Efficacy of monthly treatment with oral fluralaner (Bravecto® 1-Month) against Tunga penetrans in dogs in Brazil: a randomized, double-blind, controlled field study

Background Tungiasis is a neglected tropical disease caused by the adult female sand flea (Tunga penetrans). Dogs are considered important reservoirs of T. penetrans in Brazil. The aim of this study was to determine the monthly insecticidal efficacy of a single oral administration of fluralaner at a dose of 10–18 mg/kg (Bravecto® 1-Month, also registered as Defenza® in some countries; MSD Animal Health) in dogs naturally infested with T. penetrans. Methods This clinical trial was conducted in a rural community located in Ilhéus, Bahia, Brazil. A total of 64 dogs were selected and distributed in a completely randomized design between a treated group (TG) that received one single dose of Bravecto® 1-Month (Defenza®) and a negative control group (CG) that received no treatment. Each group was composed of 32 dogs. The evaluations took place on days 0, 7 ± 2, 14 ± 2, 21 ± 2, 28 ± 2, 35 ± 2, and 42 ± 2 post treatment, in which the dogs were inspected to evaluate the infestation stage and classify lesions associated with tungiasis. The primary efficacy was determined from the percentage of treated dogs free of fleas (stage II and III lesions) after administration of the formulation at each evaluation time. Secondary efficacy was based on the number of active lesions (stages II and III) in each group at each evaluation time. The clinical condition of the animals was defined based on the Severity Score for Acute Dog Tungiasis (SCADT), which is related to the number and severity of lesions. Results The primary efficacy of the product was greater than 95.0% from days 7 to 21 and reached 100.0% between days 28 and 42, with a significant association between treatment and infestation decline (P < 0.025) between days 7 and 42. Secondary drug efficacy was greater than 99.9% from days 7 to 21, reaching 100.0% between days 28 and 42 (P < 0.05). The treated dogs also scored lower on the SCADT than the control animals did during the entire clinical evaluation period (P < 0.05). Conclusions A single administration of Bravecto® 1-Month (Defenza®) was effective in eliminating Tunga penetrans infestations, as well as in preventing parasitism for at least 42 days after treatment. Graphical Abstract


Background
Tungiasis is a neglected tropical disease (NTD) [1,2], caused by the adult female sand flea (Tunga penetrans) [3].Dogs can also be infested by T. penetrans [4,5].Direct contact with contaminated soil predisposes the host to penetration, which usually occurs in the paw regions.After penetration, fleas hypertrophize their neosomes, mature, and eliminate their eggs, remaining in situ until their death, which occurs 4-6 weeks after penetration [4][5][6][7].In most cases, the hosts have a high parasitic load [8], predisposing them to complications such as secondary bacterial infections [9,10] that progress to deformity and/or loss of digit(s), self-mutilation, septicemia, and death in dogs [11].
The participation of dogs in the dynamics of the disease is evident in endemic locations, as these animals have a wide circulation area and cover large perimeters, spreading flea eggs in the environment and facilitating the establishment of the parasite's biological cycle [15,16].Environmental contamination caused by infested animals can be prevented by controlling the fleas in the host.This would cause a decrease in reservoirs and a subsequent reduction in prevalence rates in humans and animals [17,18].
In both humans and animals, the distal regions of the limbs are most affected by infestations because of frequent contact with contaminated soil [14].However, ectopic lesions on the lips, muzzles, elbows, mammary glands, and genitals are not rare [11].Infested dogs commonly harbor tens or hundreds of sand fleas, which favor the formation of lesion clusters and aggravate the disease [15,[17][18][19].
Currently, only a few treatments are available for canine tungiasis.The combination of 10.0% imidacloprid and 50.0%permethrin (Advantix ® ) has approximately 80.0% efficacy in treating and preventing T. penetrans reinfestations for 14 days; however, the efficacy decreases significantly to approximately 30.0% in the following week [20].In addition, subcutaneous treatment with ivermectin (Ivomec 1.0%) healed the lesions in infested Prior to the start of the study, dog owners were informed about the research objectives and methodology and provided written consent for the inclusion of their dogs.
As inclusion criteria, dogs were ≥ 8 weeks in age, had body weights ≥ 2.5 kg, had adequate temperament to allow clinical and parasitological evaluations, and presented at least one live flea at stage II or III lesion in the Fortaleza classification [3].The selected dogs underwent clinical evaluation, and only animals with no clinical signs other than tungiasis were enrolled in the study, regardless of sex or breed.Dogs treated with fluralaner less than 90 days before day 0 or with other ectoparasiticides with short acting activity within 14 days before day 0 (isoxazolines, amitraz, fipronil, macrocyclic lactones, or pyrethroids), as well as pregnant or lactating female dogs, were not allowed to participate in the trial.Throughout the study period, the dogs remained under the care of their owners and maintained their usual routines.

Study design
This field study was randomized, negatively controlled, and double-masked.On day 0, 64 dogs were randomly distributed (using a prior computer-generated list) into one of two experimental groups, each composed of 32 dogs.The dogs were implanted with numerically coded microchips for individual identification.The clinical history of each dog was recorded, and each dog underwent a complete physical examination, including ectoparasite assessment.
Study activities were distributed between the two veterinary teams.One team assigned the animals to groups, administered the treatments, and did not participate in the clinical evaluations.The other team performed the clinical evaluations and inspections of the dogs and was blinded to the treatment assignments.Dogs in the treatment group (TG) received a single oral fluralaner dose (Bravecto ® 1-Month/Defenza ® ) at the approved dose rate of 10-18 mg fluralaner/kg body weight, according to label instructions, on day 0. The dogs in the control group (CG) remained untreated.
All animals were treated on the same day, received wet commercial dog food, and the owners were not informed of the group to which their dogs were assigned.During the study, if necessary, dogs were treated for any observed secondary complications of tungiasis as the treatment presented no insecticide activity or any other routine health needs.The administration of any other parasiticidal drug or product with insecticidal activity was prohibited.In the end of the study, all 64 dogs were treated with a dose of 25-56 mg/kg of fluralaner (Bravecto ® ).
At each visit, the dogs underwent a general physical examination and detailed skin inspection.The entire body of the animal was examined, with special attention paid to the paws, limbs, tail, mammary glands, abdomen, testicles, and nose.Before the examination, the dogs' paws were cleaned using water and a brush to improve the detection of all lesion stages.The identified lesions were counted and staged according to the Fortaleza classification [3].
Severity Score for Acute Dog Tungiasis (SCADT) were also assigned to each dog to record the severity of tungiasis lesions throughout the study.Each clinical sign was scored for each affected area and the results were added to obtain the SCADT (Table 1).The maximum possible score for this classification was 27.If the SCADT score exceeded 22, the dog would be treated by surgical removal of sand fleas and excluded from the study for ethical reasons; however, this procedure was not required during the trial.All lesions were classified, quantified, photographed, and documented in the parasitological skin examination records.

Statistical analysis
Statistical analysis was performed to assess the treatment efficacy using R version 3.6.1 software, with dogs as the experimental unit.The primary efficacy assessment of the product was based on the percentage of dogs free of live fleas (stages II and III) in the treatment group.The 95.0% confidence limits for the percentage of dogs free of live T. penetrans were calculated as Wilson scoring intervals.
At each time point of post treatment evaluation, Fisher's exact test (unilateral, using Casagrande, Pike, and Smith [27] continuity correction) was used with a significance level of α = 0.025 to compare the percentage of dogs free of parasites between the TG and the CG.The resulting P values, odds ratios (OR), and 95.0% confidence limits for the OR were obtained from the PROC FREQ using the Taylor series approach.
Secondary efficacy was calculated according to the number of live fleas (stages II and III) in each group on each evaluation day by calculating geometric and arithmetic means using the following formula: where Dc is the mean number of live fleas (geometric and arithmetic) in the control group (total lesions/number of animals), Dt is the mean number of live fleas (geometric and arithmetic) in the treated group (total lesions/number of animals), and X is the experimental day.
Notably, the geometric mean is used in cases of counting zero fleas on a dog, according to the following equation: where Xg is the geometric mean and n is the number of individuals in the group.
To be considered effective, a flea product must reduce parasites by more than 90.0%[28]; therefore, this percentage was used in this study to assess formulation effectiveness.
Secondary efficacy(%) = 100 × Dc at day X−Dt at day X Dc at day X Geometric mean X g = n i=1 (X + 1) Arithmetic mean and mean SCADT scores in the two groups were compared at each post-treatment evaluation timepoint using Shapiro-Wilk test to identify the normality of data.If the data distribution was nonnormal, the Mann-Whitney U test was performed with the level of significance set to α = 0.05 (two-sided).

Results
The study dogs, aged between 3 months and 11 years, were all mixed breed, intact, and weighed between 2.5 and 33.3 kg.On day 0, before treatment, T. penetrans mean live flea counts were 10.3 ± 9.8 in treated dogs and 11.5 ± 13.9 in untreated dogs, whereas the flea count per dog was 1-94 fleas on treated dogs and 1-108 on control dogs.The study groups were comparable on day zero in terms of age, weight, sex, and SCADT distribution.The data from the animals in each group are presented in Table 2.
Three dogs died during the study, but none of the deaths were related to the administered treatment.One dog from the control group died due to the ingestion of a toxic substance applied at home by the owner, and the other two animals (one from the control group and one from the treated group) were hit by cars.However, data collected until the time of their deaths were considered in the analyses.No adverse clinical effects such as systemic allergic responses, vomiting, or diarrhea were reported.

Primary efficacy
Data related to primary efficacy (calculated based on the number of animals without fleas) are shown in Table 3 and illustrated in Fig. 1.From days 7-21, the primary efficacy remained above 95.0%,reaching 100.0%(31/31) between days 28 and 42.In the CG, the proportion of parasite-free animals did not exceed 40.0%throughout the study period.Statistical comparison of parasite-free individuals between the TG and CG demonstrated that treatment resulted in significant differences between the groups on each experimental day throughout the study from day 0 to 42 (P < 0.0001, statistical values of each evaluation day are in Table 3).Therefore, the treatment was effective in preventing T. penetrans infestation in dogs.

Secondary efficacy
The secondary efficacy (based on the number of live fleas) of fluralaner on day 7 was 99.7%, remaining above 99.0%from days 7 to 21, and reaching 100.0%from days 28 to 42.The arithmetic and geometric means were similar on all experimental days in the treated group.The arithmetic mean of live fleas was significantly low in the treated dogs (P < 0.0001, statistical values of each evaluation day are in Table 4) from days 7 to 42.The arithmetic means of live fleas in the CG varied between 3.7 and 7.1 and in the TG varied between 0.0 and 0.4, during all the experimental days of the study.
The geometric means of live flea counts for each study group at all evaluation times are shown in Fig. 2.   The presence of lesions before and after treatment was monitored using the photographic records of the enrolled dogs.Examples of these three animals are shown in Figs. 3, 4 and 5.

Total SCADT
The means of the SCADT by study day and study group as well as the amplitude ranges are shown in Table 5.The mean SCADT score differed significantly between the groups from days 7 to 42 (P < 0 0.05, statistical values of each evaluation day are in Table 5).On days 35 and 42, the mean severity scores in the treatment group were zero.

Discussion
The Bravecto ® 1-Month (Defenza ® ) formulation, with monthly indication, was chosen for the study due to its potent systemic active compound from the isoxazoline family with acaricidal and insecticidal action.The formulation is effective against infestations caused by several species of ectoparasites [29][30][31], including T. penetrans fleas [23], and has a lower cost than that of Bravecto ® chewable tablet, which is effective for 3 months.
Fluralaner is a potent inhibitor of the GABA-controlled chloride channel with specific action on arthropods, consequently causing the death of the ectoparasite [32].The systemic action of fluralaner is an important pharmacological characteristic because its effect is not affected by rain or bathing, which represents an advantage when compared to topical products with the same pharmacological action [33,34].This particularity is relevant for dogs raised in semi-domesticated areas, as they are exposed to rainwater and swim in rivers and seas, as was the case for the animals in this study.
We observed that a single oral administration of fluralaner at a dose of 10-18 mg/kg (Bravecto ® 1-Month/ Defenza ® ) effectively protected dogs against T. penetrans infestation for at least 6 weeks under field conditions in an endemic region with high infestation rates (i.e., high environmental exposure).In the first 3 weeks after treatment, the primary efficacy was greater than 95.0%, reaching 100.0%protection in the 4th week and remaining for the following 2 weeks.Notably, this is one of the first monthly formulations tested with proven efficacy against T. penetrans in dogs.Considering that the monthly formulation of fluralaner presented similar results to those achieved by the quarterly formulation for the treatment of canine tungiasis [23], we inferred that even at a low dosage, the effectiveness of the active principle in the monthly formulation would remain high.
Additionally, secondary efficacy ranged from 99.0% to 100.0%, thereby revealing the treatment's impact on the T. penetrans reproductive cycle, which was previously demonstrated in a similar article [23].The reduction in the number of flea-releasing eggs in the environment may favor the reduction of infestation in animals that share the same environment as the treated dogs.This environmental effect is a possible cause for the reduction in the number of tungiasis-positive dogs in the untreated group [35].
Furthermore, the low SCADT averages observed in the treated animals demonstrated that they showed few acute local clinical signs such as pain, edema, hyperemia, and ulceration, compared with untreated dogs, as observed in similar studies [23,35].In view of this, the present results allow us to infer that the treatment provides an improved quality of life and consequently improves the well-being of animals by eliminating sand fleas.Furthermore, no dog treated with Bravecto ® 1-Month (Defenza ® ) manifested any adverse reactions after administration, demonstrating that the formulation is safe for dogs [23,[36][37][38].
The formulation is effective in combating T. penetrans for at least 42 days; hence, we also suggest the formulation's use as a disease control tool for owners who wish to medicate their dogs for a short period of time.This will aid in treating or preventing infestations, especially in the case of tourists traveling to tungiasis-endemic areas, such as the area in this study.We emphasize that the control of tungiasis is a public health issue, especially in endemic areas of Brazil, where dogs are identified as one of the main risk factors for tungiasis.
We suggest the potential use of Bravecto ® 1-Month (Defenza ® ) in public health programs aimed at controlling tungiasis, as well as in endemic areas by dog owners.This is owing to the formulation's commercial availability and low cost compared with that of other proven effective options.

Conclusions
The Bravecto ® 1-Month (Defenza ® ) formulation, orally administered in a single dose of 10-18 mg/kg, was effective against natural T. penetrans flea infestations in dogs 7-42 days after administration.The dogs treated in this study showed an improvement in SCADT associated with tungiasis.

Table 1
Severity score for acute clinical signs of designated topographic tungiasis a Regardless of the number of foci and the size of the area involved b Three or more lesions close together (1-2 mm apart) c Mutilation of lesions indicates severe itching d For each ectopic body part involved, up to a maximum of eight ectopic sites; maximum four pointsTherefore, the maximum individual score (SCADT) for a dog was 27(23 + 4)

Fig. 1
Fig. 1 Percentage of flea-free dogs on each assessment day by study group

Fig. 2 Fig. 3
Fig. 2 Geometric means of live flea counts by study group over time

Fig. 4 Fig. 5
Fig. 4 Evolution of vital lesions in two treated dogs.a Animal 12 of the TG on day 0, with multiple lesions in stages II and III in the forepaws (arrows).b On day 7 of the evaluation, dog 12 after treatment, the footpads no longer present any vital lesions, it is possible to observe lesions in stages IV and V on the paws (arrows).c The same dog 12 on day 42 post-treatment with completely healthy front paws and no lesions caused by T. penetrans.d Animal 24 of the TG on day 0 with several vital lesions on the hind paws (arrows); e Animal 24 on day 7 of the study showing hind paws with desquamated epithelium, in the phase of re-epithelialization. f The same dog on day 42 of evaluation without the presence of lesions or scars from lesions caused by T. penetrans on the hind paws.Source: Personal archive

Table 2
Age, weight, sex, breed, flea count, and SCADT on day 0

Table 3
Number and percentage of flea-free dogs by study day and study group a CI confidence interval b Fisher's exact test, α = 0.025 c OR Odds ratio, CI Confidence Interval

Table 4
Tunga penetrans counts (percentage of efficacy and P-values) in treated and untreated dogs on all treatment days Wilcoxon test (Mann-Whitney U test), α = 0.05