ARTICLE Effective methods for tick removal: a systematic review Véronique Huygelen1, Vere Borra1, Emmy De Buck1,2, Philippe Vandekerckhove1,2,3 1 CEBaP (Centre for Evidence Based Practice), Mechelen, Belgium 2 Department of Public Health and Primary Care, Faculty of Medicine, KU Leuven, Leuven, Belgium 3 Faculty of Medicine and Health Sciences, University of Ghent, Ghent, Belgium Corresponding author: Vere Borra, CEBaP (Centre for Evidence Based Practice), Mechelen, Belgium. Motstraat 42, 2800 Mechelen, Belgium. Tel: +32 15 44 35 87; Email: Vere.Borra@rodekruis.be Short title: Effective methods for tick removal This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jebm.12257. This article is protected by copyright. All rights reserved. Vademecum - disciplinenaam 1 Abstract Aim Providing evidence-based first aid training to lay people is a cost-effective intervention. This systematic review aimed to collect the best available evidence on effective methods to remove a tick, performed by lay people. Methods A systematic search was performed searching the Cochrane Library (via Wiley), MEDLINE (via the PubMed interface) and Embase (via the Embase.com interface) to identify studies with evidence on tick removal strategies. Study selection was performed by two independent reviewers and data extraction was achieved in a tabular format. GRADE was used to determine the quality of evidence. Results Out of 2046 articles, 6 experimental animal studies were included. These studies compared different chemical treatments (gasoline, petroleum jelly, clear fingernail polish or methylated spirit) or mechanical techniques (forceps or commercial devices) for tick removal. The available studies were all of very low quality and suggest that there is limited evidence in favor of pulling with commercial devices and in favor of pulling with forceps compared to rotation with forceps to remove ticks. Conclusions Limited evidence was found in favor of pulling with commercial tick removal devices or pulling with forceps. The evidence of the included studies is of very low quality and results of these studies are imprecise due to limited sample size, large variability of results and/or lack of data. Since large high-quality studies are lacking, more rigorous studies are warranted to enable strong evidence-based recommendations. Keywords: first aid, evidence-based practice, systematic review, ticks This article is protected by copyright. All rights reserved. 2 Introduction Ixodes ticks are vectors of a broad range of pathogens of medical and veterinary importance, such as Babesia species (spp.), Borrelia spp., Anaplasma phagocytophilum, Rickettsia spp., Bartonella spp. and tick-borne encephalitis virus (TBEV). Lyme borreliosis has been reported throughout Europe and is the most common tick-borne infection (1, 2). This bacterium is transmitted to humans and other vertebrates during the blood feeding of Ixodid ticks, of which the most common in Europe is the Ixodes ricinus or sheep tick (3, 4). In Eastern Europe and Asia, including China and Japan, Ixodes persulcatus (the taiga tick) is the most important vector. In North America, Ixodes scapularis (the black-legged tick or deer tick) is the main vector on the east coast, whilst on the west coast the principle vector is Ixodes pacificus (the western black-legged tick). Despite substantial efforts to improve surveillance and control of Lyme borreliosis in recent decades, it is still the most prevalent arthropod-borne disease in Europe and the USA, with approximately 65.500 cases in Western Europe annually (5). However, this number might be largely underestimated as case reporting is highly inconsistent throughout Europe and thus many Lyme borreliosis infections are not diagnosed. A fast and complete removal (i.e. not leaving mouthparts or the hypostome in the skin of the host) of ticks is important to prevent localized infection and transmission of infectious agents. It has been shown that Borrelia requires less than 16 h of feeding for transmission to the host and the risk of transmission increases with prolonged attachment to the host (6). In literature, various techniques of tick removal by the bitten individual, or by any other layperson have been proposed using chemical or mechanical methods or a combination of mechanical removal after chemical treatment. Chemical methods (including rubbing gasoline, petroleum jelly, fingernail polish or methylated spirit over the tick‟s mouthparts) are supposed to induce the tick to detach itself from the host. Mechanical devices, such as forceps or a commercial tick removal device, are usually recommended, however they vary with regard to their mechanism of seizing and holding the tick and the manner of extracting (pulling or twisting) the tick from the skin. In making the choice for a specific technique, it is important to outweigh the ease of removal without the chance of leaving mouthparts behind. In this paper we describe the results of our systematic review of the evidence supporting effective methods of tick removal, to obtain an answer to the following PICO question: “In This article is protected by copyright. All rights reserved. 3 humans with a tick bite (P), which tick removal device (I) compared to another tick removal device (C) is most effective for laypeople to remove the tick (O)?” This systematic review was made in light of the development of evidence-based first aid guidelines for Flanders, Belgium (7). Methods This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) (8). The evidence was searched according to a strict methodology (9). No protocol was filed prior to the preparation of the manuscript. Selection criteria We included studies comprising both humans and animals with ticks attached to the skin. Interventions that were included were chemical treatment, heat, use of a forceps, use of commercial devices or other techniques with the aim of removing the ticks, limited to devices or techniques that could be performed by a layperson. The outcome measures were (complete) removal of the tick, damage to the mouth parts or mouth parts that broke off. For the study design we included: (1) experimental studies: inclusion of the following study types: (quasi or non-) randomized controlled trial, controlled before and after study or controlled interrupted time series; (2) observational studies: inclusion of the following study types: cohort and case-control studies, controlled before and after studies or controlled interrupted time series. Animal studies were included in the event that no evidence of human studies was found. Case series, cross-sectional studies, ex vivo or in vitro studies were excluded. Studies were excluded if no data were available. Only articles in English were included. Search strategy and study selection All searches were performed on 10 February 2016 within the following databases: the Cochrane Library (via Wiley), MEDLINE (via the PubMed interface) and Embase (via the This article is protected by copyright. All rights reserved. 4 Embase.com interface). All databases were searched from inception to ensure that all relevant articles would be retrieved. The development of the search strategies was performed independently by two reviewers (VH and VB). The final search strategies were combined to ensure maximal search sensitivity before the commencement of study selection. The subsequent study selection was performed independently by the same two reviewers in two phases. Initially, the titles and abstracts identified by the search were scanned for relevance to the PICO question. Next, full texts of any relevant references were obtained to assess whether the selection criteria were met. The independent results from the selection phase were compared, to ensure that all relevant studies had been identified. Finally, the references from identified studies, along with other papers citing the identified studies, were assessed for relevance. For each included study, if present in MEDLINE (via the PubMed interface), the first 20 similar articles (as suggested by PubMed) were also assessed for pertinence to the current review. Data extraction Data concerning study design, population, outcome measures, effect sizes and quality of the studies were collected. Review Manager 5 (10) was used to calculate effect sizes (risk ratios (with 95% confidence intervals) for dichotomous variables and mean differences (with 95% confidence intervals) for continuous outcomes)) if these were not reported in the study and raw data were available. A p-value < 0.05 was considered statistically significant. Quality assessment The GRADE (Grading of Recommendations Assessment, Development and Evaluation) methodology was used to assess limitations of study design for each individual study. Limitations in experimental study designs are analyzed by evaluating the presence of lack of allocation concealment, lack of blinding, incomplete accounting of outcome events and selective outcome reporting. This is followed by a quality rating of the body of evidence, which depends on study limitations, imprecision, inconsistency, indirectness and publication bias (11). As part of publication bias, we also looked at funding of the studies by companies or authors with commercial interest. The quality of the evidence can be downgraded for each of the previous quality criteria and finally results in a high, moderate, low or very low level of evidence (11). This article is protected by copyright. All rights reserved. 5 Results Study identification Figure 1 provides a flowchart of the identification and selection of studies. 2046 articles were identified with the search strategy that can be found in supplemental file 1: 209 from The Cochrane Library (via Wiley), 854 from MEDLINE (via the PubMed interface) and 983 from Embase (via the Embase.com interface). Once duplicates were removed, a total of 1246 articles were then screened by title and abstract for relevance to the PICO question. After title and abstract screening 37 studies were assessed by VH based on the full text. 15 studies were excluded based on study design, 8 on the intervention, 5 on the outcome and 3 were unavailable. Finally, 6 animal studies were included. VB assessed 33 articles for eligibility and 18 studies were excluded based on study design, 1 on the intervention, and 7 were unavailable. No additional studies were identified from cross-references of the included studies. Finally, after resolving disagreement 6 animal studies were included. Characteristics of included studies All 6 studies were non-randomized controlled animal studies. Tick removal methods were tested on various animals: sheep (12, 13), pigs (12), dogs (14-16), cats (15, 16), laboratory rabbits (17), guinea pigs or hedgehogs (16) and 2 unidentified hosts (16). In the studies the following tick species were used: Rhipicephalus sanguineus (14-16), Ixodes ricinus (12, 15, 16), Dermacentor reticulates (15, 16), Ixodes hexagonus (15), Haemaphysalis concinna (15), Dermacentor variabilis (13, 17) and Amblyomma americanum ticks (13, 17). Two studies compared topical chemical treatments (gasoline, nail polish or methylated spirit (12) or petroleum jelly, finger nail polish or 70% isopropyl alcohol (13)) or heat (a wooden kitchen match was struck, allowed to burn until red hot, then blown out and immediately held to the dorsum of the tick for 5 - 10 seconds) followed by mechanical removal (12, 13), two studies compared the use of forceps (12, 13), and four studies compared the use of This article is protected by copyright. All rights reserved. 6 commercial devices (14-17). Detailed characteristics of the included studies can be found in Table 1. Chemical treatment or heat followed by mechanical removal Successful mechanical removal following treating the ticks first with either gasoline, methylated spirit, petroleum jelly or a hot kitchen match, could not be demonstrated (12, 13) (Table 2). However, there is limited evidence in favor of applying nail polish on female ticks. It was shown that applying nail polish on female ticks (that had been attached to the skin up to 72h) resulted in a statistically significant decrease in damaged tick mouthparts after mechanical removal (12). No effect of nail polish applied to nymphs (that had been attached less than 24h to the skin) on the intactness of the mouthparts after mechanical removal could be demonstrated (12). In a different study an effect could not be demonstrated after the application of nail polish on the intactness of the mouthparts of female ticks after mechanical removal, either 12-15 h after attachment or 72-96 h after attachment to the host (13) (Table 2). Mechanical removal using forceps It was shown that pulling straight out with blunt forceps resulted in a statistically significant lower damage to the tick mouthparts compared to rotation with a blunt forceps (12) (Table 2). In a smaller study a decrease of damaged tick mouthparts or reduction in mouthparts that broke off when pulling ticks with a forceps could not be demonstrated (13). Mechanical removal using commercial devices The use of a slit and rotation (SR) device resulted in a statistically significant reduction in damaged tick mouthparts, compared to using an opposing jaws and rotation (AR) or a slit and traction device (ST) (16). Furthermore, the use of twisting devices (pen-tweezers, Tick Twister “lasso” (Trix® tick remover)) resulted in a statistically significant decrease of damaged female tick mouthparts compared to pulling devices (Adson forceps or “card” (TickPic)) (15). A reduction in damage to the hypostome or mouthparts using a forceps compared to using commercial devices for adult tick removal could not be demonstrated (14, This article is protected by copyright. All rights reserved. 7 17). For the removal of nymphs, the use of a commercial device resulted in a statistically significant reduction in mouth part damage compared to the use of forceps (Table 2) (17). Quality of included studies and body of evidence The limitations in study design of the included individual studies are detailed in Table 3. Allocation concealment was not specified in four studies (12, 13, 15, 17), in one study no raw data were provided (16). Other limitations of the included studies are that sections of the skin rather than individual ticks were treated with chemicals, and since ticks are often attached in clusters, ticks receiving different treatments were not randomized (13). Therefore, the quality of the body of evidence, initially started at a high level (because of the experimental study designs), was downgraded with one level for study limitations (-1). The quality level was further downgraded for imprecision (-1) since the sample sizes in all studies were generally small (low number of subjects), and/or because of a large variability in results and/or lack of data. Finally, the quality level was downgraded for indirectness (-1), since only animal studies were retrieved and also, studies vary with respect to different tick species, different host species and different time periods of tick attachment to the host before removal (13, 16, 17). No further downgrading was performed as there was no inconsistency or publication bias. This results finally in a very low quality level of evidence. This implies that it is difficult to infer whether the impact of these interventions would last when further research of higher quality becomes available, according to the GRADE methodology (Table 3). Discussion To our knowledge, this is the first systematic review investigating the most effective method of tick removal using chemical or mechanical techniques that can be performed by lay people. For this review, no geographical filters or time constraints were used. In the identified studies an effect of applying chemical treatment (with either gasoline, methylated spirit or petroleum jelly) or heat on nymphs or female ticks could not be demonstrated. However, This article is protected by copyright. All rights reserved. 8 there is limited evidence in favor of pulling with commercial devices to remove ticks and in favor of pulling compared to rotation with forceps. The evidence of the included studies is of very low quality and results of these studies are imprecise due to limited sample size, large variability of results and/or lack of data. Furthermore, there is limited evidence in favor of applying nail polish on female ticks (that had been attached to the skin of the hosts up to 72h) before mechanical removal of the ticks. It should be noted that this was only found in one study (12), and the effect of applying nail polish could not be demonstrated in another study (13). The reason for not being able to demonstrate an effect in the latter study could be the extent of the study (only one sheep included), or heterogeneity in tick species, hosts and times of attachment. Other limitations of the included studies are that only the outcome “number of nymphs/ticks with damaged mouthparts” was measured, and none of the studies measured “number of successful removals” or did a follow up to check if any tick borne diseases developed in the hosts. Therefore our conclusions are solely based on the findings on intact tick removal. However, it was clear that for both adult ticks and nymphs (Ixodes ricinus) pulling with a forceps resulted in less damaged mouthparts compared with rotation with a forceps (12). In a different tick species (Amblyomma americanum) the use of commercial devices resulted in less damaged mouthparts of nymphs, whereas in adult ticks this finding could not be demonstrated (17). However, not all included studies differentiated between adult ticks or nymphs (13, 16) or only used female ticks (14, 15). In these studies evidence was found in favor of pulling with a commercial tick removal device or pulling with forceps. The results of these studies are imprecise due to limited sample size, large variability of results and/or lack of data. In this systematic review only animal studies were included, and it should be noted that ticks found on pets are probably in a more advanced stage of engorgement than in humans because they might be overlooked in the hair coat when ticks are still small. Pig skin seemingly has the advantage of being currently the best model to mimic the human skin due to its similar skin morphology and physiology (18). In this search only one study was retrieved that used pigs as hosts to compare tick removal techniques (12). And thus, since experimental studies in humans might be limited due to ethical constraints, the pig might be a valuable animal model in order to design and perform better studies in order to unravel the best method for This article is protected by copyright. All rights reserved. 9 removal of ticks. The only human study on tick bites is a recently published cross-sectional study (19) comprising 160 humans with a tick bite. This study was published after our search was performed, however, since it is a cross-sectional study, it would still be excluded as described in our inclusion criteria. It must be noted that the results of this study point out that pulling (without twisting) with tweezers was the most effective methods for tick removal (19), which is in line with our conclusions. This systematic review aimed to collect the best available evidence on the most effective method to remove ticks by lay people and was made in light of the development of evidencebased first aid guidelines for Flanders, Belgium (7). Based on the evidence identified and taking into account practice considerations, the First Aid Service of Belgian Red CrossFlanders formulated draft recommendations. The evidence summaries and draft recommendations were then presented at the Medical Committee of Belgian Red CrossFlanders, deciding whether or not to recommend certain interventions, taking into account the quality of the evidence, the feasibility, the benefits and harms of the intervention, and the costs. Then final recommendations were formulated, and when appropriate, „Good Practice Points‟ were formulated. The final recommendations for tick removal in our first aid manual were formulated as follows: “Remove the tick as quickly as possible (Good Practice Point, GPP). Do not sedate the tick before removal using alcohol, methylated spirits or a lit cigarette (very low quality evidence on chemical treatments or heat). Ensure complete tick removal and avoid leaving mouthparts behind as leaving behind the tick’s mouth parts may result in a localized infection (GPP). Grasp the tick by its mouthparts as close to the skin as possible, preferably using a commercial device or forceps (very low quality evidence on the use of forceps or commercial tick devices). If using forceps grasp the tick and pull straight out (very low quality evidence on the use of forceps). If using a commercial device (type jaws) grasp the tick and pull straight out (very low quality evidence on the use of commercial tick devices). If using a commercial device (type V-shaped slot) remove tick by rotation (very low quality evidence on the use of commercial tick devices). After removing the tick, thoroughly clean the bite area with water and disinfect (GPP)”. This article is protected by copyright. All rights reserved. 10 To conclude, evidence was found in favor of pulling with a commercial tick removal device or pulling with forceps. The evidence of the included studies is of very low quality and results of these studies are imprecise due to limited sample size, large variability of results and/or lack of data. Since large high-quality studies are lacking, more rigorous studies are needed to enable strong evidence-based recommendations. Search strategy The Cochrane Library (systematic reviews and controlled trials) using the following search strategy: 1. [mh "ticks"] OR tick*:ti,ab,kw OR ixodida*:ti,ab,kw MEDLINE (via PubMed interface) for experimental and observational studies using the following search strategy: 1. "ticks"[MeSH] OR tick*[TIAB] OR ixodida*[TIAB] 2. remov*[TIAB] OR excis*[TIAB] 3. #1AND #2 Embase (via Embase.com interface) using the following search strategy: 1. 'tick'/exp OR tick*:ab,ti OR ixodida*:ab,ti 2. remov*:ab,ti OR excis*:ab,ti 3. #1AND #2 Competing interests All authors are employees of Belgian Red Cross-Flanders and receive no other funding. One of the activities of Belgian Red Cross-Flanders is providing first aid training to laypeople. This article is protected by copyright. All rights reserved. 11 Funding This work was made possible through funding from the Foundation for Scientific Research of the Belgian Red Cross-Flanders. Author Contributions All authors were involved in formulating the research question and selection criteria. VH and VB and performed the literature search, study selection and data extraction. All authors contributed to the design of the work and interpretation of the results, and have commented on drafts of the paper and approved the final version. All authors had full access to all of the data and can take responsibility for the integrity of the data and the accuracy of the data analysis. Acknowledgements The authors wish to thank Bert Avau and Hans Van Remoortel for their valuable comments and assistance in proof-reading the manuscript. References 1. Mannelli A, Bertolotti L, Gern L, Gray J. Ecology of Borrelia burgdorferi sensu lato in Europe: transmission dynamics in multi-host systems, influence of molecular processes and effects of climate change. FEMS Microbiol Rev. 2012;36(4):837-61. 2. Franke J, Hildebrandt A, Dorn W. Exploring gaps in our knowledge on Lyme borreliosis spirochaetes--updates on complex heterogeneity, ecology, and pathogenicity. Ticks Tick Borne Dis. 2013;4(1-2):11-25. 3. Linard C, Lamarque P, Heyman P, Ducoffre G, Luyasu V, Tersago K, et al. Determinants of the geographic distribution of Puumala virus and Lyme borreliosis infections in Belgium. Int J Health Geogr. 2007;6:15. 4. Smith R, Takkinen J. Lyme borreliosis: Europe-wide coordinated surveillance and action needed? Euro Surveill. 2006;11(6):E060622. 5. Rizzoli A, Hauffe H, Carpi G, Vourc HG, Neteler M, Rosa R. Lyme borreliosis in Europe. Euro Surveill. 2011;16(27). 6. Kahl O, Janetzki-Mittmann C, Gray JS, Jonas R, Stein J, de BR. Risk of infection with Borrelia burgdorferi sensu lato for a host in relation to the duration of nymphal Ixodes ricinus feeding and the method of tick removal. Zentralbl Bakteriol. 1998;287(1-2):41-52. This article is protected by copyright. All rights reserved. 12 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Borra V, Vanremoortel H, Vande veegaete A, De Buck E, Vandekerckhove P. Development of Evidence-Based First Aid Guidelines for Laypeople in Flanders, Belgium. J Epidemiol Public Health Rev. 2016;1(4):doi http://dx.doi. org/10.16966/2471-8211.124. Moher D, Liberati A, Tetzlaff J, Altman DG. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009;6(7):e1000097. De Buck E, Pauwels NS, Dieltjens T, Vandekerckhove P. Use of evidence-based practice in an aid organisation: a proposal to deal with the variety in terminology and methodology. Int J Evid Based Healthc. 2014;12(1):39-49. Review Manager (RevMan). Version 5.3 ed. Copenhagen: The Nordic Cochrane Centre; The Cochrane Collaboration; 2014. Atkins D, Best D, Briss PA, Eccles M, Falck-Ytter Y, Flottorp S, et al. Grading quality of evidence and strength of recommendations. BMJ. 2004;328(7454):1490. De Boer R, van den Bogaard AE. Removal of attached nymphs and adults of Ixodes ricinus (Acari: Ixodidae). J Med Entomol. 1993;30(4):748-52. Needham GR. Evaluation of five popular methods for tick removal. Pediatrics. 1985;75(6):997-1002. Bowles DE, McHugh CP, Spradling SL. Evaluation of devices for removing attached Rhipicephalus sanguineus (Acari: Ixodidae). J Med Entomol. 1992;29(5):901-2. Duscher GG, Peschke R, Tichy A. Mechanical tools for the removal of Ixodes ricinus female ticks--differences of instruments and pulling or twisting? Parasitol Res. 2012;111(4):1505-11. Zenner L, Drevon-Gaillot E, Callait-Cardinal MP. Evaluation of four manual tickremoval devices for dogs and cats. Vet Rec. 2006;159(16):526-9. Stewart RL, Burgdorfer W, Needham GR. Evaluation of three commercial tick removal tools. Wilderness Environ Med. 1998;9(3):137-42. Summerfield A, Meurens F, Ricklin ME. The immunology of the porcine skin and its value as a model for human skin. Mol Immunol. 2015;66(1):14-21. Akin Belli A, Dervis E, Kar S, Ergonul O, Gargili A. Revisiting detachment techniques in human-biting ticks. Journal of the American Academy of Dermatology. 2016;75(2):393-7. This article is protected by copyright. All rights reserved. 13 Table 1. Characteristics of included studies concerning removal of ticks. Author, year, Country Bowles, 1992, USA (1) Study design Population Comparison Experimental: randomized controlled trial 299 adult ticks (Rhipicephalus sanguineus) on 8 stray dogs, 135 males and 164 females. Removal of ticks using: 1. Tick Solution (n=81) 2. Standard jeweller‟s forceps (n=72) 3. Angled forceps (n=73) 4. Economy forceps (n=73) [1 nymph was collected and not included in de analysis]. De Boer, 1993, The Netherlands (2) Experimental: nonrandomized controlled trial 1060 ticks (Ixodes ricinus) applied on skin of 4 pigs (219/950 nymphs and 59/110 female ticks) 356 ticks applied on skin of 2 sheep (6/294 nymphs and 27/62 female ticks) 175 ticks were treated (130 nymphs and 45 females). Chemical treatment: 1. Application of gasoline 2. Methylated spirit on a wad or cotton wool 3. Nail polish (30-60 min). 4. No chemical treatment Removal of ticks after chemical treatment by: 1. Pulling using a blunt forceps 2. Twisting using a blunt forceps Assessment of tick removal Ticks were removed by investigators. After removal, ticks were stored in 70% isopropyl alcohol and identified according to species, sex, and relative feeding status (i.e., unfed, partially fed, engorged) and examined for damage to the tick body (i.e., damaged hypostome, perforations, broken legs). Ticks were removed by investigators. Mouthparts of extracted ticks were examined through a dissecting microscope at 10 x magnification. Nymphs subsequently were embedded in Marc-Andre mounting fluid and examined with bright light This article is protected by copyright. All rights reserved. 14 Duscher, 2012, Austria (3) Experimental: randomized controlled trial 596 ticks, 541 Ixodes ricinus, 40 Ixodes hexagonus; 2 Haemaphysalis concinna; 1 Dermacentor reticulates; 1 Rhipicephalus sanguineus; 11 undefined, sampled from 320 dogs, 198 cats, 6 hedgehogs, 1 guinea pig, 2 unidentified hosts. Of the 541 Ixodes ricinus, 527 were identified as females. Only these were used in further calculations. Removal of ticks by: 1. Twisting with pentweezers 2. Twisting with Tick Twister 3. Twisting with “lasso” (Trix® tick remover) 4. Pulling with Adson forceps 5. Pulling with 6. “card” (TickPic) illumination at 400x magnification. Missing portions of the hypostome, chelicerae, and capitulum were estimated separately. For each, the range of missing parts was 0-1; a score of 1 was given if the entire portion was missing. Ticks were removed by veterinarians and pet owners. After removal, the ticks were placed in 70 % ethanol for identification of stage, species, condition of the mouthparts, degree of squeezing was evaluated under the stereomicroscope at×7.5–40 magnification. Scoring of condition (intactness) of mouth parts of female Ixodus ricinus. 1 = intact, 4 = totally This article is protected by copyright. All rights reserved. 15 Needham, 1985, USA (4) Experimental: nonrandomized controlled trial 29 American dog ticks (Dermacentor variabilis); 22 lone star ticks (Amblyomma americanum) attached to the back of a female Dorset sheep; attachment to the host for 72-96 hours or 12-15 hours. Treatment with: 1. Petroleum jelly was applied in a generous amount to the dorsum and venter of female ticks, making certain that spiracles were covered 2. Clear finger nail polish 3. 70% isopropyl alcohol applied to the dorsum and venter with cotton swab 4. A wooden kitchen match was struck, allowed to burn until red hot, then blown out, and immediately held to the dorsum of the tick for 5 to 10 seconds. 5. No chemical treatment severed. Only female Ixodes ricinus were included in calculations. Ticks were removed by investigators. Intactness of mouthparts and cement collar. Information on how intactness of mouthparts and cement collar was assessed, was not available After chemical treatment ticks were removed mechanically by: 1. Pulling straight up with steady even pressure 2. Pulling straight up with a quick motion This article is protected by copyright. All rights reserved. 16 Stewart, 1998, Experimental: USA (5) nonrandomized controlled trial Zenner, 2006, France (6) Experimental: randomized controlled trial 148 adult ticks and 112 nymphs (Amblyomma americanum) and 82 (Dermacentor variabilis) adult ticks. Removed from laboratory rabbits 2330 h postinfestation. Veterinarians and owners removed 236 ticks attached to 46 cats and 178 dogs that were brought to 18 veterinary clinics. 193 Ixodes ricinus, 30 Dermacentor reticulates, 13 Rhipicephalus sanguineus. 91% of Ixodes ricinus, 77% of Dermacentor reticulates, 85% of Rhipicephalus sanguineus were female ticks. [Only data from removal by pet 3. Pulling the tick parallel with the skin using a steady even pressure (ventral aspect of tick up) 4. Twisting clockwise (2 to 3 revolutions) using forceps Removal by: 1. The original Ticked Off™ 2. The Pro-Tick remedy™ 3. Tick Plier or Tick Nipper™ 4. Medium-tipped tweezers. 1. Pulling the tick parallel with the skin using a device with apposing jaws and traction (AT, represented by a small surgical forceps with straight very sharp jaws (Adson forceps) 2. Rotating with a device using apposing jaws and rotation (AR) represented by a commercial product (PenTweezers, Buster) 3. Pulling with a device using slit Ticks were removed by untrained volunteers. Once the ticks were removed, they were placed in 70% ethanol for microscopic evaluation of mouthparts and associated cement. Ticks were removed by veterinarian or pet owner. Only data of removal by pet owners were extracted. Condition of mouthparts were scored from 1 to 4, 1 mouthparts intact with possible traces of tick origin cement host epidermis or both, 2 hypostome partly severed, 3 hypostome torn This article is protected by copyright. All rights reserved. 17 owners were extracted] and traction (ST) represented by a commercial product (pro-Tick Remedy, scs) 4. Rotating with a device using slit and rotation (SR) represented by a commercial product (Crochet O‟Tom, H3D). or severed at base, or 4 mouthparts totally severed. Table 2. Synthesis of findings concerning removing (parts) of ticks. Outcome Comparison Effect Size Chemical treatment or heat followed by mechanical removal Number of Gasoline vs no chemical Not statistically significant: nymphs with treatment 11/44 vs 10/29 § damaged RR: 0.73, 95%CI [0.35;1.48] mouthparts after (p=0.38) *¥ mechanical Nail polish vs no Not statistically significant: removal, <24h chemical treatment 15/34 vs 10/29 § attachment RR: 1.28, 95%CI [0.68;2.40] (p=0.43) *¥ Methylated spirit vs no Not statistically significant: chemical treatment 27/52 vs 10/29 § RR: 1.51, 95%CI [0.86;2.65] (p=0.16) *¥ Number of Gasoline vs no chemical Not statistically significant: females with treatment 20/28 vs 36/41 § damaged RR: 0.81, 95%CI [0.63;1.06] mouthparts after (p=0.1) *¥ mechanical Statistically significant: removal, up to 72h Nail polish vs no 5/12 vs 36/41 § chemical treatment attachment RR: 0.48, 95%CI [0.24;0.94] (p=0.003) * In favor of nail polish Methylated spirit vs no Not statistically significant: chemical treatment 4/5 vs 36/41 § RR: 0.91, 95%CI [0.58;1.43] (p= 0.69) *¥ #studies, # participants Reference 1, 44 vs 29 De Boer, 1993 (2) 1, 34 vs 29 1, 52 vs 29 1, 28 vs 41 1, 12 vs 41 1, 5 vs 41 This article is protected by copyright. All rights reserved. 18 Number of females (Dermacentor variabilis) with damaged mouthparts after removal with forceps, 72-96h attachment Petroleum jelly vs no chemical treatment Not statistically significant: 0/10 vs 0/7 £†§ Effect size not estimable Clear fingernail polish vs Not statistically significant: no chemical treatment 0/4 vs 0/7 £§ Effect size not estimable 70% isopropyl alcohol vs Not statistically significant: no chemical treatment 0/4 vs 0/7 £§ Effect size not estimable Hot kitchen match vs no Not statistically significant: treatment 0/4 vs 0/7 £§ Effect size not estimable Number of Petroleum jelly vs no Not statistically significant: females chemical treatment 0/4 vs 0/4 £§ (Amblyomma Effect size not estimable americanum) with Clear fingernail polish vs Not statistically significant: damaged no chemical treatment 0/4 vs 0/4 £§ mouthparts after Effect size not estimable removal with 70% isopropyl alcohol vs Not statistically significant: forceps, 12-15h no chemical treatment 0/4 vs 0/4 £§ attachment Effect size not estimable Hot kitchen match vs no Not statistically significant: treatment 0/4 vs 0/4 £§ Effect size not estimable Mechanical removal using forceps Number of Pulling straight out with Statistically significant: nymphs with blunt forceps 10/29 vs 23/34 § damaged vs RR: 0.51, 95%CI [0.29;0.88] mouthparts rotation with blunt (p=0.02) * forceps In favor of pulling straight Number of ticks Pulling straight out with Statistically significant: with damaged blunt forceps 31/53 vs 35/35 § mouthparts vs RR: 0.60, 95%CI [0.47;0.74] rotation with blunt (p<0.00001) * forceps In favor of pulling straight Number of ticks Pulling straight up with a Not statistically significant: with damaged quick motion with 7/7 vs 5/5 § mouthparts forceps vs twisting RR: 1.0, 95%CI [0.74;1.35] clockwise with forceps (p=1.0) *¥ Pulling straight up with a Not statistically significant: steady pressure with 5/5 vs 5/5 § forceps vs twisting RR: 1.0, 95%CI [0.71;1.41] clockwise with forceps (p=1.0) *¥ Pulling parallel with the Not statistically significant: 1, 10 vs 7 Needham, 1985 (4) 1, 4 vs 7 1, 4 vs 7 1, 4 vs 7 1, 4 vs 4 Needham, 1985 (4) 1, 4 vs 4 1, 4 vs 4 1, 4 vs 4 1, 29 vs 34 De Boer, 1993 (2) 1, 53 vs 35 De Boer, 1993 (2) 1, 7 vs 5 Needham, 1985 (4) 1, 5 vs 5 1, 5 vs 5 This article is protected by copyright. All rights reserved. 19 skin with forceps vs 5/5 vs 5/5 § twisting clockwise with RR: 1.0, 95%CI [0.71;1.41] forceps (p=1.0) *¥ Number of ticks Pulling straight up with a Not statistically significant: with mouthparts quick motion with 7/7 vs 0/5 § that broke off forceps vs twisting RR: 11.25, 95%CI clockwise with forceps [0.79;160.81] (p=0.07) *¥ Pulling straight up with a Not statistically significant: steady pressure with 5/5 vs 0/5 § forceps vs twisting RR: 11.0, 95%CI clockwise with forceps [0.77;158.01] (p=0.08) *¥ Pulling parallel with the Not statistically significant: skin with forceps vs 5/5 vs 0/5 § twisting clockwise with RR: 11.0, 95%CI forceps [0.77;158.01] (p=0.08) *¥ Mechanical removal using commercial devices Mouthparts intact Pulling vs twisting Statistically significant: of females ticks 57/190 vs 34/337 § (Ixodes ricinus) RR: 2.97, 95%CI [2.02;4.37] (p<0.00001) *†λ In favor of pulling Pulling with forceps vs Not statistically significant: pulling with card 26/90 vs 31/100 § RR:0.93, 95%CI [0.60;1.44] (p=0.75) *†λ Pulling with forceps vs Statistically significant: twisting with lasso 26/90 vs 11/108 § RR: 2.84, 95%CI [1.49;5.42] (p=0.002) *†λ In favor of pulling with forceps Pulling with forceps vs Statistically significant: twisting with tick twister 26/90 vs 14/108 § RR:2.23, 95%CI [1.24;4.01] (p=0.007) *†λ In favor of pulling with forceps Pulling with forceps vs Statistically significant: 26/90 vs 13/121 § twisting with penRR:2.69, 95%CI [1.47;4.94] tweezers (p=0.001) *†λ 1, 7 vs 5 1, 5 vs 5 1, 5 vs 5 1, 190 vs 337 Duscher, 2012 (3) 1, 90 vs 100 1, 90 vs 108 1, 90 vs 108 1, 90 vs 121 This article is protected by copyright. All rights reserved. 20 In favor of pulling with forceps Pulling with card vs Statistically significant: twisting with lasso 31/100 vs 11/108 § RR:3.04, 95%CI [1.61;5.73] (p=0.0006) *†λ In favor of pulling with card Pulling with card vs Statistically significant: twisting with tick twister 31/100 vs 14/108 § RR:2.39, 95%CI [1.35;4.23] (p=0.0014) *†λ In favor of pulling with card Pulling with card vs Statistically significant: twisting with pen31/100 vs 13/121 § tweezers RR:2.89, 95%CI [1.60;5.21] (p=0.0002) *†λ In favor of pulling with card Twisting with lasso vs Not statistically significant: twisting Tick twister 11/108 vs 14/108 § RR:0.79, 95%CI [0.37;1.65] (p=0.52) *†λ Twisting with lasso vs Not statistically significant: Twisting with pen11/108 vs 13/121 § tweezers RR:0.95, 95%CI [0.44;2.03] (p=0.89) *†λ Twisting with tick twister Not statistically significant: vs twisting with pen14/108 vs 13/121 § tweezers RR:1.21, 95%CI [0.59;2.45] (p=0.60) *†λ Condition of the Apposing jaws and Statistically significant: mouthparts of the rotation (AR) Data represented in graph tick vs (p<0.01) £† Slit and traction device In favor of SR device (ST) vs Slit and rotation device (SR) Damage of Tick solution vs Not statistically significant: hypostome of economy forceps 2/81 vs 2/73 § adult ticks RR: 0.90 95%CI [0.13;6.25] (Rhipicephalus (p=0.91) *¥ sanguineus) Standard jeweller‟s Not statistically significant: forceps vs economy 2/72 vs 2/73 § forceps RR: 0.90 95%CI [0.15;7.00] 1, 100 vs 108 1, 100 vs 108 1, 100 vs 121 1, 108 vs 108 1, 108 vs 121 1, 108 vs 121 1, no raw data Zenner, 2006 (6) 1, 81 vs 73 Bowles, 1992 (1) 1, 72 vs 73 This article is protected by copyright. All rights reserved. 21 Angled forceps vs economy forceps Other tick body damage Tick solution vs economy forceps Standard jeweller‟s forceps vs economy forceps Angled forceps vs economy forceps Number of Tick Solution vs removal attempts economy forceps Mouth part The original Ticked damage of adult Off™ vs medium-tipped ticks (Amblyomma tweezers americanum) The Pro-Tick remedy™ vs medium-tipped tweezers Tick Plier or Tick Nipper™ vs mediumtipped tweezers Mouth part The original Ticked damage of nymph Off™ vs medium-tipped ticks (Amblyomma tweezers americanum) The Pro-Tick remedy™ vs medium-tipped tweezers (p=0.98) *¥ Not statistically significant: 1, 72 vs 73 1/73 vs 2/73 § RR: 0.50 95%CI [0.05;5.40] (p=0.57) *¥ Not statistically significant: 1, 81 vs 73 5/81 vs 1/73 § RR: 4.51, 95%CI [0.54;37.68] (p=0.16) *¥ Not statistically significant: 1, 72 vs 73 3/72 vs 1/73 § RR: 3.04, 95%CI [0.32;28.56] (p=0.33) *¥ Not statistically significant: 1, 72 vs 73 1/73 vs 1/73 § RR: 1.00, 95%CI [0.06;15.69] (p=1.00)* ¥ Statistically significant: 1, 81 vs 73 § 3.1 vs 1.1 MD: 2.0 ££ (p<0.05) Not statistically significant: 1, 55 vs 42 Stewart, 1998 0/55 vs 1/42 § (5) RR: 0.26, 95%CI [0.01; 6.13] (p=0.40) *¥ Not statistically significant: 1, 21 vs 42 3/21 vs 1/42 § RR: 6.00, [0.66;54.24] (p=0.11) *¥ Not statistically significant: 1, 30 vs 42 0/30 vs 1/42 § RR: 0.46, 95% CI [0.02;10.98] (p=0.63) *¥ Statistically significant: 1, 29 vs 19 9/29 vs 18/19 § RR:0.33, 95%CI [0.22;0.57] (p<0.0001) * In favor of Ticked Off Statistically significant: 1, 36 vs 19 10/36 vs 18/19 § RR: 0.29, 95%CI [0.17;0.50] (p<0.00001) * In favor of Pro-Tick remedy This article is protected by copyright. All rights reserved. 22 Tick Plier or Tick Nipper™ vs mediumtipped tweezers Mouth part damage of adult ticks (Dermacentor variabilis) Statistically significant: 10/28 vs 18/19 § RR: 0.38, 95%CI [0.23;0.63] (p=0.0002) * In favor of Tick Plier or Tick Nipper The original Ticked Not statistically significant: Off™ vs medium-tipped 0/20 vs 1/18 § tweezers RR: 0.30, 95%CI [0.01;6.97] (p=0.45) *¥§ The Pro-Tick remedy™ Not statistically significant: vs medium-tipped 0/25 vs 1/18 § tweezers RR: 0.24, 95%CI [0.01;5.66] (p=0.38) *¥ Tick Plier or Tick Not statistically significant: Nipper™ vs medium0/19 vs 1/18 § tipped tweezers RR: 0.32, 95%CI [0.01;7.30] (p=0.47) *¥ 1, 28 vs 19 1, 20 vs 18 1, 25 vs 18 1, 19 vs 18 SD: standard deviation, CI: confidence interval, RR: risk ratio * Calculations done by the reviewer(s) using Review Manager Software £ No raw data/SD‟s available, effect size and CI cannot be calculated ££ No SD‟s available, CI cannot be calculated ¥ Imprecision (large variability of results) † Imprecision (lack of data) § Imprecision (limited sample size or low number of events) λ data extracted from graph. Table 3. Limitations of study design included studies. Author, Year Bowles, 1992 (1) De Boer, Lack of allocation concealment No, random selection was used to determine the order in which the devices were used. Unclear, not Lack of blinding Yes, but not possible Yes, but Incomplete accounting of outcome events No Selective outcome reporting No, the target outcomes were reported Other limitations No No, the target Sections of the skin This article is protected by copyright. All rights reserved. 23 1993 (2) specified. not possible. outcomes were reported. Dusher, 2012 (3) Yes, but not possible. No No, the target outcomes were reported. Needham, 1985 (4) The device was chosen by participants in random order. Unclear, not specified. No Stewart, 1998 (5) Unclear, not specified. Zenner, 2006 (6) No, the veterinarian used an intervention grid that randomly selected the tick removal device to be used. Yes, but not possible. Yes, but not possible. Yes, but not possible. No, the target outcomes were reported. No, the target outcomes were reported. No, the target outcomes were reported. No Unclear, no raw data. rather than individual ticks were treated with the chemicals, because the ticks are often attached in clusters. Ticks receiving different treatments were not randomized. Several tick species were included in the study. Several tick species were included in the study. Several tick species were included in the study. This article is protected by copyright. All rights reserved. 24 This article is protected by copyright. All rights reserved. 25