Antiparasitic Activity of Tea Tree Oil (TTO) and Its Components against Medically Important Ectoparasites: A Systematic Review

Ectoparasites are pathogens that can infect the skin and cause immense pain, discomfort, and disease. They are typically managed with insecticides. However, the fast-emerging antimicrobial resistance and the slow rate of development of new bio-actives combined with environmental and health concerns over the continued use of neurotoxic insecticides warrant newer and alternative methods of control. Tea tree oil (TTO), as an alternative agent, has shown remarkable promise against ectoparasites in recent studies. To our knowledge, this is the first systematic review to assess preclinical and clinical studies exploring the antiparasitic activity of TTO and its components against clinically significant ectoparasites, such as Demodex mites, scabies mites, house dust mites, lice, fleas, chiggers, and bed bugs. We systematically searched databases, including PubMed, MEDLINE (EBSCOhost), Embase (Scopus), CENTRAL, Cochrane Library, CINAHL, ScienceDirect, Web of Science, SciELO, and LILACS in any language from inception to 4 April 2022. Studies exploring the therapeutic activity of TTO and its components against the ectoparasites were eligible. We used the ToxRTool (Toxicological data reliability assessment) tool, the Joanna Briggs Institute (JBI) critical appraisal tools, and the Jadad scale to assess the methodological qualities of preclinical (in vitro and in vivo) studies, non-randomised controlled trials (including cohort, case series, and case studies), and randomised controlled trials, respectively. Of 497 identified records, 71 studies were included in this systematic review, and most (66%) had high methodological quality. The findings of this review revealed the promising efficacy of TTO and its components against ectoparasites of medical importance. Most importantly, the compelling in vitro activity of TTO against ectoparasites noted in this review seems to have translated well into the clinical environment. The promising outcomes observed in clinical studies provide enough evidence to justify the use of TTO in the pharmacotherapy of ectoparasitic infections.


Introduction
Neglected tropical diseases (NTDs) are a group of communicable diseases that affect nearly two billion people worldwide and kill over 500,000 people annually [1,2]. They are endemic to impoverished communities living in low-and middle-income countries (LMICs), and are increasingly being recognised as the emerging causes of cardiovascular diseases (CVDs) in these countries [1,3]. CVDs are the leading cause of death worldwide, and >80% of these deaths occur in LMICs, with rheumatic heart disease (RHD) remaining a substantial preventable cause of cardiovascular disability and death [4,5]. About 95% of RHD cases occur in LMICs [4]. Scabies, myasis, tungiasis, and other ectoparasites have also been added recently to the global NTD portfolio [6].
Ectoparasites are pathogens that usually infect the skin of humans or other host organisms [7]. While temporarily blood-sucking arthropods (e.g., mosquitoes) are considered ectoparasites, the term is mainly used to refer to parasites such as mites, lice, fleas, and bedbugs that live on or in the skin [7,8]. Ectoparasites can cause serious diseases either directly by sucking blood or indirectly as vectors of infectious diseases, collectively posing a serious threat to human health and a significant burden to the global economy [7,9]. Among ectoparasitic diseases, scabies, demodicosis, headlice, and tungiasis are known as ectoparasitic diseases of medical importance as they cause substantial human morbidity [7,8]. Ectoparasitic diseases can be sporadic, endemic, or epidemic, depending on the type and place of living [10]. For example, in Australia, although the prevalence of scabies in the general population is low, the condition is hyperendemic in rural remote Aboriginal communities [11]. Similarly, about 80% of vulnerable children from Kenya and almost all indigenous peoples in the Amazon rainforest are impacted by tungiasis and head lice, respectively [10,12].
Over the years, several insecticides and pesticides have been successfully used to treat ectoparasitic infestations; however, as with other antimicrobial agents, overuse of these agents has led to the development of resistance, which is a worrisome public health concern [13,14]. As a result, screening plant products, with a key focus on secondary plant metabolites such as essential oils (EOs), has become important in the search for alternative therapeutic solutions [15][16][17][18][19]. EOs have traditionally been used for centuries for the treatment of ectoparasitic infestations-this is because of their antiparasitic, antibacterial, and/or anti-inflammatory properties [14,20]. However, most EOs have weak to moderate antimicrobial activities and are overshadowed by more active synthetic agents in practice [16]. In fact, only a few of them produce broad activity against a wide range of microbes. Tea tree oil (TTO), the EO obtained from Melaleuca alternifolia, is one such EO with potent and broad antimicrobial properties [16,21,22].
TTO contains approximately 100 compounds. Among the components of TTO, terpinen-4-ol (T4O) γ-terpinene, α-terpinene, 1,8-cineole, and terpinolene are the main bioactive, and most abundant, components. T4O and α-terpineol have been identified as the components most responsible for TTO's antimicrobial activity. These components have been standardised for TTO quality control by the industry, as per the International Organization for Standardization standard (ISO 4730) [21,22]. TTO possesses a unique combination of potent acaricidal, insecticidal, antibacterial, wound healing, antioxidant, and anti-inflammatory effects [22]. As a result, it has long been explored as a topical treatment for a variety of ectoparasite infestations, including head lice, scabies, and demodicosis, with good safety and efficacy data [22,23]. It is known for its potent activity as a bactericide (at 0.002-2%), including against methicillin-resistant S. aureus (MRSA), and as an anti-inflammatory agent (≤0.125%). Bacterial secondary infection and inflammation are both often associated with ectoparasitic infections [21,22]. The leaves of Melaleuca alternifolia have been used as bush medicine for different skin diseases by Australian Aboriginals, and the steam distilled oil has been used widely by Australian communities for more than 90 years [23]. TTO is an active ingredient in products registered in the UK's Medicines and Healthcare products Regulatory Agency and listed on the Australian Register of Therapeutic Goods.
The mechanism by which TTO produces its antiparasitic effect has not been fully elucidated. However, its miticidal effect is partly attributed to the anticholinesterase activity of T4O, 1,8-cineole, γ-terpinene, α-terpinene, and -cymene, which can cause lethal muscular contraction and spastic paralysis of the parasite (Figure 1) [24][25][26]. TTO's anticholinesterase inhibition is shown to be more potent than that of the individual components [26], suggesting a synergistic effect of the components responsible for its antiparasitic activity [28][29][30]. The combined action of multiple active ingredients may reduce the potential for development of resistance to TTO, as multiple simultaneous mutations would be required to overcome all the actions of the individual components [22]. In lice, TTO is shown to cause bulging of respiratory spiracles that might lead to suffocation ( Figure 2) [31]. Given ectoparasite infestations progress to inflammatory skin reactions and secondary bacterial complications [9,13,33], TTO could be a good fit in managing associated comorbidities and secondary complications, attributed to its anti-inflammatory, antimicrobial, and wound-healing properties.
While several systematic reviews [34,35] and narrative reviews [21,[36][37][38][39] have explored the antibacterial, anti-inflammatory, antifungal, and antiviral activities of TTO, few have comprehensively investigated its antiparasitic activity. One narrative review [29] summarised the studies evaluating TTO against Demodex mites and five [28,30,[40][41][42] systematically reviewed clinical studies assessing TTO and other anti-Demodex agents. To our knowledge, this is the first systematic review of preclinical (in vitro and in vivo) and clinical studies exploring TTO and its components against medically important ectoparasites, including mites (Demodex, scabies, and house dust), lice, fleas, chiggers, and bed bugs. These ectoparasites cause extensive morbidity to humans by either directly feeding on the host or causing allergic reactions and other serious diseases [7,9]. A review of this nature can help establish the evidence base for the efficacy and safety of TTO and its components against these ectoparasites, and inform clinical practice and direct future studies in this space.

Study Design
Initial searches revealed that published studies varied considerably in terms of study interventions, duration of treatment, participants, study design, study outcome measures, and follow-up durations, making a meta-analysis impossible. Hence, narrative-style data synthesis was employed to systematically organise, present, and appraise preclinical and clinical data.

Search Strategies and Selection Criteria
This systematic review was registered with the International Prospective Register of Systematic Reviews (PROSPERO; CRD42020212037) and is reported according to the Preferred Reporting Items for Systematic Reviews and Metanalyses (PRISMA) statement (Table S2, pp. [3][4] [43]. Two researchers (S.A.B. and W.T.) independently searched for in vitro, in vivo, and clinical studies exploring the use of TTO against the selected medically important ectoparasites using combinations of the terms "tea tree oil", "Melaleuca alternifolia oil", parasites, "ectoparasitic infestations", mites, "mite infestations", scabies, blepharitis, Pyroglyphidae, Trombiculidae, Pediculus, "lice infestations", Phthirapteran, flea, "flea infestations", Siphonaptera, Tunga, tungiasis, and "bed bugs". The databases searched were PubMed, MEDLINE (EBSCOhost), Embase (Scopus), CENTRAL (Cochrane Central Register of Controlled Trials), Cochrane Library, CINAHL (Cumulative Index to Nursing & Allied Health Literature), ScienceDirect, Web of Science, SciELO (The Scientific Electronic Library Online), and LILACS (Latin America and Caribbean Health Sciences Literature). Searches were performed without language restrictions from database inception to 12 November 2020. The search was then updated on 4 April 2022, using the same search terms and 12 new records targeting Demodex mites (in vitro (n = 2), randomised controlled trials (RCTs, n = 4), quasi-experimental (n = 2), cohort, case series, and cases studies (n = 1 each)) were identified and included in the review. The full search strategy is summarised in Supplementary Materials (Table S1, pp. 1-2). Grey literature was searched in Australian Tea Tree Industry Association (ATTIA) database. Additional searches were performed in Google and Google Scholar, and reference lists of included papers were manually screened to target articles potentially missed during the main search.
To perform the screening, the records obtained from the search results were exported to Covidence (Veritas Health Innovation, Melbourne, Australia) [44]. After duplicates were removed, two researchers (S.A.B. and W.T.) independently screened the titles and abstracts of the records for relevance and reviewed the full-text articles for eligibility. Any disagreements between the two researchers were resolved via discussion. Articles published in languages other than English were translated by Google Translate.
The RCT [86] compared the cure rates of TTO (5%) cream and a combination of TTO and permethrin (5% each) cream with permethrin (5%) cream in pediatric scabies patients (n = 72). The TTO (5%) cream demonstrated higher efficacy (54%) than the combination cream (20.8%) and the active control groups (16.7%) (p < 0.05). The study also reported a minor skin irritation associated with TTO use, although this was not statistically different from the combination and active control groups (p > 0.05). The two case studies (n = 1 each) [113,118] explored a combination of topical therapy (5% TTO in 25% benzyl benzoate lotion) with oral ivermectin in crusted scabies patients, with both showing a 100% mite eradication rate.

Insecticidal Effect of TTO and Its Components against Fleas
Four studies (in vitro [95,104] and in vivo [96,105]) explored the insecticidal activity of TTO solution alone [96] and in combinations with other EOs [95,104,105] against dog and cat fleas (Table 8). We did not find studies exploring TTO and its components against human, rat, and sand fleas.

Discussion
This is, to our knowledge, the first systematic review to rigorously assess all preclinical and clinical studies exploring the antiparasitic activity of TTO and its components against medically important ectoparasites. Our findings reveal several studies reporting promising acaricidal and insecticidal efficacy for TTO and its components. In addition, TTO and its components demonstrated significant improvement in ectoparasite-related symptoms.

Acaricidal Activity of TTO and Its Components against Mites
Mites are small arthropods of the family of Acarina. Demodex, scabies, and chiggers mites are the primary mite species of medical and/or public health importance [7].
Our review found that TTO and its components (mainly T4O) can completely eradicate Demodex mites and reduce mite-related symptoms without any serious AEs, as evidenced by multiple studies (Tables 1 and 2). The efficacy reported in clinical studies was consistent with the in vitro results of laboratory-based studies and results reported for veterinary Demodex mites (D. canis), where the survival time of mites was 8-100 min for TTO (3.125-100%) [122]. Demodex mites (Demodex folliculorum and D. brevis) are the most common permanent ectoparasites in humans, infesting the pilosebaceous unit of the face and scalp skin to cause demodicosis [7,123]. They invade the base of the eyelashes, eyelash follicles, and sebaceous and meibomian glands, causing Demodex blepharitis (chronic ocular inflammation), cylindrical dandruff, disorders of the eyelash, meibomian gland dysfunction, lid margin inflammation, conjunctival inflammation, and corneal lesions [33,123,124]. Although there is limited evidence on Demodex mite-bacteria interactions, studies report that infestation with these mites causes bacterial infections either through transferring symbiotic bacteria living inside (e.g., Bacillus oleronius) or on the surface (e.g., Streptococci and Staphylococci) of the mites, or promoting bacterial invasion from the surrounding environment [33,125,126]. Currently, there is no standard drug treatment for demodicosis; however, existing treatment approaches include various oral (e.g., ivermectin and metronidazole) and topical (e.g., pilocarpine gel, metronidazole ointment, lindane lotion, permethrin cream, benzyl benzoate lotion, and TTO) treatments [28,30,40]. Topical (cream or eye drops) and oral antibiotics are also usually given together with the anti-Demodex drugs to treat the associated bacterial infections [125,127]. Among these treatments, TTO (T4O as a primary active ingredient) is considered the most promising treatment of Demodex blepharitis [30]. The promising preclinical and clinical findings for TTO and its components (5-50%) in this review further justify their current and future use as mainstay Demodex treatments. In addition, the antibacterial property of TTO holds tremendous potential in reducing the burden of bacteria associated with Demodex mite infestation compared with the currently used Demodex treatments. However, there is a lack of evidence on head-to-head comparisons of TTO and its components with currently used Demodex treatments, necessitating the need for well-designed studies to inform clinicians of the most efficacious and safe therapeutic options.
Although there are few preclinical and clinical studies assessing TTO against scabies mites, the in vitro evidence shows that TTO-based treatments alone or in combination with other agents (i.e., benzyl benzoate and permethrin) are more lethal to human scabies mites than standard scabies treatments. Equally promising activity was also reported in an animal study involving pigs infested with sarcoptic mange mites (Sarcoptes scabiei var. suis), with TTO (100%) killing 98.5% of the mites [128]. Scabies mites (Sarcoptes scabiei var. hominis) cause scabies in humans, a contagious parasitic skin disease affecting over 300 million people worldwide [129,130]. Scabies mites enter the body by burrowing into the skin [7]. As they burrow into the skin, they release antigens, including scabies mite inactivated protease paralogues (SMIPPs) and scabies mite serpins (SMSs) in their saliva and faecal matter which trigger inflammatory and immune (allergic) reactions towards the mites and their products [129]. The host rapidly develops intense itching and scratching causing skin abrasion or cracks [7,129,131]. The skin cracks serve as an entry point for pathogenic bacteria (e.g., Staphylococcus aureus and group A beta-haemolytic streptococci, [GAS])) leading to secondary bacterial infections, including potentially fatal systemic complications such as sepsis, post-streptococcus glomerulonephritis (APSGN), acute rheumatic fever (ARF), chronic kidney disease (CKD), and RHD [129,131]. The SMIPPs and SMSs are also suggested to contribute to the growth and survival of bacteria (e.g., in patients' blood), possibly contributing to the potentially fatal disease sequalae [129,131]. Standard treatments for scabies include oral ivermectin, topical permethrin, and topical benzyl benzoate, and most of these treatments are potentially hazardous and associated with side effects, including severe skin irritation, headache, and nausea [130,132,133]. Furthermore, emerging drug-resistance of scabies mites is suggested as a critical failing of current treatments that demands the development of alternative scabies treatments [13,130,132]. Although additional RCTs are needed to confirm reported findings, the studies included in this review indicate promise for TTO-based formulations in the future of scabies treatment. Importantly, the results from a Phase II randomised controlled trial (n = 200) exploring the efficacy of TTO (5% v/w) gel in Australian Aboriginal settings are likely to provide additional insight into the utility of TTO for scabies treatment [130].
TTO showed significant in vitro activity against house dust mites, indicating TTObased formulations could provide an effective control mechanism for these mites (Table 5). House dust mites (Dermatophagoides farinae and D. pteronyssinus) are a group of mites naturally associated with the dust and debris inside houses [9]. Although free-living, they are known to cause severe allergic diseases, including asthma, atopic dermatitis, and perennial rhinitis in humans [7,134]. Alongside meticulous hygiene, synthetic acaricides, such as benzyl benzoate, dibutyl phthalate, N, N-diethyl-meta-toluamide (DEET), and pirimiphos-methyl, have been used to control house dust mites [134]. However, similar to other treatments, these agents are associated with several drawbacks, including potent toxicity, damage to household contents, and widespread development of resistance of the mites against these treatments [83]. The promising in vitro findings for TTO warrant further well-designed and controlled studies with a head-to-head comparison of TTO with currently used treatments for house dust mites.

Insecticidal Activity of TTO and Its Components against Lice
Both preclinical and clinical studies considered in this review revealed promising ovicidal and pediculicidal activities for TTO against head and body lice. Several reports from laboratory and animal studies investigating TTO (1-20%) for the treatment of Bovicola ocellatus lice-infested donkeys [135][136][137][138] and Bovicola ovis lice-infested sheep [139,140] also showed promising efficacy with mortality rates in the range of 78-100%, verifying the findings from human studies. Three species of lice are known to parasitise humans: the head louse (Pediculus humanus capitis), the body louse (P. humanus humanus), and the crab or pubic louse (Pthirus pubis) [9,141]. Body lice are widely considered of public health importance because they transmit typhus fever, relapsing fever, and trench fever [141]. Infestation with body lice is associated with poor hygiene and precarious living condi-tions [141]. In contrast, headlice, the lice species most commonly found in humans, affect individuals irrespective of hygiene and living conditions [9,141]. Head lice prevalence is believed to be increasing steadily across the globe; while some extrapolate the annual occurrence to be hundreds of millions of cases, the Centres for Disease Control and Prevention (CDC) provides estimates of 6-12 million cases per annum in the USA alone [142]. Although there are no reported cases of human disease transmission linked with headlice [141], secondary bacterial infections (Staphylococcus aureus and GAS) can occur from constant scratching as a result of allergic reactions induced by lice saliva [141,143,144]. Several conventional pediculicides are currently available for pediculosis treatment [141]. However, their widespread use increased the development of resistant lice, driving the need for newer treatment alternatives with minimal potential for resistance [13,24,145,146]. The preclinical and clinical data from this review suggest that TTO is highly likely to be an effective lice treatment. Apart from its ovicidal and pediculicidal activities, TTO possesses good antibacterial and anti-inflammatory properties, offering additional benefits in potentially preventing the disease sequelae linked to secondary bacterial infections. In general, an effective lice treatment must possess activity against both lice and their eggs to break the parasite's life cycle, with no requirement of repeated applications for drugs with additional ovicidal activity [146]. Given its pediculicidal and ovicidal effects, along with good antibacterial and anti-inflammatory activities and safety profile, it is reasonable to consider TTO, in a suitable pharmacological formulation, as a potential headlice treatment.

Insecticidal Activity of TTO and Its Components against Fleas
A few studies included in this review described promising insecticidal activity of TTO against cat and dog fleas. TTO was also found to have additional beneficial properties in reducing secondary infection and promoting the healing of scratches associated with flea infestation [96]. Fleas are small, wingless bloodsucking insects with a characteristic jumping movement [147]. The most important species are the rat flea (Xenopsylla cheopis), human flea (Pulex irritans), and cat flea (Ctenocephalides felis) [7,134]. A flea bite can lead to irritation, serious discomfort, and, most importantly, can be a means of pathogen transmission [148]. Rat fleas can cause plague and flea-borne typhus, while cat flea, the most abundant ectoparasite of cats and dogs, can cause cat scratch disease, flea allergic dermatitis, and tapeworm [134,148]. Sand fleas (Tunga penetrans) cause tungiasis, a WHO classified neglected epidermal parasitic skin disease, by burrowing into animal and human skin [149,150]. They secrete proteolytic enzymes to break the upper layer of the skin, which results in an inflammatory skin response by the host. As a result, patients develop intense itching and scratching that promotes the entry of pathogenic bacteria (such as Streptococcus sp. and Staphylococcus sp.) through the skin cracks [143,151]. Adult sand fleas frequently contain Wolbachia bacteria, which are known to infect many insect species [152]. Although the precise mechanism is yet to be determined, Wolbachia antigens are released following the death of the parasite, and these appear to play a key role in initiating severe localised inflammation commonly seen in patients with tungiasis [143]. Tungiasis inflicts pain and suffering on millions of people, particularly children with prevalence rate of up to 80%, living in sub-Saharan Africa, Latin America, and the Caribbean, leading to substantial human consequences, including childhood disability, stigma, and low quality of life [149,150]. Due to resistance development, many available flea treatments fail to eliminate flea infestation [134,148]. These treatments are also potentially hazardous to humans and must be applied by qualified personnel, requiring additional expense, which could be unaffordable to people living in resource-constrained settings [134]. For sand fleas, in particular, there is currently no proven, standard treatment. In desperation for relief, affected individuals physically extract the embedded flea using unhygienic sharp instruments, which can lead to severe inflammation and bacterial superinfections [153]. Other than several anecdotal or undocumented claims in tungiasis endemic settings, no study was found investigating TTO for tungiasis. TTO's unique parasiticidal, antibacterial, and anti-inflammatory properties indicate its tremendous potential for reducing the severity of tungiasis and its complications. An exploratory tungiasis trial (ACTRN12619001610123) [154] has been planned to investigate the safety and efficacy of a TTO (5% v/w) gel formulation. Results from this investigation are likely to provide key evidence on the future place of TTO in the treatment of tungiasis.

Safety and Patient Compliance of TTO and Its Components
Our review found no report of severe AEs or systemic reactions in the included clinical studies. Studies reported either no AEs or only mild to moderate skin irritation, suggesting the use of TTO and its components did not raise serious safety concerns. Multiple clinical studies investigating TTO against bacterial and fungal infections [155][156][157][158][159][160][161] also reported no or low risk of adverse skin reactions when TTO is formulated in a suitable pharmaceutical base at concentrations ≤25%. Regarding acceptance and compliance, multiple Demodex studies reported that treatments with TTO and its components were well-accepted, preferred over other available treatments, and there was good compliance by users [61,69,121]. Similarly, a report from a RCT [162] in children (mean age 6.3 + 5.1 years) with viral molluscum contagiosum demonstrated that TTO (75%) was well tolerated in the 30-day treatment period. From the preceding, TTO and its components appear to be sufficiently safe and acceptable to users to warrant further evaluation against these ectoparasites in well-designed RCTs.

Pharmaceutical Dosage Forms of TTO and Its Components
In addition to safety and efficacy, the nature of a pharmaceutical formulation and its ease of administration are crucial aspects to consider while devising a pharmacotherapy for ectoparasitic infections, because formulations can play a significant role in determining patient uptake. In this review, all but three in vitro studies [31,103,115] investigated diluted and undiluted solutions of TTO or its components. For Demodex mites, weekly eyelid scrubbing with TTO (50%) sterile wipes followed by daily eyelid scrubbing with TTO (5-10%) shampoo or ointment were the most explored treatments, and the two-time scrubbing practice seems to relate to the site of the Demodex mites, which usually reside at the base of the eyelashes and eyelash follicles [28]. The mechanical agitation from the weekly scrubbing is suggested to stimulate Demodex mites embedded inside the skin to move out to the surface and make it possible for the daily application to kill the mites before they start mating [97]. Innovative formulation designs involving nanoparticulate delivery mechanisms have demonstrated improved activity of TTO when compared to the conventional TTO formulations against bacteria. Such delivery methods could be valuable for Demodex treatment, as it can enhance the stability of TTO in the dosage form, control its release rate, and improve its penetration into the hair follicles [29,39,163,164]. Given that different formulations have the potential to influence the ocular exposure time and volume of product delivered to the eyelids [112], a comparative study of the most widely used anti-demodectic formulations is needed.
The reviewed scabies studies investigated lotion and cream formulations, while the headlice studies investigated solutions, shampoos, gels, and lotions/sprays. Heukelbach et al. (2008) [115] performed a head-to-head comparison of in vitro pediculicide efficacy of TTO (5%) gel and TTO (10%) and lavender oil (1%) lotion, reporting significantly higher efficacy for the gel (96%) compared with the lotion (17%). This finding is consistent with another in vitro pediculicide study [31] reporting similar efficacy (96%) for TTO (5%) gel formulation. Evidence [53,67,115] indicates that the presence of ethanol, as a solvent, in TTO formulation and enhanced skin partitioning from a lipophobic formulation base may contribute to superior results as opposed to involving solvents, such as acetone, or lipophilic carrier oils, such as coconut oil or sunflower oil. These findings suggest the need for formulation optimisation, and the likely impact it could have on bioactivitysuch effects could include improved partitioning onto to the application site from the formulation base, prolonged skin contact time, and enhanced permeability of TTO into the parasite exoskeleton [67,115]. Gel formulations are generally most preferred for hair-bearing areas, such as the scalp [115,165]. They are likely to offer ease of application (less messy), better coverage, and enhanced skin partitioning and contact time, allowing the drug to permeate more effectively into the parasite [115,165]. In recent times, a new dimeticone gel-based formulation for headlice treatment has been developed to improve the formulation characteristics of the previous products (e.g., lotion) [166,167]. Similarly, a gelbased formulation may prove successful for TTO in ectoparasitic infestation treatment. In sum, there is a lack of head-to-head comparisons of different formulations for ectoparasite treatments, and further RCTs are required to inform efficacy, safety, and user preference.

Strengths and Limitations
This is the first systematic review to comprehensively analyse and summarise the antiparasitic activity of TTO and its components against mites, lice, and fleas to inform future researchers and clinicians. It rigorously assessed all preclinical and clinical studies exploring the acaricidal and insecticidal activity of TTO and its components. However, interpretation of the findings of this review should consider its limitations, including the narrative approach employed to review the available data. Heterogeneity in the study designs, their evaluation methods, outcome measures, and study periods precluded a metaanalysis. Most of the included clinical studies are non-RCTs (observational studies), limiting the quality and generalisability of reported findings. That said, well-designed observational studies are categorised as level II or III evidence, and they can still play an important role in informing RCTs in terms of hypothesis generation, refining research questions, and defining clinical conditions [168]. Different methodologies were used for some of the ectoparasites in the in vitro studies, suggesting the results from these studies should be explored in clinical practice with caution. However, from the methodological assessment results, most studies were found to be reliable enough to provide complementary evidence for the clinical efficacy results. Also, the studies sourced TTO from different providers in different countries, and the use of TTO with a low content of the main active components such as T4O, α-terpineol, γ-terpinene, and 1,8-cineole may have potentially had an impact on the study findings. To reduce the compositional variation from various factors, including the extraction methods, geographical locations, and harvest times [83], researchers should use oil that meets the International Standard ISO 4730 ("Oil of Melaleuca, terpinen-4-ol type") [21].

Conclusions
The findings of this review show that TTO and its components are a promising treatment option for a range of ectoparasitic infections caused by mites, lice, and fleas. The compelling in vitro activity of TTO against ectoparasites has translated well into advanced investigations with promising outcomes observed in clinical studies, providing enough evidence to make recommendations for their clinical application. Also, most of the studies included in this review had high reliability and methodological quality. We found no study exploring TTO and its components against bed bugs, chigger mites (red bugs), and sand fleas. Given the promising activity of TTO and its components against similar ectoparasites, this review alerts researchers in this space to further explore the untapped potential use of TTO and its components as an alternative treatment against such parasites.
Ectoparasite infestations are usually associated with skin inflammation and secondary bacterial complications. Impetigo, a superficial paediatric bacterial infection, also occurs secondary to scabies in high-burden settings. Unresolved impetigo infections lead to serious sequelae with substantial morbidity and mortality, ranging from abscesses or bone infections and blood poisoning resulting in kidney and heart disease including acute post-streptococcal glomerulonephritis, acute rheumatic fever, and RHD.
Considering the unique therapeutic attributes of TTO, such as antimicrobial, antipruritic, anti-inflammatory, and wound-healing effects, TTO could be an excellent alternative option to tackle neglected skin ectoparasitoses and associated bacterial and inflammatory complications, particularly in light of rapidly emerging global antimicrobial resistance crisis. This is particularly important in high-burden settings, where potentially fatal se-quelae to ectoparasitoses arise from a complex interplay between environmental factors, bacterial pathogens, and skin parasites. TTO has been used widely over several decades with no evidence of resistance. The clinical decision on the use of TTO and its components against the discussed ectoparasites depends on multiple factors, such as efficacy, safety, duration of treatment, cost, ease of administration, and treatment acceptability. Further large-scale and high-quality RCTs can provide deeper insight into the therapeutic use of TTO for Demodex, head lice, and scabies infections.