This was an assessor-blind, randomised, parallel group, comparative study. The study population was elementary school-aged children (aged 4 yrs to 12 yrs) in Queensland, Australia, with live eggs of head lice in their hair. After written, informed consent had been provided, subjects were screened by visual inspection for the presence of putatively live eggs of head lice in their hair. The hair was divided into six sections. Each section of hair was examined for the presence of putatively live eggs by an expert technician - a magnifying glass was not needed. Eggs were classified as putatively alive if they were attached to a hair shaft within about 1 cm of the scalp and if they were not black (dead) nor white to translucent (hatched). Putatively live eggs were counted with a hand-operated click-counter. Subjects with at least 20 putatively live eggs in their hair were randomised to one of the three treatment groups. Subjects with scalp disease, or a history of allergies, and those who were treated with a pediculicide, or hair dye or bleach in the four weeks prior to the trial, were excluded from the trial.
Criterion for a live egg, a dead egg and hatched egg
Each egg was examined individually, within 2 hrs of collection of being removed from the head, with a stereo light microscope of 16 power to check for eggs that looked like they were alive with the naked eye, but were in fact were dead or had hatched. None of the eggs that were classified as alive by their appearance and close proximity to the scalp (within 1 cm) were found to be either dead or had hatched when examined with a microscopy.
Live egg: the egg was less than about 1 cm from the scalp; and the operculum was closed; and the egg had a uniform ovoid shape; and the egg had a uniform density and appearance (may have had an "eye spot" but this depended on the age of the egg. (Refer to Sonnberg et al for some excellent colour pictures of eggs of P. capitis)).
Dead egg: the egg was misshapen, shrivelled, indented or irregular in shape; or the egg had a non-uniform density with parts of the egg clear whereas other parts of the egg were opaque.
Hatched egg: operculum was open and nymph was not in the egg.
Collection, transport and incubation of eggs
Hair shafts with eggs attached less than 1 cm from the scalp were cut with hairdresser's scissors. The hair shaft was then put in a plastic tube (Corning 50 mL Centrifuge Tubes; CentriStar™ Cap-Polypropylene-Sterile). All of the pre-treatment hair shafts from one subject were put into one plastic tube whereas the hair shafts collected post-treatment were all put into a separate tube. The tubes were sealed with screw caps, put in a polystyrene container to minimize temperature variation, and taken to the laboratory. Then the pre-treatment and post-treatment eggs were counted and placed in separate Petri dishes in an incubator at 29 to 30°C and 70% relative humidity. Eggs were put into the incubator within 4 hrs of collection. Eggs were held in the incubator for 14 days.
Randomisation and blinding
Eligible subjects were randomly assigned to be treated with one of the three pediculicides, by a computer generated code with blocked randomisation (groups of six). This trial was assessor-blind. The person applying the treatment could not be blinded because the three pediculicides were easily identifiable by their physical attributes and "feel"; however, the person (MA) who classified eggs as either alive, dead or hatched was blinded to the treatment. Analysts were blinded to the treatment group until after the analyses.
Treatments and criteria for evaluation of efficacy
Enrolled subjects were treated once with one of the three pediculicides, according to the manufacturers' instructions. The louse-combing procedure normally used in combination with the TTO/LO and the "suffocation" pediculicide would have confounded our study, so it was not done. After the treatment, at least l0 live eggs, but more eggs if more than 10 eggs were present, were taken from the hair of each enrolled subject, as for the pre-treatment eggs. The pre-treatment and post-treatment eggs were examined again with the microscope on Day 14 to determine whether the eggs had hatched, partly hatched or had not hatched at all. Partly hatched eggs were classified as unhatched eggs; in these cases the partly hatched nymph was always dead. The number of hatched (empty) and unhatched eggs was counted on Day 14. The proportion of eggs that had hatched after 14 days was compared in the pre-treatment and post-treatment groups of eggs. The primary outcome measure was percent (%) ovicidal efficacy for each treatment-group (1 - [Post-Treatment Hatching Rate/Pre-Treatment Hatching Rate] × 100%). Hatch-Rate = number of eggs that had hatched divided by the number of live eggs that had been collected. Characteristics of the subjects and their hair were recorded: hair type (whether curly, straight or wavy), hair colour (whether black, blonde, brown, red), hair length (cm), subject gender and school attended (subjects attended three different schools in western Brisbane). Grade at school was a surrogate for subject age. Subjects were from eight school grades: "preparation year" (ca. 5 years) and grades 1 (ca. 6 years), 2 (ca. 7 years), 3 (ca. 8 years), 4 (ca. 9 years), 5 (ca. 10 years), 6 (ca. 11 years), and 7 (ca. 12 years).
Dosage and dosage regimen
The dose and method of application was that recommended by the manufacturers: all three pediculicides were applied for 10 minutes. After the EO/LTTO and the TTO/LO were applied, the hair was covered with a plastic ("shower") cap as per the manufacturers instructions. The EO/LTTO was washed from the hair with regular shampoo whereas the TTO/LO and the "suffocation" pediculicide were washed from the hair with tap-water. Then the hair was dried with a towel.
Criteria for evaluation of safety (tolerance)
Safety was evaluated by the incidence and severity of Adverse Events (AEs), and the likelihood in the opinion of the Investigator (SCB), that those AEs were caused by the pediculicides. The scalps of subjects were examined after the treatments and subjects were asked "was the hair treatment okay?" to elicit responses. The incidence and severity of adverse events was compared among treatment groups. Fisher's Exact Test was used to compare the proportions of subjects who reported an AE among the three treatment groups.
In our statistical analyses, the egg was the investigative unit whereas whether the egg hatched or not was the outcome. We used a framework of Generalised Estimating Equations (GEE) so that we could take into account the: (i) different numbers of eggs collected, pre-treatment (10 to 22 eggs) and post-treatment (10 to 41 eggs), from the 92 subjects; (ii) the three hair types; (iii) the four hair colours (iv) hair length (in cm); (v) the eight different age classes; (vi) gender; and (vii) the three different schools attended (populations). Logistic regression models were fitted to the data. For the comparison of the eucalyptus oil and lemon tea tree oil pediculicide with the "suffocation" pediculicide the formula was Logit(Y) = 1.8651 + (Treatment * 0.1749) + (Time * -2.8370) + (Interaction * 2.5796) whereas for the comparison of the eucalyptus oil and lemon tea tree oil pediculicide with the melaleuca oil and lavender oil pediculicide the formula was Logit(Y) = 2.1182 + (Treatment * -0.0782) + (Time * -2.1334) + (Interaction * 1.8761). The "fixed effects" were the pediculicide, whether the egg was collected pre-treatment or post-treatment, and the interaction of these two "fixed effects", whereas the "random effect" was the subject from whom the eggs were collected. Since the pre-treatment and post-treatment eggs were collected from the same individual subject, the model was a model of repeated measures.
This trial was conducted in compliance with the World Medical Association Declaration of Helsinki; the requirements of the National Statement on Ethical Conduct in Research Involving Humans and ICH E6 Guidance for the Industry; Good Clinical Practice: Consolidated Guidance; the National Privacy Principles and relevant State/territory laws. The trial activities were approved by the Medical Research Ethics Committee of the University of Queensland and all parents/guardians provided written informed consent.