The Attractivity of the Head Louse, Pediculus humanus capitis (Pediculidae: Anoplura) to Isolated Compounds and Blends of Human Skin.

Human head lice Pediculus humanus capitis De Geer (Phthiraptera: Pediculidae) are ectoparasites that cause pediculosis, a global scale disease mainly found in school-age children. Previous works from our laboratory found nonanal, sulcatone, and geranylacetone as the main human scalp volatile components, and individually evaluated their attraction to head lice using an olfactometer. In this work, we compared how their blends at different concentrations attract head lice, and how their blended effect compares to the effect of isolated compounds. At the concentrations evaluated, individual components did not show attraction towards head lice, but a ternary mixture of them was attractive. Moreover, a solvent extract from the human head scalp was analyzed by GC-MS, finding that tetradecanoic acid, palmitic acid, oleic acid, palmitoleic acid, and squalene are the most abundant components. Attraction to these individual compounds at natural concentrations was tested by bioassays in a circular experimental arena. No attraction was observed when the components were tested individually, but when they were evaluated as a blend they attracted head lice. This work presents new information about how chemical signals are attractive at certain concentrations and proportions. This information could be used to better understand communication mechanisms in head lice and for the development of louse repellents.

Sensory features of the human louse antenna: new contributions and comparisons between ecotypes.

Body and head lice are known to be exclusive ectoparasites of human hosts. Current genomic and transcriptomic data suggest that both louse types represent ecotypes of the same species. They exhibit morphological and physiological differences that probably arose from living in different biotopes. Sensory traits represent suitable candidates to be affected by environmental heterogeneity. Therefore, through scanning electron microscope studies, this study analysed and compared the sensory structures of both ecotypes' antennae. Seven types of sensory structures were identified in both ecotypes: bristles, tuft organs, pore organs, single pore and three morphotypes of sensilla basiconica. Notably, the single pore and the morphotypes of sensilla basiconica were described for the first time in the body louse antenna. This study's comparative analysis mainly revealed size differences across the sensory structures of the ecotypes. Bristles of the flagellomere 2 of the body louse antenna were longer than the head louse bristles. In addition, the pore organs of the head louse antenna presented a higher diameter than those of the body louse. The possible relevance of size differences regarding the biotopes exploited by the body louse and the head louse is discussed. Yet, physiological studies may help to fully understand the phenotypical differences of both ecotypes.

Head Louse Feces: Chemical Analysis and Behavioral Activity.

Human head lice Pediculus humanus capitis (De Geer) (Phthiraptera: Pediculidae) are insect parasites closely associated with humans, feeding on the blood of their hosts and causing them skin irritation and probable secondary infections. Despite being a severe nuisance, very few studies have reported on intraspecific chemical communication in head lice. Here, we evaluated the attractive response of head lice to the volatile compounds and solvent extracts from their feces. We also chemically analyzed the main volatile components of these feces and those of the feces' extracts. Head lice were attracted to the methanol extract of their feces but not to the hexane or dichloromethane extracts, suggesting the polar nature of bioactive chemicals present in head louse feces. Follow-up chemical identifications, in fact, showed the presence of hypoxanthine, uric acid, and another purine tentatively identified as either guanine or iso-guanine. Additionally, head lice were significantly attracted by volatiles emitted from samples containing feces. The volatiles emanated from feces alone contained 19 identified substances: 2-pentanone, hexanal, heptanal, 3-methyl-3-buten-1-ol, octanal, sulcatone, nonanal, acetic acid, 2-ethyl-1-hexanol, decanal, 1-octanol, butyric acid, 1-nonanol, hexanoic acid, octanoic acid, 2,6-dimethyl-7-octen-2-ol, 2-undecanone, geranylacetone, and hexadecane. The major compounds found were decanal, nonanal, hexanal, and acetic acid, together representing approximately 60% of the identified compounds. This work represents the first chemical evidence of intraspecies communication among head lice. The results support the existence of active substances present in the feces of P. humanus capitis that may be involved in its aggregation behavior.

Modelling the association between deltamethrin resistance in Triatoma infestans populations of the Argentinian Gran Chaco region with environmental factors.

In Latin America, Triatoma infestans is the main vector of the protozoan Trypanosoma cruzi, causal agent of Chagas disease. This blood-sucking triatomine is widely distributed in the Gran Chaco ecoregion, where chemical control has failed because of the evolution of resistance to pyrethroid insecticides. Recently, we described a deltamethrin high resistant focus in Güemes Department (Chaco province) characterized by susceptible populations, populations with low resistance (without field control failures) and some of the populations with the highest resistance level detected. This toxicological heterogeneity could be a result of non-homogenous insecticide pressure and be influenced by environmental factors. The present study evaluated the association of deltamethrin resistance ratios (RR50s) of T. infestans populations with explanatory variables extracted from the WorldClim dataset and constructed from information of National Chagas Program actions during 2005-2015. Control actions were distributed throughout the analyzed period, representing a homogeneous selective pressure. The average percentage of total positive houses was 33.66%. Models that included temperature and precipitation indicators presented 65% explanation. When village size variables where added, the explanatory power reached 70%. This observational result suggests that the climate may favor directly or indirectly the development/selection for resistance, representing a valuable tool to understand the occurrence of resistance that could increase the Chagas disease in the Gran Chaco.

Response of Pediculus humanus capitis (Phthiraptera: Pediculidae) to Volatiles of Whole and Individual Components of the Human Scalp.

The head louse Pediculus humanus capitis (De Geer) (Phthiraptera: Pediculidae) is a cosmopolitan human ectoparasite causing pediculosis, one of the most common arthropod parasitic conditions of humans. The mechanisms and/or chemicals involved in host environment recognition by head lice are still unknown. In this study, we evaluated the response of head lice to volatiles that emanate from the human scalp. In addition, we identified the volatile components of the odor and evaluated the attractive or repellent activity of their pure main components. The volatiles were collected by means of Solid Phase microextraction and the extract obtained was chemically analyzed by gas chromatograph-mass spectrometer. Twenty-four volatile were identified in the human scalp odor, with the main compounds being the following: nonanal, sulcatone, geranylacetone, and palmitic acid. Head lice were highly attracted by the blend human scalp volatiles, as well as by the individual major components. A significant finding of our study was to demonstrate that nonanal activity depends on the mass of the compound as it is repellent at high concentrations and an attractant at low concentrations. The results of this study indicate that head lice may use chemical signals in addition to other mechanisms to remain on the host.

Demographic effects of deltamethrin resistance in the Chagas disease vector Triatoma infestans.

Triatoma infestans (Heteroptera: Reduviidae) Klug is the main vector of Chagas disease in Latin America. Resistance to deltamethrin was reported in Argentina and recently associated with reproductive and longevity trade-offs. The objectives of the present study were to describe the demographic consequences of deltamethrin resistance in T. infestans and to establish possible target stages for chemical control in susceptible and resistant colonies. A stage-classified matrix model was constructed based on the average stage length for susceptible, resistant and reciprocal matings' progeny. The differences between colonies were analysed by prospective and retrospective analysis. The life table parameters indicated reduced fecundity, fertility and population growth in resistant insects. The retrospective analysis suggested the latter was associated with lower reproductive output and increased fifth-instar nymph stage length. The prospective analysis suggested that the adult stage should be the main target for insecticide control. Although, fifth-instar nymphs should also be targeted when resistance has been detected. The presented results show demographic effects of deltamethrin resistance in T. infestans. While the older stages could be the main targets for chemical control, this approach is impeded by their higher tolerance to insecticides. It is concluded that the different mode of action insecticides would be more effective than a dose increase for the control of deltamethrin-resistant T. infestans.

Geographical Variation of Deltamethrin Susceptibility of Triatoma infestans (Hemiptera: Reduviidae) in Argentina With Emphasis on a Resistant Focus in the Gran Chaco.

Chagas disease is one of the most important parasitic infections in Latin America. The main vector of the protozoan Trypanosoma cruzi in America is Triatoma infestans, a blood-sucking triatomine bug who is widely distributed in the Gran Chaco ecoregion. Control programs in endemic countries are focused in the elimination of triatomine vectors with pyrethroid insecticides. However, chemical control has failed in the Gran Chaco over the last two decades because of several factors. Previous studies have reported the evolution of different levels of resistance to deltamethrin in Tri. infestans Recently, very high resistance has been found in the central area of the Argentine Gran Chaco. However, the origin and the extension of this remarkably resistant focus remain unknown. The aim of this study was to evaluate the geographical variation of deltamethrin susceptibility of Tri. infestans in different endemic provinces of Argentina, with emphasis in the center of the Argentine Gran Chaco ecoregion where this main vector has not been reduced. Populations of Mendoza, San Juan, Santiago del Estero, and Tucumán provinces were all susceptible. Resistant populations were only detected in the province of Chaco, where a mosaic resistant focus was described at the Güemes Department. It was characterized into three pyrethroid resistance categories: susceptible, low, and highly resistant populations. We found the populations with the highest resistance levels to deltamethrin, with resistant ratios over 1000.

Examining Mechanisms of Pyrethroid Resistance in Eggs of Two Populations of the Chagas' Disease Vector Triatoma infestans (Hemiptera: Reduviidae).

Chagas disease is a zoonosis transmitted to man by blood-sucking triatomine bugs found in the Americas. Triatoma infestans (Klug, 1834) is the main vector of Chagas' disease in Argentina. The control of this illness relies heavily on vector control through the use of insecticide. However, resistance to pyrethroid insecticides associated with ineffective field treatments has been increasingly reported in T. infestans from Argentina and Bolivia. There are few reports on the expression and causes of resistance in eggs of resistant populations, and even fewer studies on insecticide resistance throughout embryonic development. In this study, we explore the biochemical and molecular mechanisms potentially associated with the deltamethrin resistance assessed in the developing eggs of the Argentinean (Campo Largo) and Bolivian (Entre Ríos) T. infestans populations.We found measurable activity of monooxigenases and pyrethroid esterases throughout embryonic development. The pyrethroid esterase activity grew steadily throughout development in all the studied populations and was highest in eggs 12 d old. Mean enzyme activity increased from 13.6 to 16.3 and 22.2 picomol 7-hydroxycoumarin/min (7-OHC) in eggs of 4-, 7-, and 12 d old from the susceptible reference bug colony. Mean activity of resistant populations increased from 16.0 to 25.9 picomol 7-OHC/min in eggs of 4- to 12 d old in Entre Ríos population, and from 15.9 to 28.9 picomol 7-OHC/min in Campo Largo population. Molecular analysis of susceptible and resistant developing eggs detected L1014F mutation in both resistant populations, but no L925I mutation was found in any of the studied populations.Higher esterase activity and L1014F presence justify the resistance to pyrethroid throughout developing eggs of both studied T. infestans populations. The description of resistance profiles including resistance mechanisms involved will allow a rational design of campaigns for the control of Chagas disease transmission.

Effect of Environmental Conditions and Toxic Compounds on the Locomotor Activity of Pediculus humanus capitis (Phthiraptera: Pediculidae).

In this work, we evaluated the effect of environmental variables such as temperature, humidity, and light on the locomotor activity of Pediculus humanus capitis. In addition, we used selected conditions of temperature, humidity, and light to study the effects of cypermethrin and N,N-diethyl-3-methylbenzamide (DEET) on the locomotor activity of head lice. Head lice increased their locomotor activity in an arena at 30°C compared with activity at 20°C. When we tested the influence of the humidity level, the locomotor activity of head lice showed no significant differences related to humidity level, both at 30°C and 20°C. Concerning light influence, we observed that the higher the intensity of light, the slower the movement of head lice. We also demonstrated that sublethal doses of toxics may alter locomotor activity in adults of head lice. Sublethal doses of cypermethrin induced hyperactivated responses in adult head lice. Sublethal doses of DEET evocated hypoactivated responses in head lice. The observation of stereotyped behavior in head lice elicited by toxic compounds proved that measuring locomotor activity in an experimental set-up where environmental conditions are controlled would be appropriate to evaluate compounds of biological importance, such as molecules involved in the host-parasite interaction and intraspecific relationships.

Fenitrothion: an alternative insecticide for the control of deltamethrin-resistant populations of Triatoma infestans in northern Argentina.

Deltamethrin-based campaigns to control Triatoma infestans (Klug) (Hemiptera: Reduviidae) have decreased in success as a result of the development of insecticide resistance. We compared the in vitro effects of the pyrethroid deltamethrin and two doses of the organophosphate fenitrothion, presented on different materials, on T. infestans from La Esperanza, Argentina. Laboratory tests demonstrated a decrease in susceptibility to deltamethrin in the field population [LD50 : 30.32 nanograms per insect (ng/i)] compared with the reference population (LD50 : 0.13 ng/i), giving a high resistance ratio of 233.42. By contrast, similar susceptibility to fenitrothion was assessed in both the field and reference populations (LD50 : 21.65 ng/i and 21.38 ng/i, respectively). The effectiveness of the formulated insecticides varied according to the surfaces to which they were applied. The application of fenitrothion formulations to glass or brick resulted in mortality of 90-100%. The application of fenitrothion formulations to wood or mud caused mortality in the range of 6.7-56.7%. Resistant insects presented low mortality when exposed to the deltamethrin formulation and high mortality when exposed to fenitrothion formulations. Moreover, the insecticides demonstrated residual activity only when applied to glass. The present work demonstrates that fenitrothion is an alternative to pyrethroids for the management of deltamethrin-resistant insects in La Esperanza. However, this effectiveness is not sustained over time.

Scientific evidence of three different insecticide-resistant profiles in Triatoma infestans (Hemiptera: Reduviidae) populations from Argentina and Bolivia.

Triatoma infestans (Klug, 1834) (Hemiptera, Reduviidae) is the main vector of Chagas disease in the southern cone South America. Chemical control to the vectors appears to be the best option to reduce the incidence of the disease. However, since 2002, high resistance to insecticides that correlated with field control failures was detected in T. infestans from Argentina and Bolivia. In this paper, we analyzed three T. infestans populations whose pyrethroid-resistance had been recently detected, and we defined at least three resistant profiles according to the toxicological and biochemical characteristics of the studied resistant populations. The resistance profiles were identified as Ti-R1, Ti-R2, and Ti-R3, corresponding to the Argentinean Acambuco, and the Bolivians Entre Ríos and Mataral. Ti-R1 exhibited nymphs and eggs with medium resistance level to deltamethrin (RR = 32.5 and 28.6; respectively). Pyrethroid-esterases played a relevant role in deltamethrin resistance. Ti-R2 exhibited nymphs with high resistance to deltamethrin (RR = 173.8) and low resistance to fipronil (RR = 12.4). Pyrethroid-esterases were involved in resistance. Moreover, eggs showed medium resistance level to deltamethrin (RR = 39.1). Ti-R3 had nymphs with low resistance to deltamethrin (RR = 17.4), and medium resistance to fipronil (RR = 66.8). Pyrethroid-esterases showed increased activity, and eggs possessed low resistance to deltamethrin (RR = 8.4). The characterization of the resistance to pyrethroid in these T. infestans populations from Argentina and Bolivia do not permit the generalization of three forms of resistance profile. So far as we appear to know, the forms of mechanisms and their frequencies reported here are selected independently, so additional sites might well show additional combinations of resistance mechanisms and their frequencies.

Insecticidal activity of individual and mixed monoterpenoids of geranium essential oil against Pediculus humanus capitis (Phthiraptera: Pediculidae).

The major components of geranium (Geranium maculatum L.) oil and their mixtures were tested against female Pediculus humanus capitis De Geer (Phthiraptera: Pediculidae). Chemical analysis by gas chromatography coupled to mass spectrometry revealed four major constituents: citronellol (38%), geraniol (16%), citronellyl formate (10.4%), and linalool (6.45%) (concentration expressed as percentage of total). Topical application demonstrated that the most potent component was citronellol and geraniol, with LD50 values 9.7 and 12.7 microg/insect, respectively. Linalool and Citronellyl formate were less toxic with LD50 values 24.7 and 38.5 microg/insect, respectively. Toxicity of these four major constituents in the same proportion as the natural oil, was greater than whole oil and each individual component. Removal of any four constituents produced a decreased in effectiveness. The absence of citronellol caused the greatest decrease in toxicity (DL50 from 2.2 to 10.9 microg/insect), leading us to conclude that this constituent is the major contributor to oil toxicity. The knowledge of the role of each constituent in the toxicity of the whole oil gives the possibility to create artificial blends of different constituents for the development of more effective control agents.

Cholinesterase activity during embryonic development in the blood-feeding bug Triatoma patagonica.

Triatoma patagonica Del Ponte (Hemiptera: Reduviidae), a vector of Chagas' disease, is widely distributed in Argentina and is found in sylvatic and peridomiciliary ecotopes, as well as occasionally in human dwellings after the chemical control of Triatoma infestans. Anti-cholinesteratic products can be applied in peridomiciliary areas and thus knowledge of cholinesterase activity during embryonic development in this species might contribute further information relevant to effective chemical control. Cholinesterase activity was characterized by reactions to eserine 10(-5) m, to increasing concentrations of substrate and to varying centrifugal speeds. Acetylcholinesterase activity was detected on day 4 and was significant from day 5. A reduction in cholinesterase activity towards acetylthiocholine (ATC) was observed on days 9 and 10 of development. Cholinesterase activity towards ATC and butyrylthiocholine (BTC) in homogenates of eggs was inhibited by eserine 10(-5) m. The shape of the curve indicating levels of inhibition at different concentrations of ATC was typical of acetylcholinesterase. Activity towards BTC did not appear to be inhibited by excess substrate, which parallels the behaviour of butyrylcholinesterases. Cholinesterase activity towards ATC was reduced in supernatant centrifuged at 15 000 g compared with supernatant centrifuged at 1100 g. The cholinesterase system that hydrolyzes mainly ATC seems to belong to the nervous system, as indicated by its behaviour towards the substrates assayed, its greater insolubility and the fact that it evolves parallel to the development of the nervous system. Knowledge of biochemical changes associated with the development and maturation of the nervous system during embryonic development would contribute to the better understanding of anti-cholinesteratic compounds with ovicidal action that might be used in control campaigns against vectors of Chagas' disease.

New findings of insecticide resistance in Triatoma infestans (Heteroptera: Reduviidae) from the Gran Chaco.

Chagas disease is a major health concern in Latin America, and Triatoma infestans (Klug, 1834) is responsible for the majority of cases of Chagas disease in the continent. After the discovery of deltamethrin highly resistant populations in the neighboring areas to Yacuiba (Bolivia) and Salvador Mazza (Argentina), we studied T. infestans populations north and southward, with the aim of describing the range of the resistant area. In addition, tests were conducted to describe the susceptibility to fipronil in deltamethrin-resistant populations. Tarija populations were highly resistant to deltamethrin, showing that the resistant area is greater than previously reported. Argentinean and Paraguayan populations were susceptible or presented moderate to low levels of deltamethrin resistance. Resistance to fipronil was found in Bolivian populations, although this insecticide was effective against Argentinean populations. This study shows that the Argentinean and Paraguayan populations are currently under successful control with deltamethrin. However, continuous resistance monitoring is necessary. We found that fipronil is a viable option for Argentina, whereas the resistance found in Bolivia implies that new formulations are needed to control T. infestans in Bolivia. Further research is required to find new alternatives of control in those areas that are currently suffering from high infestation rates.

Pediculus humanus capitis (head lice) and Pediculus humanus humanus (body lice): response to laboratory temperature and humidity and susceptibility to monoterpenoids.

Human pediculosis is produced by Pediculus humanus humanus (Linnaeus 1758) and Pediculus humanus capitis (De Geer 1767). Laboratory-reared body lice, susceptible to insecticides, were used as reference in toxicological studies on head lice. In this work, we evaluated the survival of both subspecies at different temperatures and relative humidities and we propose the optimal conditions for comparative bioassays. Moreover, we used these conditions to test the activity of three monoterpenoids against both lice. The results showed differential response to changes in temperature and humidity between both organisms. The survival of body lice ranged between 83% and 100% and was not affected for the tested conditions. The survival of head lice depended on temperature, humidity, and exposure time. The optimal conditions for head lice were 18 masculineC and 97% relative humidity at 18 h of exposition. The insecticidal activity of three monoterpenoids (pulegone, linalool, and 1,8-cineole), evaluated according the selected conditions by topical application, showed no significant differences between males of body and head lice. To conclude, as head lice required more special laboratory conditions than body lice, the optimal head lice conditions should be used in both organisms in comparative bioassays. Body louse is an appropriate organism for testing products against of head louse.

Anticholinesterase and pediculicidal activities of monoterpenoids.

The repetitive and inadequate application of pediculicidal products frequently results in the development of resistance to these compounds. Essential oils are a promising alternative to synthetic insecticides, although their mode of action remains to be explored. It has been proposed that one possible target of the essential oils is the inhibition of acetylcholinesterase (AChE). The role of monoterpenoids as possible AChE inhibitors and their relationship with the toxicity was investigated both in vitro and in vivo. Inhibition of electric eel AChE activity showed that the most effective inhibitor was 1,8-cineole with IC(50) 6 x 10(-3) M. The inhibition of AChE activity of head louse homogenate by 1,8-cineole showed IC(50) 7.7 x 10(-2) M. The intoxication symptoms of head lice exposed to vapors of 1,8-cineole was recorded before the in vivo head louse AChE inhibition assay. No correlation was found between neurotoxic symptoms and inhibition of AChE activity.

Ovicidal and adulticidal effects of monoterpenoids against permethrin-resistant human head lice, Pediculus humanus capitis.

The improper use of pediculicides containing permethrin has led to the development of resistance. Thus, new alternatives for control are needed. Plant-derived insecticides are attractive alternatives to common chemical insecticides because most of them are environmentally friendly and non-toxic to mammals. The toxic activity of 23 monoterpenoids belonging to several chemical classes was tested against the eggs of permethrin-resistant head lice, Pediculus humanus capitis De Geer (Phthiraptera: Pediculidae). Significant differences in ovicidal action were observed among the tested substances. The most effective chemicals were hydrocarbons and ethers, followed by ketones, alcohols, phenols and esters. A linear relationship between egg mortality and knockdown time (KT(50)) on adults by the tested components revealed that most of the components were effective on both egg and adult stages. The monoterpenoids described herein are good candidates as effective pediculicides.

Permethrin-resistant head lice (Anoplura: Pediculidae) in Argentina are susceptible to spinosad.

The insecticidal activity of spinosad was evaluated against susceptible and permethrin-resistant human lice. In a permethrin-susceptible strain of the body louse, Pediculus humanus humanus L. (Anoplura: Pediculidae), the toxicity of spinosad was similar to that established for permethrin, with an LD50 value of 1.2 ng/insect and 2.4 ng/insect, respectively. Topical application of spinosad to populations of permethrin-resistant head louse, Pediculus humanus capitis De Geer (Anoplura: Pediculidae), showed that susceptibility to spinosad was independent of resistance to permethrin. The effectiveness of spinosad against human lice and the low mammalian toxicity reported in the literature suggest that spinosad could be useful for the management of permethrin-resistant human lice.

Increased monooxygenase activity associated with resistance to permethrin in Pediculus humanus capitis (Anoplura: Pediculidae) from Argentina.

We studied the profile of permethrin resistance in populations of head lice infesting children 6-12 yr old in schools and their homes in and around Buenos Aires, Argentina. Five permethrin-resistant populations with different levels of resistance were collected: Hogar Loyola (HL), Republica de Turquia (RT), Hogar Mitre (HM), Guardia de Honor (GH), and Ricardo Guiraldes (RG). One susceptible population, Bandera Argentina (BA), also was collected. Their level of resistance was evaluated, and results showed resistance ratios of 13 for HL, 16 for RT, 22 for HM, 61 for GH, and 69 for RG. To elucidate the possible involvement of the cytochrome P450 monooxygenase system in conferring permethrin resistance, ethoxycoumarin-O-deethylase (ECOD) activity was measured in abdomens of individual third instars and adults by using a fluorometric assay. The ECOD activity was lower in the susceptible BA population (4.7 ng per louse) than in the resistant ones (13.7 ng per louse for RG, 12.3 ng per louse for GH, 8.6 ng per louse for RT, and 8.2 ng per louse for HL). ECOD activity was significantly correlated with the level of resistance in the field populations (r = 0.97, P = 0.0009), suggesting a role for cytochrome monooxygenase P450 system in permethrin resistance by head louse, Pediculus humanus capitis De Geer.

Prevalence and levels of permethrin resistance in Pediculus humanus capitis De Geer (Anoplura: Pediculidae) from Buenos Aires, Argentina.

Permethrin has been used extensively for control of Pediculus humanus capitis De Geer (Anoplura: Pediculidae) in Argentina since 1990, resulting in the development of resistance to this and other pyrethroids. This resistance was first detected in some field populations in 1997. A survey for resistance in Buenos Aires in 2001 revealed significant resistance levels in lice on children at 24 of 26 (92.3%) schools. When compared with a previously unexposed reference population, resistance ratios (RRs) obtained by exposing the insects to filter papers impregnated with permethrin ranged from 2 to 60 in 10 (39%) of the schools. RRs in the remaining 14 (61%) populations were too great to not be measured with the filter paper method (RR > 88.7). As an alternative, we used topical applications of 0.1 microl of acetone solution of permethrin on the dorsal abdomen of adults and third instars. This topical method, which has not been previously reported for head lice, was capable of quantifying higher levels of resistance. Highly resistant populations had RRs from 162.5 to 655.2. When applied to populations with low and intermediate levels of resistance, results from the filter paper and topical application methods were highly correlated, and RRs from topical application were higher than those from the filter paper method. Results from the combination of the two methods indicated that head lice among Buenos Aries school children are highly resistant to permethrin, and the resistance is widespread.