Comparison of the Results of Ultrasound-Guided Using Surface Probe and Non-Ultrasound-Guided Botulinum Toxin Injection into the Internal Anal Sphincter in Children with Constipation-Randomized Clinical Trial.

INTRODUCTION
Constipation, as a common disease, is the reason for 3% and 10–25% of patients to refer to pediatricians and pediatric gastroenterologists, respectively. Constipation is divided into organic and functional groups according to the cause. Functional constipation includes 95% of patients with constipation; while the remaining 5% has an organic cause for their symptoms.[12]
In this regard, among the most common treatments for constipation are training evacuation of the bowel, prescribing high-fiber diets, and using laxatives. A combination of evacuation of bowel training and laxative prescription is typically effective in treating children; however, some patients do not indicate any effective recovery, are resistant to treatments, and have constant symptoms.[3] Therefore, researchers are looking for novel and effective treatment methods in this respect.
As the failure of the internal anal sphincter (IAS) relaxation instead of anal spasm can play a role in the output performance of the anus in evacuating and causing constipation,[4] the injection of botulinum toxin (BTX) into the IAS has been offered as one of the new treatments for chronic constipation in children.[356] Clostridium botulinum is a strong bacterial neurotoxin that acts at the neuromuscular junction and blocks the release of acetylcholine from the presynaptic cholinergic nerves.[789] At present, the injection of this toxin is used for treating various diseases such as strabismus, muscle spasms, a number of digestive disorders including esophageal achalasia, non-relaxation of the sphincter of Oddi in biliary dyskinesia, treatment of pain in chronic anal fissure, treatment of pain following hemorrhoidectomy, and persistence of constipation after a pull-through surgery in patients with Hirschsprung’s disease.[78910]
Despite the existence of many pieces of evidence regarding the effectiveness of the IAS injection of BTX in the treatment of many of the above-mentioned diseases, its effectiveness depends on the correct injection technique and its dose such that the injection should be exactly at the site of the IAS.[7] Consequently, an ultrasound (US)-guided injection with a rectal probe has been suggested to increase the accuracy of determining the injection site.[5]
One of the initiative studies on BTX injection as a treatment for achalasia used US-guided endoscopy to directly inject BTX into the lower esophageal sphincter muscle. This treatment was more effective than the endoscopic injection of BTX without visualization of direct tissue layers.[6] Currently, the use of US in the implementation of anorectal procedures has been investigated in adults and children in many studies.[111213] It has been revealed that US-guided BTX injection is safe and effective for functional bowel obstruction and can avoid the need for definitive surgery.[5] However, it is worth considering that the use of US requires a pediatric rectal probe, which is expensive and less available. In addition, this technique is less employed in developing countries. Hence, this study aimed at comparing the results of US-guided using surface probes and non-US-guided BTX injection into IAS in children with constipation.

MATERIALS AND METHODS
The current study was a single-blind randomized clinical trial. The study population included all children over three years of age with constipation that referred to the Colorectal Clinic of Imam Hossein (AS) Hospital, Isfahan University of Medical Sciences in 2019–2020.
Inclusion criteria comprised children over three years of age with constipation, failure to respond to medical treatment and lifestyle changes proposed by gastroenterologists, absence of Hirschsprung’s typical appearance in a barium enema, presence of ganglion cells in the rectal biopsy, lack of contraindications to BTX, and presentation of written consent to participate in the study.
It should be taken into consideration that the contraindications of BTX consisted of previous allergic reactions, infection, and inflammation in the target site, neuromuscular junction diseases such as myasthenia gravis, use of aminoglycosides, penicillamine, quinine, and calcium channel blockers.
Moreover, the patient was excluded from the study if surgery was needed to treat the patient in cases such as constipation caused by Hirschsprung’s disease with long segments or positive pathology in terms of Hirschsprung’s disease with short segments. Due to the small size of the population, all 40 eligible patients were included in the study.
After obtaining the code of ethics 40 children were entered the study that had already undergone a rectal biopsy and had a pathological result of the presence of ganglion cells in the posterior rectal muscle. The mentioned patients were divided into two groups using random allocation software. The patients’ age and gender were recorded at the beginning of the study.
BTX (disport type A) injection was performed by a single expert anesthetist for all patients under general anesthesia in a lithotomy position using a black spinal needle. In the first group, the US-guided BTX injection into the IAS was performed using a surface probe and with the presence of a radiologist. To do so, a HONDA ELECTRONICS HS-2600 ultrasound machine using a 7.5 MHz surface probe in the anus was employed to determine the IAS at three, six, and nine o’clock in the lithotomy position and 25 units of BTX were injected in each site. It should be noted that the injection at 12 o’clock was avoided due to the presence of the urethra.
In the second group, non-US-guided BTX injection was performed only by touching with the index finger, and the procedure was similar to that followed in the first group in terms of the amount, injection site, and type of BTX. Due to the different injection procedures followed in the two study groups, the researcher was aware of the type of intervention in each of the two groups. However, to avoid any bias in collecting the data, the evaluator checking the patients’ condition in the subsequent follow-ups was not aware of the type of intervention in the two groups.
After two weeks, which is the time of the peak effect of BTX, it was time to refer to the clinic, and the patients were called the previous day to be reminded about their visit. The history of the children’s conditions including the number of bowel movements a week (two or less bowel movements), large diameter stool each time, touching a large amount of feces in the rectum during examination, soiling at least once a week (in a child that has been properly trained), hard stool or pain during the bowel movement, and behaviors during which the child did not want to defecate was taken from patients’ parents. Then, based on the ROME III criteria for diagnosing constipation in children, if children under four years old had two or more of the above-mentioned criteria for at least one month, and if children over four years old had two or more of the above-mentioned criteria for at least two months, the diagnosis of constipation was confirmed. Otherwise, recovery was considered in these patients. Moreover, the occurrence of fecal incontinence in the studied children was also recorded separately.

Data analysis
Finally, the collected data were entered into SPSS software (ver. 26). Data were presented as means ± standard deviation or frequency (percentage). The Chi-squared test and the independent samples t-test were used to compare the frequency distribution of qualitative variables and the mean of the quantitative variables between the two groups, respectively. A significance level of less than 0.05 was considered in all analyses.

RESULTS
In this study, out of 20 patients in the US-guided group with a mean age of 4.85 ± 1.60 years, 13 (65%) and 7 (35%) were male and female, respectively. Moreover, out of 20 patients in the non-US-guided group with the mean age of 5.50 ± 1.76 years, 12 (60%) and 8 (40%) were male and female, respectively (P value < 0.05) [Table 1].
After BTX injection, the US-guided group had a constipation recovery of 95% and 95% in the second week and the first month, respectively. The mentioned finding was significantly more than the constipation recovery in the non-US-guided group with 70% and 75% in the second week and the first month, respectively (P value < 0.05). In the third month after BTX injection, although the recovery status was still higher in the US-guided group, as compared with the non-US-guided group, this difference was not statistically significant (P value > 0.05) [Table 2].
The occurrence of fecal incontinence was not reported in any of the children in the US-guided group; however, the non-US-guided group had fecal incontinence in the second week (45%), the first month (40%), and the third month (30%) after BTX injection. This difference was statistically significant between the two groups (P value < 0.05) [Table 3].

DISCUSSION
The results of the present study indicated that the US-guided BTX injection using surface probe had a greater effect on the constipation recovery than the non-US-guided BTX injection such that this effect was significant in the second week and the first month. In the third month, although the frequency of constipation recovery in the US-guided BTX injection group was more than the other group, this difference was not significant.
Consistent with the findings of the present study, Church et al.[5] showed that US-guided BTX injection into IAS relieved obstructed bowel movement caused by Hirschsprung’s disease and IAS achalasia. In this study, the recovery was reported to be 76% and 65% in the US-guided and non-US-guided groups, respectively. However, this difference was not significant in the mentioned study due to the small sample size. Although the patients in our study were children with constipation without the Hirschsprung’s origin, both studies indicated the positive effect of BTX injection on children’s bowel movements.
The results of Randhawa et al.’s[14] study on the effect of BTX injection guided by Endoscopic Ultrasound (EUS) for anal fissure resistant to treatment also indicated that six patients had excellent, one patient had moderate, and one patient had no response to treatment within two months after BTX injection. Therefore, they generally concluded that EUS-guided BTX injection into the IAS can be a promising technique for patients with anal fissure resistant to treatment with pain.
Another study also investigated the BTX injection on 73 children with HD or IAS achalasia and figured out that 89% of initial clinical recovery was obtained two weeks after the intervention, and 53.4% of patients had excellent treatment outcomes in the long term.[15] Although their study did not address the significance of employing the US in determining the exact injection site, they reported that the duration of the effect of BTX injection was three months on average. In the present study, a difference was found between the two procedures of BTX injection in the second week and the first month; however, no difference was found in the injection procedure (with or without US) on the effectiveness of the two groups’ three months after the intervention. In other words, it can be said that the positive results of US-guided BTX injection were more visible in the short term so in the long term, the procedure of injection and correct determination of the injection site does not affect the recovery of the patient’s bowel movements.
In addition, a meta-analysis reviewing previous literature aimed at evaluating the two techniques of posterior IAS myectomy and intrasphincteric BTX injection andreported that frequent bowel movements were more regular with myomectomy, as compared to BTX with similar complications, constipation, and soiling rates. However, the subsequent need for surgical treatment with BTX injection was higher.[16] This approach is not far-fetched. It may be argued that the goal of BTX injection into IAS is not a definitive treatment management, but a temporary relief because obstructive bowel movements associated with HD often improve over time.[17]
It should be taken into consideration that numerous factors are involved in the effectiveness of BTX injection. For example, the storage conditions of BTX, which must be followed by the cold chain, should be considered; otherwise, the onset of the effect will be delayed and it will have less or no effect. Furthermore, the patient’s metabolism, which is effective in the duration and extent of the effect of BTX, should be taken into account. Other factors comprise the amount of the use of the injected muscle, the correct injection site, the dose of BTX that is probably more effective in younger children, and the heating of the injection site that causes BTX to spread to nearby muscles. Also, the duration of the Botox effect is between 3 to 6 months. In addition, it should be noted that BTX injection into the IAS is not a first-line treatment for functional obstructed bowel movements and constipation. Rather, medically, primary treatments include rectal washing with or without systemic antibiotics, diet modification, stool softeners, laxatives, and behavioral therapy.[1819] Our patients were no exception to this rule, and when the aforementioned treatment options failed, intrasphincteric BTX injection was recommended. At present, what is of particular importance for researchers is that, in addition to the effectiveness of the treatment, the least complications are looked for to avoid endangering the patients.
In this regard, the results of this study in examining the complications caused by this intervention indicated that in the second week and the first and third months, the occurrence of fecal incontinence as a complication caused by BTX injection was less than 50% and reported only in the non-US-guided group. In other words, it seems that if the BTX injection is performed using the US by superficial probe, it will not be associated with any complications due to the high accuracy in determining the injection site.
Chumpitazi et al.[15] also reported 9.5% of temporary fecal incontinence in their study. Although the percentage of reporting fecal incontinence was higher in our study, it seems that the percentage of this complication can be reduced to zero by determining the exact injection site.
The results of another study investigating the EUS-guided BTX injection into the anal fissure resistant to treatment did not indicate any complications caused by this intervention.[14] Besides, Ravichandran et al.’s[20] study figured out that all non-US-guided injections were finally followed by a more definitive method, while 50% of patients undergoing US-guided injections in the 87-week follow-up did not need a more definitive surgery.
It is crucial to emphasize that the pharmacokinetics of BTX are highly dependent on the injection site, and we hypothesize that improving the accuracy of determining the injection site by limiting the toxin through natural tissue planes will take advantage of this fact.
The small sample size, the lack of long-term follow-up (more than three months), and the examination of patients’ need for more definitive surgical treatments after this injection (such as myomectomy, appendicostomy, or colostomy) can be some of the limitations of this study. However, the clinical trial nature of this study, the injection of US-guided BTX with a surface probe, and the implementation of this study only on children with chronic constipation without Hirschsprung’s origin can be considered as its strengths and add to the accuracy and validity of this study. It is suggested to address the effect of various techniques of BTX injection in various diseases such as IAS achalasia, pain treatment in chronic anal fissures, after hemorrhoidectomy surgery, and constipation after surgery in HD patients in future studies. In addition, examination of the average dose and number of BTX injections in different age ranges can be illuminative in this regard.

CONCLUSION
The findings of the present study revealed that the US-guided BTX injection by using a superficial probe, as compared with non-US-guided BTX injection, had a greater effect on constipation recovery and did not cause any complications (fecal incontinence).

Ethics approval and consent to participate
This study received the Isfahan University of Medical Sciences (Approval code: IR.MUI.MED. REC.1398.249), written consent from the eligible children’s parents to enter the study, and the clinical trial code (Code: IRCT20230704058661N1).

Conflicts of interest
There are no conflicts of interest.