Introduction ORIGINAL PAPER

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1 Parasitol Res (2017) 116: DOI /s ORIGINAL PAPER Randomized, investigator-blinded, controlled clinical study with lice shampoo (Licener ) versus dimethicone (Jacutin Pedicul Fluid) for the treatment of infestations with head lice Margit Semmler 1 & Fathy Abdel-Ghaffar 1 & Falk Gestmann 2 & Mohammed Abdel-Aty 3 & Ibrahim Rizk 4 & Saleh Al-Quraishy 5 & Walter Lehmacher 6 & Norman-Philipp Hoff 7 Received: 11 March 2017 /Accepted: 24 April 2017 /Published online: 9 May 2017 # Springer-Verlag Berlin Heidelberg 2017 Abstract The present clinical trial was conducted to obtain additional data for the safety and efficacy of a head lice shampoo that is free of silicone compared with an anti-head lice product containing dimethicone. Both products act by a physical mode of action. This randomized, investigator-blinded, controlled clinical study was conducted between July and November 2016 in households of two villages (Abou Rawash and Shandalat) in Egypt. Children older than 2 years with an active head lice infestation were treated with either a shampoo-based head lice treatment containing neem extract (Licener ) or dimethicone (Jacutin Pedicul Fluid) on day 1 and additionally on day 9. Assessment for living lice by combing was conducted before and 1 2 h after treatment and on days 5 and 13. The main objective was to demonstrate a cure rate of the test product of at least 85% after a single application (day 5 and 9). Secondary objectives were to scrutinize patient safety and satisfaction as well as cure rates on day 13 after two treatments and the evaluation of ovicidal and licicidal * Falk Gestmann Gestmann@alphabiocare.de Department of Zoology, Faculty of Science, Cairo University, Giza, Egypt Alpha-Biocare GmbH, Neuss, Germany El-Santa Medical Province, Ministry of Public Health, Cairo, Egypt El-Fath Clinical Hospital, Kafr El-Sheikh, Egypt Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia Institute of Medical Statistics, Informatics and Epidemiology, University of Cologne, Cologne, Germany Clinic for Dermatology, Heinrich-Heine-University, Düsseldorf, Germany efficacies of the products. Sixty-one children in the test-group (Licener ) and 58 children in the reference group (Jacutin Pedicul Fluid) were included in this study. The test product and the reference product were very well tolerated. Both products exceeded the objective of cure rates of over 85% after single treatment (test group 60/60 = 100%; 95% CI = %; reference group 54/57 = 94.74%; 95% CI = %; p = 0.112; CI by Clopper-Pearson) and after two treatments (test group 58/58 = 100%; 95% CI = %; reference group 52/54 = 96.30%; 95% CI = %; p = 0.230) with higher cure rates and non-inferiority for the test product. The combined success rate shows significant superiority of the test product against the reference product (test group 58/ 58 = 100%; 95% CI = %; reference group 49/ 54 = 90.7%; 95% CI = %; p = 0.024). The test product showed higher ovicidal efficacy than the reference product. Thus, the present study demonstrates that a single treatment with a head lice product like Licener can be sufficient to eliminate a head lice infestation. Keywords Head lice. Treatment. Shampoo. Pediculus humanus capitis. Clinical trial. Clinical study. Randomized. Blinded. Controlled Introduction The infestation with head lice (Pediculus humanus capitis) is one of the most common parasitic infections in children worldwide (Mehlhorn 2012). In contrast to body lice (Pediculus humanus corporis) which can act as vectors for agents of serious diseases such as Rickettsia prowazekii, Borrelia recurrentis and Bartonella quintana (Badiaga and Brouqui 2012; Raoult et al. 2006), head lice are considered mostly harmless. However,

2 1864 Parasitol Res (2017) 116: constituents from the saliva of the head lice can cause intense local reactions to the skin such as papules and redness of the scalp (Mehlhorn 2012; Mumcuoglu et al. 1991). For the treatment of pediculosis, a variety of products is available. Their effect is given either physically (medical devices) or as an insecticidal effect (medical products). Lice have developed resistances against many insecticides that had been or are still used for the treatment of head lice (Downs et al. 1999; Gallardoetal.2012; Mehlhorn 2012). In addition, some products contain mineral or synthetic oils which make them highly flammable when mixed with dry hair (Abdel-Ghaffar et al. 2010; Dorge et al. 2017; Gallardo et al. 2012). Moreover, several products need very long application times (up to 8 h) and at least a second application after 1 week or even a third application after 2 weeks is necessary because many formulations do not kill the eggs. For the patient, it is therefore advantageous if a remedy for head lice is applied only once and that the formulation does not present high health hazards. This study aims for the assessment of the efficacy and safety of a head lice treatment in a shampoo basis containing neem extract compared to a treatment that consists of dimethicone under close to real conditions. Both products act by physical means (suffocation) on hatched lice and eggs and are applied forashortperiodof10min. Material and methods Participants The participants of this study were recruited from households in two villages (Abou Rawash and Shandalat, Egypt) by scientists of the University of Cairo. Children of both sexes with active head lice infestations (minimum 5 living lice plus 5 apparently intact eggs) and a minimum age of 2 years were eligible to participate. The patients and the mothers were comprehensively informed about the study and had to give oral and written consent to participate. In addition, the patients and the mothers had to agree to not to apply any other product against head lice and not to use any shampoo that contains dimethicone (polydimethylsiloxane) and neem extract and not to bleach and colour the hair or apply permanent wave products during the study period. Patients matching the following criteria were excluded: Large injuries or disease, especially of the scalp, which would make another preferable medical care necessary (e.g. impetigo, eczema), chronic scalp disorders (e.g. psoriasis, chronic dermatosis of unknown origin) and secondary infections of the scalp Hypersensitivities or allergies to one or more ingredients of the test product or the reference products Skin allergies, multiple drug allergies or multiple allergies to cosmetic products Pregnancy or lactation period Participation in other clinical trials in the last 30 days Treatments with antibiotics (e.g. co-trimoxazole, trimethoprim, tetracycline) or anthelmintics (e.g. ivermectin, levamisole, albendazole) in the last 30 days or imminent and other topical or systemic drug treatments, which in the investigators view would bias the study results Contraindication as per the use of information of the test or reference products Employees of the sponsor (Alpha-Biocare GmbH, Neuss, Germany), other organization that is involved in the study and members of the study team or are directly connected to the study and/or their close family (spouse, parent, child, biological or adopted siblings) Data collection The hair length of the patients was recorded with a 5-point scale: very short (up to 2 cm), short (up to 7 cm), chin length, shoulder length and very long. The appearance of the hair was recorded as smooth/not curly, slightly curled and heavily curled. Additionally, basic biometric data, the general condition, the treatment history with pediculicides and concomitant medications were recorded. The patients and mothers were asked to fill in a questionnaire regarding their satisfaction with the product. Test and reference products The test product (Licener ; Pronova Laboratories B.V., Muiden, the Netherlands) consists of a debitterized neem extract (Azardirachta indica) in a shampoo basis that is applied to dry hair and washed out with lukewarm tab water after a 10- min application. In accordance with the recommendation by the manufacturer, 1 bottle (100 ml) per person and treatment was applied. The reference product (Jacutin Pedicul Fluid; Almirall Hermal GmbH, Reinbek, Germany) consists of a moderately high viscous dimethicone that is applied to dry hair but is washed out after a 10-min application, then combing with a lice comb and washing out with warm water and the patients regular shampoo. As recommended by the manufacturer, 50 ml was used for short hair, 75 ml for shoulder long hair and 100 ml for more than shoulder long hair. According to the manufacturers, both products act by physical means (suffocation) on hatched lice and enter the eggs. Initial intensity of infestation Assessment of infestation with head lice was done by visual inspection by the investigator. If necessary, the dry hair was detangled by help of a coarse comb (tine spacing >3 mm). The

3 Parasitol Res (2017) 116: hair of the head was divided into four parts and initially examined with a magnifying glass for the presence of nits/eggs that had a maximum distance to the scalp of about 1 cm. The hair of all four parts of the head was then combed with a lice comb starting from the scalp. Each quadrant was combed six times. Lice found by combing were left in the hair to not bias the infestation. The intensity of infestation was assessed as follows: Mild at least 5 and up to 10 living lice and at least 5 eggs Intermediate more than 10 and up to 30 living lice and at least 5 eggs Severe over 30 living lice and at least 5 eggs Follow-up assessment The hair was divided into four parts, and each quadrant was combed less frequent (3 times) with a lice comb to keep the efficacy/bias by combing as low as possible. Obtained head lice were stripped into a white plastic tray and examined for viability. The viability of the lice was primary determined by their mobility. Motionless lice were additionally analysed for peristaltic movements of the intestine. In the absence of both criteria, the louse was assessed as not viable. Motionless lice with existing peristalsis of the intestine were further examined every hour for 3 h and were counted as not vital in the absence of both indicators or immobility after tactile stimulation. In addition, lice were categorized by their stage of development (juvenile stages I to III, adult) and sex. Follow-up assessment was carried out after treatments and on day 5 between the two treatments. Vital lice found directly after treatment were assessed as failure of treatment. Treatment and visit schedule Infested patients were treated on day 1 and on day 9 (±1 day) with the test product or the reference product by the instructed mothers. One to 2 h after application of the products, the patients were examined for the presence of living head lice by a blinded investigator. On days 5 and 9, further follow-up examinations of the scalp and the hair of the patient were carried out to determine whether any adult or juvenile head lice were present. After the second treatment, the final examination took place on day 13 (±2 days). The treatment schedule is based on the calculations of Lebwohl et al. (2007). Assessment of re-infestations and ovicidal failure The algorithm for the assessment of re-infestations was adapted from the study of Burgess et al. (2007). Briefly, a re-infestation on day 5, day 9 or day 13 was assumed, when up to two vital stage III juveniles or adults were found but the patient had been free of living lice at the examinations on previous days. The quantity 2 is an interpretation of Boccasional^ and must not be exceeded (Burgess et al. 2012). The occurrence of juvenile stages I and II during assessments on day 5, day 9 and day 13 was taken as evidence that not all eggs were killed and was assessed as ovicidal failure. In addition, no further juveniles or adult lice should be found on day 13, neither adults nor stage III juveniles at day 9. Endpoints of the study The primary endpoint of the study was the success rate (absence of living lice) after a single treatment assessed at day 9 immediately prior a second treatment. Secondary endpoints were the success rate after two treatments assessed at day 13 and the evaluation of the patient safety and satisfaction. Sample size As shown in previous studies (Abdel-Ghaffar et al. 2016; Ihde et al. 2015), success rates for both groups of treatments (efficacies) were expected to be very high; at least 50 patients were planned for each study arm. Because of possible dropouts, the total number of patients to be recruited was set to 120. Randomisation and blinding According to the manufacturer s instructions, all other infested persons of the same household were treated at the same time. To exclude the likelihood of confusion of products in presence of both products in the same household, all infested household members were treated with the same product. Therefore, the randomization was performed per household/family as cluster randomization. Due to the different application modes of the two tested products (shampooing and washing versus applying, shampooing and washing), it was not possible to blind the patient; therefore, the investigator had to be blinded. The handing out and supervised application of the products was done by one investigator (team), and the control for the presence of living lice 1 2 h after treatment was done by another investigator (team) who had no knowledge which product had been applied. It was proceeded in the same manner during all other assessments on day 5, day 9 and day 13. The investigators received sealed emergency envelopes to unblind themselves if necessary. Statistical analyses Primarily, the success rate of the test product was evaluated with a 95% confidence interval (95%-CI) by Clopper-Pearson. Then, the success rate of the reference product was evaluated by a 95%- CI by Clopper-Pearson. Then, a 95%-CI was calculated for the difference between the success rates of the test and reference

4 1866 Parasitol Res (2017) 116: Fig. 1 CONSORT diagram. ITT intention to treat, PP per protocol products. An efficacy of the investigational medical device is implied when the lower limit of the Clopper-Pearson 95%-CI is above 85% and the lower limit of the 95%-CI of the difference in success rates (test minus reference product) is above the noninferiority margin of 15%. Additionally, the success rates of the two arms of the study were compared with Fisher s exact test. All other variables were analysed descriptively with standard methods. The data of the patient satisfaction assessment were analysed by Wilcoxon test. As usual, in non-inferiority studies, the primary analysis is based on the per-protocol-set. In the additional intention-to-treat analysis, the endpoints for dropouts were assessed by the last-observation-carried-forward method. reference group) were included (Fig. 1). Five children did not have an active head lice infestation and thus could not be included. In both study arms, 44 girls were included plus 17 boys in the test group and 14 boys in the reference group. The median age in the test group was 7 and 6 years in the reference group (total 7 years). The majority of patients had shoulder long (30.3%) or longer hair (42.2%) mostly of the curly type. The distribution of the hair length and the curliness is given in Table 1. The intensity of infestations was comparable between both study arms. The distribution into the different intensity categories is given in Table 2. All other baseline data of both study arms were also statistically comparable. Results Baseline data One hundred twenty-four children from 38 families were screened from which 119 (61 in the test group and 58 in the Success rates after a single treatment An overview of the course of the study is given in Fig. 1.After the first treatment (day 1), vital lice (stage I juveniles) were isolated only from one patient in the reference group (1.7%) and none in the test group. This finding of a single louse was assessed as failure of treatment. Some lice that were 1 2 h

5 Parasitol Res (2017) 116: Table 1 Baseline data Group Licener Jacutin N % N % Number of patients Age [years] Sex Male Female Hair length Very short Short Chin length Shoulder length Very long Curliness Smooth/not curly Slightly curled Heavily curled after treatment combed out from patients in the test group showed peristalsis of the intestines but were not motile as described in another study (Abdel-Ghaffar et al. 2016). This was also noticeable in some lice from patients in the reference group. The movements of the intestines, however, stopped about 1 h later, and the lice obtained from the test group were dead and appeared considerably shrunk compared to the lice obtained from the reference group. At the control visit on day 5, all 61 patients in the test group remained free of lice, while in the reference group, two of 58 (3.4%) patients showed vital lice. Because one patient in each study arm refused the combing with a lice comb, they were dropped out. With one reinfestation in each study arm for the primary endpoint (success rate after a single treatment assessed at day 9), 60 patients in the test group and 57 patients in the reference group were assessable. Success rates and corresponding confidence intervals are given in Table 3: the success rate in the test group was Table 2 Intensity of infestation Intensity of infestation Group 60/60 = 100%. With a confidence probability of 95%, it is shown that the test product has a success rate of at least 94.0%. The success rate in the reference group was 54/ 57 = 94.74%. With a confidence probability of 95%, it was shown that the reference product has a success rate of at least 85.4%. The exact Fisher test showed a two-sided p = The observed difference between the success rates of the test and reference products were d = 5.26% in favour of the test product. With a confidence probability of 95%, the test product is maximally 1.68% worse and maximally 14.37% better than the reference product. The test product was therefore found to be non-inferior to the reference product. Success rates after two treatments Three patients in the reference group refused a second treatment and were dropped out. Immediately after the second treatment, all patients (60/60) in the test group and all patients in the reference group (54/54) were free of vital lice. With 2 more dropouts in the test group and 1 re-infestation in each study group for the secondary endpoint (success rate after 2 treatments assessed at day 13), 58 and 54 patients were assessable. The success rates and corresponding confidence intervals are given in Table 3. The success rate after two treatments in the test group was 58/58 = 100%. With a confidence probability of 95%, it was shown that the test product has a success rate of at least 93.84%. The success rate in the reference group was 52/54 = 96.30%. With a confidence probability of 95%, it was shown that the reference product has a success rate of at least 87.25%. The exact Fisher test showed a two-sided p = The observed difference between the success rates of the test and reference products is d = 3.70% in favour of the test product. With a probability of 95%, the test product is maximally 4.62% worse and maximally 13.84% better than the reference product. The non-inferiority of the test product versus the reference product was shown. All living lice found in the reference group on day 5, day 9 and day 13 were, except for two re-infestations, assessed as ovicidal failures of treatment per the assessment algorithm. For the intention-to-treat analysis, we have 61/61 (100%) and 55/58 (94.8%) successes for the primary endpoint and 61/ 61 (100%) and 56/58 (96.6%) successes for the secondary endpoint; these results are very similar with the per-protocol analysis. Licener Jacutin Combined success after single and dual treatment N % N % Mild Intermediate Severe Total Fifty-eight and 54 patients could be assessed at the beginning of day 9 and at day 13. In the test group, 58 patients had no lice at both days (Table 3). In the reference group, 2/54 patients had infestations at day 9 and 3/54 patients had infestations at day 13; thus, 5 failed to have success at day 9 (prior second treatment) or day 13, i.e. 49/54 had success at prior the second

6 1868 Parasitol Res (2017) 116: Table 3 Success rates after single and dual treatment Group Licener Jacutin Assessment Success/total % (95%-CI) Success/total % (95%-CI) P value 95%-CI for difference Day 9 60/ ( %) 54/ ( %) % Day 13 58/ ( %) 52/ ( %) % Combined 58/ ( %) 49/ ( %) % treatment at day 9 and day 13. The success rate in the test group was 58/58 = 100%. With a confidence probability of 95%, it was shown that the test product has a success rate of at least 93.8%. The success rate in the reference group was 49/ 54 = 90.7%. With a confidence probability of 95%, it was shown that the reference product has a success rate of at least 79.7%. The Fisher exact test shows a significant superiority of the test product versus the reference product with a two-sided p = The observed difference between the success rates of the test and reference products is d = 9.26% in favour of the test product. With a probability of 95%, the test product is minimally 1.13% better and maximally 13.84% better than the reference product. Therefore, the non-inferiority of the test product versus the reference product was shown. The primary and secondary efficacy endpoints were additionally analysed stratified by sex. There were no relevant differences between girls and boys (data not shown). Analyses of therapy, adverse events, compliance and sensitivity No adverse or serious adverse events occurred during this trial. The 7 dropouts (3 in the test group and 4 in the reference group) were not associated with a failure of treatment. In most cases (n = 5), the rigorous combing was felt unpleasant and there was no will for further participation. In two cases, the mother refused further participation, since both of her children had become lice free. Since the dropout rates were small and balanced between the groups, all dropouts gave no indication for a missing efficacy. There is no evidence for a bias and no need for sensitivity analyses. The omission of the dropouts from the statistical assessments should induce an underestimation of the efficacy rates, or for the test group induce only a slightly higher precision. Patient satisfaction The results of the questionnaires that were filled in after the end of the therapy are given in Table 4. The overall response to most of the questions was that the patients/mothers preferred the test product relative to the reference product. However, the smell of the reference product was better rated than the smell of the test product. Discussion This study was done to obtain additional data on efficacy and safety of a head lice treatment on shampoo basis containing Table 4 Result of the patient satisfaction questionnaire Licener (N =58) Jacutin(N = 54) Wilcoxon test Mean Median sd Mean Median sd P value Are you overall satisfied with this anti-lice product? Would you say the anti-lice product: Is easy to spread? Smells good? Allows quick treatment? Is easy to wash out? Works well? Is well tolerated? Would you recommend this anti-lice product? After the end of the therapy, patients/mothers were asked to fill out a questionnaire with Likert-scaled values from 1 = I totally agree to 6 = I totally disagree N number, sd standard deviation

7 Parasitol Res (2017) 116: neem extract compared to one based on dimethicone and both being applied for 10 min under close to real conditions in households. These conditions included for both study arms combing of the hair with a lice comb as specified by both manufacturers which contributes to efficacy as shown by Hill et al. (2005) but is clearly not efficient to eliminate an infestation with head lice (Gallardo et al. 2013; Kurt et al. 2015) and in one study has led to a mortality of hatched lice of up to 11% (Goates et al. 2006). The actual risk of biasing the efficacies on hatched lice by combing is negligible since both products showed 100% efficacies in vitro (Abdel- Ghaffar et al. 2016; Abdel-Ghaffar et al. 2012; Abdel- Ghaffar and Semmler 2007; Abdel-Ghaffaretal.2010). While in a previous study (Abdel-Ghaffar et al. 2016) the feasibility under close to real conditions has been proven, the present study highlights the high efficacy and safety of a special shampoo for the treatment against head lice (Licener ) compared to an over-the-counter product containing dimethicone (Jacutin Pedicul Fluid). The analysis of the cure rates at different time points and after single and two treatments did clearly show that already a single treatment with Licener efficiently overcomes an active head lice infestation regardless of the intensity of infestation. The superiority of the test product is mainly based on the fact that it kills all lice stages including unhatched eggs when applied undiluted in accordance with the use instruction as shown in other studies (Abdel-Ghaffar et al. 2012; Abdel-Ghaffar et al. 2010). Some ovicidal efficacy may be contributed by combing with a lice comb that is mandatory for most head lice treatments and is demanded by many public institutions; an ovicidal mode of action is apparently not sufficiently inherent in the reference product. This was the main reason for the occurrence of vital lice on different occasions even after two treatments, despite that combing was performed equally in both study arms. A lack of sufficient ovicidal efficacy was recently shown in a controlled clinical trial (Ihde et al. 2015) for another pediculicide consisting of a single-stage dimethicone that is also applied for only 10 min which makes a second or even more treatments necessary. The same was shown for the short-term application of a high concentration dimethicone formulation which additionally contains the ovicidal substance alpha-terpineol (Yang et al. 2009) in vitro which also lacks a sufficient ovicidal efficacy (Strycharz et al. 2012). Other preparations containing 92% dimethicone showed only good ovicidal efficacy in an in vivo trial after 8 h of applications on the patient s head (Heukelbach et al. 2008). Although no adverse events occurred, it is noticeable from the patient questionnaire and during the trial that the dimethicone product is very sticky and very difficult to wash out, which makes it inconvenient for the patient. Besides the better-rated smell of the odourless dimethicone product, the neem-based shampoo was clearly better rated with respect to efficacy, convenient use and overall satisfaction, because no additional washing step is necessary. Re-infestations might occur at any occasion after a treatment as it was shown in the present and in previous trials with neem extract-based shampoo and dimethicone as a fluid (Abdel-Ghaffar et al. 2016; Ihde et al. 2015) but also tocopheryl acetate spray, permethrin cremes and mineral oilbased shampoo (Burgess et al. 2013; Wolfetal.2016). Without inherent repellency of existing head lice treatments andaslongasheadlicerepellentsfailtoshowefficacyinvitro (Semmler et al. 2012) and in vivo (Burgess et al. 2014), only educational efforts might lower the risk of re-infestations after a successful treatment (Moshki et al. 2017). For the present study, the visit schedule with examinations on days 5 and 13 after treatments on days 1 and 9 prove to be effective to detect interim re-infestations while the estimated number remains unclear and is variable for every trial. While some guidelines for clinical trials with head lice treatments have been proposed (Barker et al. 2012), these guidelines still appear to lack generalizability and international applicability so that ultimately every study design must relate to all previous studies and local regulations. Other authors (Do-Pham et al. 2014) specified more precise criteria based on an international expert survey that were considered in the design of the present study. Conclusions The present study demonstrates that a single treatment with a head lice treatment shampoo (Licener ) for only 10 min can be sufficient to overcome a head lice infestation. Moreover, this treatment is well tolerated and because of its shampoo basis, it is easy to wash out making it convenient for the patient. Acknowledgements We thank Hennig Arzneimittel GmbH & Co. KG, Flörsheim, Germany, for providing the sufficient bottles of Licener to carry out the test and Alpha-Biocare GmbH, Neuss, Germany, for sponsoring this study. Compliance with ethical standards Conflict of interest F. Gestmann is an employee of the sponsor (Alpha- Biocare GmbH, Neuss, Germany). M. Semmler, F. Abdel-Ghaffar, M. Abdel-Aty, I. Rizk, S. Al-Quraishy and N.P. Hoff took part in the study design and implementation of at least one previous study with the test product. The other authors have declared that they have no conflict of interest regarding the content of this article. Ethics The study was conducted under local institutional review and in accordance with the Declaration of Helsinki (last revision in October, 2013 by the 64th General Assembly in Fortaleza, Brazil) and the European standard DIN EN ISO 14155: (Clinical investigation of medical devices for human subjects Good clinical practice) and the GCP Regulation. This is a study of CE-marked medical devices, which were not modified and not used outside their CE mark intended purpose and does therefore not require ethic approval which is also applicable for studies in Egypt.

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