Efficacy and Safety of Neem Seed Extract Compared with Clotrimazole in Tinea Corporis / Cruris : A Randomized Controlled Trial

Introduction: Tinea corporis and tinea cruris are common superficial fungal infections. Neem (Azadirachta indica) seed extract has shown antifungal properties in vitro, but no clinical studies have been done for superficial dermatophytoses. This study compared the efficacy and safety of 5% neem seed extract in cream base with 1% clotrimazole cream when used twice daily for four weeks, in the treatment of localized tinea corporis and/or tinea cruris. Materials and Methods: This is a randomized, double blind, clinical trial. Patients with localized tinea corporis and/ or tinea cruris were evaluated at baseline, week 1, week 2 and week 4. Clinical, mycological and effective cure rates of both treatment groups were determined. The patients’ post treatment overall satisfaction scores and the incidence of adverse effects were also documented. Results: Sixty patients were recruited, and 30 patients were randomized to each treatment arm. There were no significant differences in post treatment scores (p value=0.221). Effective cure, defined as the combination of marked clinical improvement and mycological cure, was seen in 20/30 (66.67%) in the neem seed extract group and 24/30 (80%) in the clotrimazole group (relative risk = 1.67, 95% confidence interval = 0.69 – 4.0). Post treatment overall satisfaction scores were comparable (p value = 0.333). Two patients experienced adverse effects in the neem seed extract group, while there were no adverse effects in the clotrimazole group. Conclusion: Efficacy and safety of 5% neem seed extract in cream base is comparable to 1% clotrimazole cream for the treatment of localized tinea corporis and/or tinea cruris.


Introduction
T inea cruris and nea corporis are common infec ons caused by the dermatophytes like Trichophyton tonsurans, Trichophyton rubrum and Microsporum species.2][3] Clinically, superfi cial dermatophytoses are characterized by annular, scaly lesions with central clearing.While nea cruris presents at the groin, nea corporis may aff ect any other part of the body. 4The diagnosis of superfi cial dermpatophytosis can be done by clinical examina on, potassium hydroxide examina on and fungal cultures. 5,6eatment of nea corporis and nea cruris varies according to the areas involved.For localized infec on, topical therapy is recommended.Systemic therapy may be indicated for extensive skin infec on, immunosuppression, resistance to topical an fungal NJDVL.Vol 16, No.1, 2018 therapy, and comorbidi es of nea capi s or nea unguium. 7otrimazole is widely used for the treatment of superfi cial dermatophytosis. Neem (Azadirachta indica) belongs to family milliaceae (mahogany), and has been studied for various therapeu c proper es including an -bacterial, anfungal and wound healing eff ects. 10Sulfur-containing compounds (cyclic trisulfi de and tetrasulfi de), gedunin and nimbidin are components of neem shown to have an fungal proper es in vitro. 11The mechanism of ac on is not well understood, but it is postulated that neem seed extracts may inhibit proteases which are needed in fungal physiology and development. 12 the best of our knowledge, this is the fi rst clinical study comparing neem seed extract with an azole in the treatment of nea corporis and/or nea cruris.

General Objec ve:
To compare the effi cacy and safety of 5% neem seed extract in cream base with clotrimazole 1% cream when used twice daily for four weeks, in the treatment of nea corporis and/or nea cruris.

Specifi c Objec ves:
1. To compare the post treatment clinical severity scores of both groups.2. To compare the propor ons of pa ents with marked clinical response a er 4 weeks of treatment in both groups.3. To compare the propor ons of pa ents with mycological cure rates a er 4 weeks of treatment in both groups.4. To compare the propor ons of pa ents with eff ec ve cure (marked clinical response and mycological cure) a er 4 weeks of treatment in both groups.5. To compare the pa ents' overall sa sfac on scores at the end of treatment.6.To compare the incidence of adverse eff ects in both treatment groups.

Study design
This is a randomized, double blind, controlled trial comparing the effi cacy and safety of 5% neem seed extract cream and clotrimazole 1% cream when applied twice daily for the treatment of nea corporis and/or nea cruris.The study was performed in accordance with the Declara on of Helsinki and Good Clinical Prac ce Guidelines.Informed consent from the pa ents was likewise secured prior to treatment, a er each subject was briefed regarding the nature of the study.

Randomiza on, treatment alloca on, and blinding
The study sta s cian generated a list of random numbers using the table of random numbers.An assigned resident, who was blinded to the codes, allocated the treatments randomly using the list and dispensed the packaged jars accordingly.The codes were not disclosed to the inves gators un l the end of the study.

Materials
A previous in vitro study by Natarajan et al 12 determined that the minimum inhibi on concentra on of neem seed extract against dermatophytes was 15 μg/ ml.Based on this, a concentra on of 5% neem seed extract was determined for this study.The neem seed extract was compounded in cream base by a registered industrial pharmacist from the University of the Philippines, Manila.Clotrimazole 1% cream was obtained from a local pharmacy (Dermskin, Philippines).The two study creams were similar in appearance and were packed in iden cal jars and were coded A or B by the same industrial pharmacist.

Interven on
The pa ents in both groups were instructed to apply their assigned cream on aff ected areas twice daily for four weeks.The selec on of a four-week treatment period was based on standard therapy for superfi cial fungal infec ons with most currently available topical an fungals. 14The pa ents were also instructed not to apply any other topical, or take any oral medica ons.

Clinical assessment
The pa ents were evaluated at baseline, and at 1, 2, and 4 weeks by the primary inves gator.The primary endpoints of the study were the propor on of pa ents who demonstrated marked clinical response, mycological cure and eff ec ve cure.Secondary endpoints included the clinical severity scores throughout treatment, pa ents' overall sa sfac on scores post-treatment and the incidence of adverse events during the study.
Clinical severity scores were determined through an evalua on of scaling, erythema, burning, and itching.These were assessed using a four-point scale of 0 to 3 (0 = absent, 1 = mild, 2 = moderate, 3 = severe).The scores for each parameter were added to give the total clinical score.A marked clinical response was considered to be a reduc on of ≥ 2 in the clinical score or a final value of zero.All clinical evalua ons were done by the same blinded evaluator.Potassium hydroxide examina ons were done by the same medical technologist for all pa ents.A mycological cure was defi ned as a nega ve KOH smear.Eff ec ve cure was defi ned to be the occurrence of both a marked clinical response and a mycological cure.Clinical evalua on was done at baseline, week 1, week 2 and week 4 of treatment, while KOH examina ons were done at baseline, week 2 and week 4. Monitoring of adverse events and digital photography were also done during every visit.
Overall sa sfac on scores were obtained post treatment by asking each pa ent to assess his or her overall sa sfac on using a four-point scale of 0 to 3 (0 = worse, 1 = the same, 2 = somewhat improved, 3 =markedly improved).

Stopping guidelines
The study was stopped in pa ents who experienced burning, erythema, or pain on applica on.Appropriate rescue treatment and monitoring were delivered.These pa ents were considered as withdrawals from the study.Those who did not comply with the treatment regimen, or those who used other topical medica ons, were also withdrawn from the study.Dropouts were defi ned as those who did not follow up within two weeks and whose outcome was unknown by the end of the study period.

Sample size
Based on the 2013 census of the Jose R. Reyes Memorial Medical Center Dermatology Outpa ent Department, there were 386 cases of nea corporis and 290 cases of nea cruris seen out of a total of 30,328 pa ents.Sta s cal computa ons indicated a minimum of 60 pa ents to achieve a 95% level of confi dence, precision of 5%, with allowance for a 20% dropout, power of 80%, 0.05 signifi cance level and equal number of cases and controls.

Data processing and sta s cal analysis
For demographic characteris cs, student's t-test was used for con nuous variables and Pearson's chi-square test for categorical data.Repeated measures analysis of variance was used to compare clinical severity scores while student's t-test was used to compare post-treatment overall sa sfac on scores.Treatment eff ects such as rela ve risk, risk reduc on and number needed to treat were also computed.Sta s cal analyses were performed using STATA Version 10 (Stata Corp LP, College Sta on, TX, USA).An inten onto-treat analysis was used for all pa ents included who received at least one dose of treatment.Test results that produced p values of <0.05 were regarded as sta s cally signifi cant.

Results
Of the 72 individuals who were screened, 60 met the entry criteria and were randomized to treatment (neem seed extract group, n=30) and control (clotrimazole, n=30) groups.Of these pa ents, two were withdrawn from the treatment group because of adverse eff ects.There were no dropouts in both groups.Because an inten on-to-treat analysis was performed, all 60 pa ents were included in the full analysis (Figure 1).
The baseline characteris cs of the study popula on are summarized in Table 1.No sta s cally signifi cant diff erences were noted between the two groups based on age, sex, aff ected site, and baseline clinical scores.
Both treatment groups showed decreasing clinical severity scores throughout treatment (Figure 2).By week 2 of treatment, 25/30 (83.33%) pa ents in the neem seed extract group and 28/30 (93.33%) in the clotrimazole group had marked clinical response.A er 4 weeks of treatment, all the pa ents in both groups had marked clinical response.Post treatment clinical severity scores were comparable in both groups (p value = 0.221).There is no sta s cally signifi cant diff erence between two groups.
By the end of the treatment, 20/30 (66.67%) in the neem seed extract group and 24/30 (80%) in the clotrimazole group had eff ec ve cure of their disease {Rela ve risk (RR) = 1.67, 95% confi dence interval (CI) = 0.69 -4.0).Rela ve risk reduc on (RRR) for failed outcomes (those who did not achieve eff ec ve cure) was 67% (RRR = 0.67, 95% CI = 0.31 -3.0).The number needed to treat revealed that 8 pa ents were required to be treated with neem seed extract cream to demonstrate eff ec ve cure.(Table 3) Post treatment overall sa sfac on scores were comparable in both groups (p value = 0.333) (Table 4).Two pa ents experienced adverse eff ects in the neem seed extract group, while there were no adverse eff ects documented in the clotrimazole group.

Discussion
Both neem seed and leaf extracts have demonstrated an fungal eff ects, but the former has a lower minimum inhibitory concentra on and is more readily available. 13Neem seed extracts have also been used for scabies, 14 pediculosis capi s, 15 and acne with minimal to no side eff ects.A study published in 2015, compared the effi cacy of neem seed oil, terbinafi ne cream and a combina on of both in treatment of dermatophytosis.It was concluded that neem seed oil is equally eff ec ve as terbinafi ne cream in the treatment of dermatophytosis. 16 our study, both the neem seed extract and clotrimazole groups signifi cantly improved the clinical symptoms of the pa ents.The improvement with the use of neem seed extract may be a ributed to its eff ect on fungal proteases, which are considered as virulence factors because they are needed by fungi for adherence and penetra on. 9In addi on, proteases may also induce infl ammatory responses by altering the permeability of the epithelial barrier and by induc on of pro-infl ammatory cytokines through proteaseac vated receptors.These enzymes may further contribute to infl amma on through interac ons with the kinin system as well as the coagula on and fi brinoly c cascades.Their eff ect on the host protease-an protease balance results from ac va on of endogenous proteases and degrada on of protease inhibitors. 17Therefore, the clinical improvement with the use of neem seed extract may be explained by the preven on of microbial spread and inhibi on of infl ammatory mediators associated with the inhibi on of dermatophyte proteases.
Although the majority of the pa ents in the neem seed extract group did not experience adverse eff ects, two pa ents developed irrita on, erythema and pruritus on applica on.Possible causa ve components in neem include triterpenoid compounds (such as azadirach n and nimbin), and coumarins. 18Future studies may focus on diff erent concentra ons of neem seed extract to determine if a lower concentra on may have fewer side eff ects and s ll maintain effi cacy.Also, a comparison with other exis ng topical an fungals may

Table 1 :
Demographic characteris cs of the study popula on.

Table 2 :
Number of pa ents showing marked clinical response, mycological and eff ec ve cures.

Table 3 :
Table showing rela ve risk calcula on

Table 4 :
Overall sa sfac on scores at the end of the treatment.