Lateral internal sphincterotomy can be safely offered as first-line therapy in selected patients pharmacologically naive with no underlying FI (ASCRS grade 1A) [3]. Although its healing rate ranges from 88 to 100%, anal incontinence after surgery occurs frequently (8–30%) and is permanent in some patients [3, 20]. The risk of permanent incontinence dictates the need for a treatment with no persistent negative effects, and local BT injection seems to be a good candidate since it is a less invasive and safe procedure [16]. The healing rate with BT at 2 months was as low as 29.2% and reached values up to 96% [2, 16, 17, 21]. In a voluntary survey addressed to all ASCRS members, 89.4% of respondents injected 50–100 U of BT to treat AFs [22]. Studies conducted thus far are heterogeneous regarding techniques, BT doses injected, follow-up time, and results [2, 17], leading to a recommendation based on moderate-quality evidence (ASCRS grade 1B) [3]. In fact, in a retrospective study conducted by Brisinda et al. in 1,003 patients with symptomatic chronic AF, the dosage of toxin and the site of injection had an impact on the healing rate [21]. The dose significantly correlated with healing at 2 months, with 29.2%, 75%, 79.3%, and 83.9% healed patients treated with 15 U, 20 U, 30 U, and 50 U of BT, respectively (p < 0.001) [21]. Of note, a high-dose circumferential chemodenervation-IAS approach using 100 U of BT yielded a healing rate of 90.7% at 3 months [23]. Moreover, healing had a significant correlation with the site of injection as well (healing rates at 2 months were 59.2% and 82.1% in the posterior and anterior injection, respectively, p < 0.001) [21]. Recurrence of healed AF with BT was 55% at 3 years [24] and mean resting anal pressure at 2 months post-BT injection (71.1 ± 16.2 mmHg) was significantly reduced when compared to baseline mean value (96.1 ± 18.0 mmHg; p < 0.001) [25]. A higher incidence of treatment failures was observed in men, which may be attributed to the presence of greater sphincter hypertrophy during baseline physical examinations and increased hypertonia in initial manometry. It may be advisable to consider increasing the dosage for these patients. Treatment with BT resulted in a significant improvement in both pain intensity score (variation: -4.2 ± 2.9; p < 0.001) and pain post-defecation score (variation: -5.1 ± 3.0; p < 0.001) [26]. Another study established an 82% rate of reduction in pain scores defined as minimum reduction in discomfort of 50% in AF patients treated with BT [16]. In our study, the healing rate measured at the 2-year visit was 83.9%. Given the entire population (N = 49), healing rate at the last monitoring visit was 65.3%. Also, improvement/healing rate was maintained over 70% during follow-up visits starting at 1 month. All those values are within the range of healing rates at 2 months obtained in previous studies (range: 29.2%-96%) [2, 16, 17, 21]. Therefore, our results suggest that the efficacy is maintained at 2 years. However, follow-up was difficult due to COVID-19 pandemic and only 31 patients out of the initial 49 attended the 2-year visit. Although some studies suggest that dose and site of injection do not have an influence on BT injection outcome [27, 28], the only differences in treatment between our study and the one by Arroyo et al. [24] were precisely dosage and site of injection. A mean dosage of 40.5 U was used in the present study, administered in both lateral commissures, and in the posterior one, while Arroyo et al. used 25 U injected in both lateral commissures, and in the anterior commissure, yielding a higher recurrence rate at 24 months (55%) [24]. A recent study comparing sites of BT injection found no differences in long-term healing rates while showing differences in postoperative pain scores that yielded better patient satisfaction [29]. In accordance with other studies [24,25,26], all pressures measured in the manometry (anal resting pressure, voluntary squeeze pressure, and pressure during VM) were significantly reduced compared to baseline both at first- and third-month visits. Our results show a greater reduction in voluntary contraction pressures (related to the external sphincter) at 3 months. Moreover, basal pressure values (related to the internal sphincter and therefore to fissure healing) also improved. This demonstrates that, even if BT is injected at the intersphyncteric level, its paralyzing effect is observed in both sphincters. On this basis, it could be theorized that, if the aim of the treatment is to relax only the internal sphincter, BT could be injected into the muscular mass of the IAS and not at the intersphincteric level. Those results agree with the literature where BT injection induces an improvement in pain under several assessment techniques [16, 30]. Regarding safety, the main AEs previously reported were temporary incontinence, perianal hematoma and thrombosis, thrombosis of external hemorrhoids, prolapse of internal hemorrhoids, and perianal abscess [17]. In the present study, only 7 patients (14.3%) reported AEs, all of which have been previously described. Finally, incontinence was assessed in our study with the Wexner scale score. In accordance with previous studies where incontinence was reported in 5% of patients at 2 months but was not present at 6 months anymore [24], there was a slight increase in incontinence at the first month post-injection that was not further maintained.

Study limitation was the monocentric design which could induce selection bias and limit the external validation so that the results must be interpreted carefully. Furthermore, the heterogeneity and small sample size limit both the theoretical and statistical robustness of the study, particularly with regard to the logistic regressions, as these factors may compromise both the external and internal validity of the findings. Additionally, the effectiveness of the treatment must be interpreted with caution due to the absence of a control group.

In conclusion, this study provides further evidence to support the use of incoBoNT/A injections to treat AFs that persist after treatment with the conservative approach and topical nitrates/CCB. BT injection is a non-invasive treatment that should be considered before other invasive treatments due to its efficacy and safety which includes no permanent impairment. However, on the basis of the mentioned limitations, further research involving more homogeneous populations is necessary to draw more definitive conclusions regarding the treatment's efficacy.