tests because pyrethroid-based compounds are often
estimation approach to account for clustering. combined with PB in French over-the-counter pe-
Prevalence of head lice infestation (3.98%, 95% CI: diculicides. Thus results of ex vivo tests reflect the
3.45-4.50) estimates from our analysis are smaller effects of both components and not only those of
than the infestation rate among children reported permethrin alone. Lee et al12 have shown that the
from surveys conducted across Europe (ie, 8.3% in mortality and knockdown responses to permethrin
the United Kingdom21 or 14.4% in Denmark16), but of susceptible and resistant head lice was signifi-
they are in accordance with what has been previously cantly synergized by PB. These authors suggested
shown in urban France.19,22 A comparison between that the effects of PB, which acts as a cytochrome
these results is hard to make because variation in P450 mono-oxygenase enzyme inhibitor, are indic-
head lice infestation rates across different surveys ative of the role in resistance of enhanced oxidative
may also be due to different methods used to diag- metabolism. Our results were consistent with their
nose active pediculosis. Indeed, it was shown that data as we observed a susceptibility to the combina-
visual inspection underestimated the prevalence of tion of 0.3% permethrin and 1% PB in the ex vivo test
active infestation in comparison with systematic wet (near 85% of mortality) that was much higher than
combing.20 Our study was not designed to measure what could be inferred from the sole analysis of the
head lice infestation; the visual screening completed kdr gene typing (near 98% of lice having homozy-
with combing of the hair was chosen as a compro- gous kdr mutations). In addition, our results were
mise between a sensitive and a practical and fast consistent with those of Yoon et al28 who found that
method to detect infestation and collect head lice. two products containing 0.3% pyrethrin and PB were
The pyrethrins and pyrethroids are among the efficacious (62.2%-84.4% mortality assessed on the
major currently available pediculicides on the mar- 8th day post-treatment in a model mimicking the
ket. Their extensive use as over-the-counter pedicu- exposure to these products on the human scalp) on
licides has been rapidly followed by resistance. strains having kdr mutant genotypes.
Pyrethroid resistance in head louse populations ap- Head lice having different kdr-like genotypes may
pears to be widespread in various countries but varies live together on the head of a subject.19,22 In an
in intensity and is not yet uniform.23 The uncontrolled attempt to evaluate this diversity, 10 children were
use of pyrethroids may have been a key factor for the randomly selected among heavily infested children
selection of lice having homozygous kdr mutations. A and 10 randomly selected lice per head were ana-
recent article suggested that lice from countries that lyzed. We did not observe any mix of genotypes in
J AM ACAD DERMATOL
VOLUME 67, NUMBER 6
these children. These results do not exclude the
possibility of a combination of different genotypes in
other children.
Detecting resistance through bioassay-based
methods is often arduous29 and the lack of standard-
ization of assays makes it difficult to compare results
from different studies.30 In addition, slower kill times
or knockdown responses with permethrin that were
observed in bioassays for resistant lice were not
necessarily predictive of clinical failure as the insec-
ticide killed all the lice, albeit more slowly, at the end
of the test. In clinical practice, longer exposure times
could result in relatively good efficacy, even on
resistant head lice. Likewise, the high occurrence
of the kdr mutant allele has to be interpreted with
caution as T917I-L920F mutations in the kdr gene
may not correlate with treatment failure in prospec-
tive studies. A recent study including a limited
number of samples in Germany reported that the
presence of kdr mutant allele was not associated
with clinical failure of pyrethroids.26 Further clinical
trials are required to document the relevance of kdr
genotyping as predictive of the clinical outcome of
pyrethroid-based treatment. Nevertheless, the very
high proportion of kdr mutant allele in our sampled
population, showing a strong selection of mutant
head lice by use of pyrethroids, and the availability of
other options for treatment, would advocate for the
abandonment of pyrethroids as head lice treatment,
at least in the studied geographic area. Alternative
therapeutic strategies include medical devices (bug
busting) and non-insecticidal pediculicides, such as
dimethicone or oil-based compounds. Recently, oral
ivermectin has been shown to be a therapeutic
option for difficult-to-treat head lice.31
Ex vivo assays showed complete efficacy of mal-
athion in our series after 1 hour of exposure. At
shorter exposure times, the efficacy was not total but
appeared higher than with the permethrin plus PB
formulation. Ex vivo assays were not performed
under standardized conditions of temperature or
hygrometry, but the use of a control group (head lice
collected from the same head and exposed to a saline
solution), showing negligible mortality in untreated
head lice, enabled the validity of the results. No
molecular marker of resistance to malathion is cur-
rently available and consequently only clinical data
could confirm these results. Malathion appeared to
have retained its efficacy in France, as shown by a
recent controlled trial comparing oral ivermectin to
0.5% malathion lotion in four countries including
France.31 Given these results, it seems that formula-
tions containing malathion can still be recommended
as pediculicides for schoolchildren of Paris. The
absence of malathion resistant head lice in Paris is
Bouvresse et al 1149
not sufficient to draw any conclusion for other
populations (for instance in other French cities).
This large-scale population-based survey of the
occurrence of insecticide-resistant head lice indi-
cates a major insecticide pressure. Further studies are
necessary to identify all contributors to pyrethroid
resistance, such as head louse attributes and possible
host factors. Long-term surveillance of insecticidal
resistance could be important to guide therapeutic
strategy for head lice.
We acknowledge and express our thanks for the valu-
able technical advice provided by Dr Arnaud Fontanet on
the statistical aspects of this study. We thank Zohra Hassani
and Aur
elie Delos for technical assistance. The authors
thank Kim Sholt Larsen (KSL consulting) for providing
some of the lice-combs. We are grateful for the advice on
study design from Professor Alfred Spira and Ian Burgess.
We would like to express our gratitude to all the children
and their parents as well as the federations of parents
(PEEP-F
ed
eration des Parents dEl eves de lEnseignement
Public- and FCPE -F
ed
eration des Conseils de Parents
dEl
eves) for their participation in this study. The authors
are also indebted to the DASES (Direction de lAction
Sociale, de lEnfance et de la Sant
e), especially to Dr
Genevi eve Richard and Dr Francoise Delbard, and head
teachers and staff of Paris elementary schools .We thank
Alain Lhostis, Alain Claquin and Jean-Marie Le Guen
(Mairie de Paris).
REFERENCES
1. Chosidow O. Scabies and pediculosis. Lancet 2000;355:819-26.
2. Avis du CSHP de France du 27 juin 2003, relatif a la conduite a
tenir devant un sujet atteint de p
ediculose du cuir chevelu.
Ann Dermatol Venereol 2004;131:1122-4.
3. Chosidow O, Chastang C, Brue C, Bouvet E, Izri M, Monteny N,
et al. Controlled study of malathion and d-phenothrin lotions
for Pediculus humanus var capitis-infested schoolchildren.
Lancet 1994;344:1724-7.
4. Rupes V, Moravec J, Chmela J, Ledvinka J, Zelenkova J. A
resistance of head lice (Pediculus capitis) to permethrin in
Czech Republic. Cent Eur J Public Health 1995;3:30-2.
5. Burkhart CG, Burkhart CN. Clinical evidence of lice resistance to
over-the-counter products. J Cutan Med Surg 2000;4:199-201.
6. Kristensen M. Identification of sodium channel mutations in
human head louse (anoplura: Pediculidae) from Denmark.
J Med Entomol 2005;42:826-9.
7. Mumcuoglu KY, Hemingway J, Miller J, Ioffe-Uspensky I, Klaus S,
Ben-Ishai F, et al. Permethrin resistance in the head louse
Pediculus capitis from Israel. Med Vet Entomol 1995;9:427-32, 447.
8. Pollack RJ, Kiszewski A, Armstrong P, Hahn C, Wolfe N, Rahman
HA, et al. Differential permethrin susceptibility of head lice
sampled in the United States and Borneo. Arch Pediatr
Adolesc Med 1999;153:969-73.
9. Picollo MI, Vassena CV, Casadio AA, M
aximo J, Zerba EN.
Laboratory studies of susceptibility and resistance to insecti-
cides in Pediculus capitis (Anoplura; Pediculidae). J Med
Entomol 1998;35:814-7.
10. Tomita T, Yaguchi N, Mihara M, Takahashi M, Agui N, Kasai S.
Molecular analysis of a para sodium channel gene from
pyrethroid-resistant head lice, Pediculus humanus capitis
(Anoplura: Pediculidae). J Med Entomol 2003;40:468-74.
1150 Bouvresse et al
11. Hunter JA, Barker SC. Susceptibility of head lice (Pediculus
humanus capitis) to pediculicides in Australia. Parasitol Res
2003;90:476-8.
12. Lee SH, Yoon KS, Williamson MS, Goodson SJ, Takano-Lee M,
Edman JD, et al. Molecular analysis of kdr-like resistance in
permethrin-resistant strains of head lice, Pediculus capitis.
Pestic Biochem Phys 2000;66:130-43.
13. Yoon KS, Symington SB, Lee SH, Soderlund DM, Clark JM.
Three mutations identified in the voltage-sensitive sodium
channel alpha-subunit gene of permethrin-resistant human
head lice reduce the permethrin sensitivity of house fly Vssc1
sodium channels expressed in Xenopus oocytes. Insect Bio-
chem Mol Biol 2008;38:296-306.
14. Izri MA, Briere C. First cases of resistance of Pediculus capitis
Linn
e 1758 to malathion in France. Presse Med 1995;24:1444.
15. Downs AMR, Stafford KA, Hunt LP, Ravenscroft JC, Coles GC.
Widespread insecticide resistance in head lice to the
over-the-counter pediculocides in England, and the emer-
gence of carbaryl resistance. Br J Dermatol 2002;146:88-93.
16. Kristensen M, Knorr M, Rasmussen AM, Jespersen JB. Survey of
permethrin and malathion resistance in human head lice
populations from Denmark. J Med Entomol 2006;43:533-8.
17. Downs AMR, Stafford KA, Harvey I, Coles GC. Evidence for
double resistance to permethrin and malathion in head lice. Br
J Dermatol 1999;141:508-11.
18. Gao JR, Yoon KS, Frisbie RK, Coles GC, Clark JM. Esterase-me-
diated malathion resistance in the human head louse, Pedic-
ulus capitis (anoplura: Pediculidae). Pestic Biochem Phys 2006;
85:28-37.
19. Durand R, Millard B, Bouges-Michel C, Bruel C, Bouvresse S, Izri
A. Detection of pyrethroid resistance gene in head lice
in schoolchildren from Bobigny, France. J Med Entomol
2007;44:796-8.
20. Mumcuoglu KY, Friger M, Ioffe-Uspensky I, Ben-Ishai F, Miller J.
Louse comb versus direct visual examination for the diagnosis
of head louse infestation. Pediatr Dermatol 2001;18:9-12.
21. Thomas DR, McCarroll L, Roberts R, Karunaratne P, Roberts C,
Casey D, et al. Surveillance of insecticide resistance in head lice
J AM ACAD DERMATOL
DECEMBER 2012
using biochemical and molecular methods. Arch Dis Child
2006;91:777-8.
22. Durand R, Bouvresse S, Andriantsoanirina V, Berdjane Z,
Chosidow O, Izri A. High frequency of mutations associated
with head lice pyrethroid resistance in schoolchildren from
Bobigny, France. J Med Entomol 2011;48:73-5.
23. Gao JR, Yoon KS, Lee SH, Takano-Lee M, Edman JD, Meinking
TL, et al. Increased frequency of the T929I and L932F muta-
tions associated with knockdown resistance in permethrin
resistant populations of the human head louse, Pediculus
capitis, from California, Florida and Texas. Pestic Biochem Phys
2003;77:115-24.
24. Hodgdon HE, Yoon KS, Previte DJ, Kim HJ, Aboelghar GE, Lee
SH, et al. Determination of knockdown resistance allele
frequencies in global human head louse populations using
the serial invasive signal amplification reaction. Pest Manag Sci
2010;66:1031-40.
25. Yoon KS, Gao JR, Lee SH, Clark JM, Brown L, Taplin D.
Permethrin-resistant human head lice, Pediculus capitis, and
their treatment. Arch Dermatol 2003;139:994-1000.
26. Bialek R, Zelck UE, F olster-Holst R. Permethrin treatment of
head lice with knockdown resistance-like gene. N Engl J Med
2011;364:386-7.
27. Lee SH, Gao JR, Yoon KS, Mumcuoglu KY, Taplin D, Edman JD,
et al. Sodium channel mutations associated with knockdown
resistance in the human head louse, Pediculus capitis (de
Geer). Pestic Biochem Phys 2003;75:79-91.
28. Yoon KS, Strycharz JP, Gao JR, Takano-Lee M, Edman JD, Clark
JM. An improved in vitro rearing system for the human head
louse allows the determination of resistance to formulated
pediculicides. Pestic Biochem Phys 2006;86:195-202.
29. Clark JM. Determination, mechanism and monitoring of
knockdown resistance in permethrin-resistant human head
lice, Pediculus humanus capitis. J Asia Pac Entomol 2009;12:1-7.
30. Elston DM. Drug-resistant lice. Arch Dermatol 2003;139:1061-4.
31. Chosidow O, Giraudeau B, Cottrell J, Izri A, Hofmann R, Mann
SG, et al. Oral ivermectin versus malathion lotion for
difficult-to-treat head lice. N Engl J Med 2010;362:896-905.