Efficacy of a Single Dose versus Triple Dose Regimen of Mebendazole against Hookworm Infection among School Children: A Randomized, Single blinded Trial.

Background : The current control efforts against soil transmitted infection focused on and transmission of dose of mebendazole and albendazole regimen. Single dose mebendazole is one of extensively applicable drug regimen as a preventive chemotherapy in hookworm endemic areas. However, nowadays, studies reveal single dose treatment regimen has poor and unsatisfactory efficacy status against hookworm infection. We evaluated the efficacy status of single dose (500mg) versus triple dose (100mg) of mebendazole against hookworm infection among school aged children. Methods : This randomized, single-blinded clinical trial took place in a primary school on Burie and Debre Elias towns, Northwest Ethiopia among school-aged children (6-14). Using simple randomization, eligible hookworm positive children were randomly allocated (1:1) to either a single dose or triple dose of mebendazole arm. Stool samples were collected at baseline and follow-up period (14-21 days after treatment) for McMaster analysis. The primary and secondary outcome measures in this study were cure rate (CR) and egg reduction rate (ERR), respectively. Results were displayed using tables and figure. Independent t test was used to compare group means, logistic regression was used to calculate odds ratio (OR), and P-value < 0.05 at 95% CI was considered for statistical significance. Result: 109 children were allocated for each treatment arm and 103 children were completed the drug efficacy follow up study. Cure rate against hookworm was significantly higher in triple dose (96.1%) than in single dose (30.8%) with (OR=55.125; 95% CI: 11.92-254.9; P < 0.001). Egg reduction rate against hookworm infection in triple dose (99.5%) was also significantly higher than single dose (68.9%) with difference t (101) =5.38; 95% CI 230.95-505.36; P < 0.001. Conclusion : Single dose regimen of mebendazole for the treatment of hookworm infection showed poor efficacy, while triple dose revealed satisfactory efficacy. Therefore, we recommend for giving special emphasis on current deworming program which implemented an imminent need to revise the treatment guideline and give special emphasis on the current deworming programs. Moreover, we recommend conducting further study on the efficacy of single and triple dose regimen of mebendazole against hookworm using molecular diagnostic techniques to explore and investigate the parasitic infection other than larval/oval stage of the parasite. And also better to undergo further investigation on species identification with respect to efficacy status of the drug.


3
Hookworms are nematodes belonging to the family Ancylostomatidae. It has two major genera that accounts almost all human infection, Necator (Necator americanus) and Ancylostoma (Ancylostoma duodenale) (1). They affect most impoverished people in the world (2). It is among the neglected tropical disease which poses a global disease burden by infecting over 576 million people around the world. The disease burden predominantly associated with anemia particularly in children and which causes physical and intellectual growth retardation (3).
Moreover, it has considerable public health importance in low and middle income countries with an estimated 439 million people were infected and causing 3.2 million disability-adjusted life years (DALYs) (4)(5)(6). China and Sub Saharan Africa (SSA) are regions where highest prevalence of hookworm infection occur with nearly 200 million infections found in each country. It also occurs in almost half of SSA's poorest people, estimated with 117 million infected individuals, including 40-50 million school-aged children and 7 million pregnant women (5,7). Besides, it causes 1.6 million DALYs and US$139 billion annual productivity losses in this region (29,30). Because of its' high transmission potential and regardless of immune response produced by infected individual, the adult worm reside in the gut for many years (10).
In general, over 267 million preschool-age children and over 568 million school-age children live in areas where these parasites are intensively transmitted and are in need of treatment and preventive interventions (11).
Nowadays, control efforts for hookworm infection are implementing to reducing morbidity and infection intensity through periodic mass drug administration of affected populations with anthelminthic drugs in frame work of preventive chemotherapy (12,13).
Single dose mebendazole (500 mg) is one of extensively used and recommended benzimidazole (BZ) class of drugs under the list of world health organization (WHO) essential medicine for treating and controlling helminthic infection in the endemic areas (14). This controlling effort implement without prior information on the diagnosis status of individual and efficacy status of the drug. As a result, current literatures showed complete cure of hookworm in not achieved with any drug used as a single dose and variable efficacy profiles were observed in terms of cure rate (CR) and faecal egg reduction 4 rate (ERR) against hookworm infection (15).
For instance, based on a recent randomized clinical trial conducted in Tanzania, triple dose (100 mg per day over three consecutive days) of mebendazole treatment shows satisfactory efficacy status against hookworm infection (CR = 98%), while single dose of mebendazole failed to achieve complete cure of this infection with (CR = 13%) (16). Moreover, a study conducted on the efficacy status of single dose mebendazole in terms of faecal egg count reduction rate from six soil transmitted helminthes endemic countries among school children revealed ranged from 65.4% − 95% and with pooled result 80.6% (17). And also, other studies revealed inconsistent and unacceptable efficacy status of single dose mebendazole in terms of CR ranged from 17.6% − 58.5% for treating hookworm infection (14,(18)(19)(20). Generally, this predominantly used drug as a preventive chemotherapy gave unsatisfactory results for clearing hookworm infections in different region throughout the world, despite its relatively cheap cost and wide availability (21).
Furthermore, A triple dose (100 mg per day over three consecutive days) mebendazole is among the recommended and widely used anthelminthic drug for treating hookworm and other soil transmitted helminthic infection throughout the world (22,23). However, this regimen is widely applicable, only few studies were conducted to evaluate its efficacy status against hookworm infection.
The reported result showed varied efficacy status in terms of CR ranging from 26-97.9% and ERR from 85-100% (16,20,24). This inconsistent efficacy status of the drug did not allow concluding on its treatment effect. Therefore, this varied and unsatisfactory efficacy status of mebendazole, especially the single dose which currently underway as a public health intervention mechanism against STH are enforced us for evaluating mebendazole for treating hookworm infection. Furthermore, based on electronic data search, this is the first randomized, clinical trial for evaluating the efficacy status of single dose versus triple dose regimen of mebendazole for treating hookworm infection in the study site and another endemic area in Ethiopia.

Methods
This randomized, single-blinded clinical trial was conducted at Burie and Debre Elias towns' primary schools, Northwest Ethiopia among school-aged children (6)(7)(8)(9)(10)(11)(12)(13)(14). Before the initiation of the study, Prior to participant enrolment, all parents/ legal guardians' of children whose aged 6 -14 years attending the primary school of Debre Elias and Burie town were invited to inform about the objective, the purpose, the procedure of the study, and the potential risk and benefit which that happened in the participant by the research team at school. Parents/ legal guardian of the participant had the chance to ask any questions which need clarification or create any confusion related to the study before they were asked whether they wanted their child to be part of the study or not willing. After all, parents/ legal guardians who permit their child to include in the study were asked to give a written informed consent and verbal assent from each participant.
Moreover, for those parents/legal guardians who unable to read and write were asked to give thumbprint after reading all information which available in the informed consent form by the data collector.
The desired sample size for this study was calculated based on WHO recommended guideline for anthelminthic efficacy assessment study (25). It recommend a sample of 50 positive participants were desired for each parasite tested, therefore based on the guideline a minimum of 100 eligible hookworm positive individual were needed for this study. Based on the guideline to achieve the minimum required sample size, we screened 300 school aged children based on the following assumption: we assume that the prevalence of hookworm infection in the target population is 50% due to lack of recent publication at the study site and with 80% compliance rate at a two-sided 5% statistical significance level. Moreover, 20% as a non-response rate was added by consider the potential loss to follow up.

Participant data and stool sample collection procedure
After obtaining written informed consent from participants' parent/legal guardian and verbal assent from each participant age, sex grade level and other relevant participant data were recorded using 6 WHO prepared protocol for the evaluation of anthelminthic drug efficacy form. Then specific identification number was given to each participant who had permission to participate in the study.
Afterward, each participant received a sterile stool containers labeled with their unique identification number and were asked to provide approximately 10gm of fresh fecal specimen. Children were reminded to avoid any contamination of specimen and specimen were transferred to the nearby health center laboratory in Debre Elias and hospital laboratory in Burie within an hour after the sample collected. McMaster concentration technique, which is the standard reference method for evaluating drug efficacy in Veterinary Parasitology and has recently been evaluated for human helminthes was used for this study (25,26). Hookworm positive children, who were healthy; based on medical history, physical examination, vital signs, hemoglobin level, participants who had signed informed consent by parents/ legal guardians and verbal assent from each participant age <18 years) according to Ethiopian regulation, participants did not took any anthelminthic with in past 4 weeks, females with negative pregnancy test age >12 years, participant who had hemoglobin level >8g/dl, and children who were able to chew the drug were subjected for randomization in the allocated treatment arm.
But those individual who has taken any form of medication containing mebendazole or any other treatment for STH infection within 30 days of entry into the study, who had diarrhea at the time of the first sampling, who experience a severe concurrent medical condition, participant with haemoglobin level below 8g/dl, with known history of allergic reaction to mebendazole, and participants infected with other parasitic infection were excluded from the allocated treatment.

Randomization and drug administration
Finally, eligible hookworm positive children were randomly assigned with (1:1) ration either to the single dose (500mg) or triple dose regimen of mebendazole (100 mg twice a day for three consecutive days) arm of the study. Randomized children were neither stratified by age nor sex or by any means of personal parameter before randomization. Using simple randomization lottery techniques, 55 eligible children were categorized into single dose arm, whereas 54 eligible children in triple dose mebendazole arm. The drug which administered for the study were found in the local market triple dose (100 mg twice a day for three consecutive days) (WORMIN tab) and single dose

Data entry and analysis
Data was entered to Epi-data software to check data completeness and clearance then 8 transferred to SPSS version-23 for statistical analysis. The baseline characteristics of the study population were summarized using frequencies, mean and standard deviation. Only participants who complete baseline and follow up data were included to determine the treatment efficacy. Infection intensity with hookworm were grouped in to light, moderate and heavy infections, according to WHO guideline for soil transmitted helminthes (27).
CR and ERR were used to assess the efficacy of the drug based on the following mathematical calculation. CR means the proportion of individual hosts positive for parasites who become parasitologically negative after treatment (28). Whereas ERR is the arithmetic mean egg count at baseline of the treatment group minus mean egg count at the end of treatment period divided by the mean egg count at the baseline and express it in percent (29).

ERR=100% × 1-arithmetic mean (post-intervention FEC)
Arithmetic mean (pre-intervention FEC) Confidence interval for ERR was calculated using bootstrap re-sampling method with 5000 iterations. Independent t-test was used to compare group means, whereas CRs were compared by calculated Odds Ratio (OR) using logistic regression. For all statistical analyses a P-value of 0.05 was considered as the limit for statistical significance.  table 1). In terms of ERR the triple dose with arithmetic mean (ERR= 99.5%) was also significantly more effective than single dose (ERR = 68.9%) with difference t (101) =5.38; 95% CI 230.95-505.36; p <0.001.

Discussion
The current STH control strategy in Ethiopia, relies on periodic administration of single dose albendazole and mebendazole among WHO recommended anthelminthic drugs (30). Moreover, triple dose mebendazole was used for treatment hookworm infection and other STH infection (23) The effectiveness of both doses of mebendazole assessed through either CR or ERR following drug administration (31). But studies which conducted before revealed varied efficacy status and its inconsistency make a challenge on treatment recommendation. In this study, head to head comparison of triple versus single doses of mebendazole for the treatment of hookworm infection may provide clear and applicable results, since single and triple doses of mebendazole are widely used drugs for treating STHs with variable efficacy status in endemic countries.
This study clearly showed that triple dose is significantly more effective at clearing of hookworm infection than single dose of mebendazole (96.1% vs. 30.8) with (OR = 55.125; 95% CI: 11.92-254.9; P < 0.001). It indicated that participants who treated with triple dose mebendazole had 55 times more likely to cured from hookworm infection than single dose of mebendazole. This significant efficacy variation between the two mebendazole doses might be due to extensive and frequent uses of single dose regimen of mebendazole for deworming program unlike triple dose mebendazole. As a result of this frequent and wide-range use of single dose may raise concern on drug resistance or tolerance.
In this study almost all children who were infected by hookworm were cured (CR = 96.1%) with the ERR 99.5% following administering triple dose of mebendazole. This is strongly in agreement with other RCT studies conducted in Tanzania with CR = 98% and ERR = 100% (16). But it is considerably higher than study reported in China (CR = 58.5) (20) and Brazil (CR = 58.5%) (32). This result variability between our result and summary estimate of other randomized control trial could be due to several factors such as the diagnostic technique, the sample size variation, the age of study participants, the parasite strain, the location of study where it was conducted. For instance, both of the two studies which compared with our finding applied Kato-Katz diagnostic techniques and the one which is conducted in Brazil used Hoffmann's spontaneous sedimentation techniques in addition to Kato-Katz techniques. Besides, both studies were carried out in all age group and with a sample size of 41 and 83 participants in the treatment arm (20,32).
The present study also showed that the efficacy status of single dose mebendazole for the treatment of hookworm infection in terms of CR and ERR were30.8% and 68.9%, respectively. This ERR status indicates that single dose of mebendazole had unsatisfactory efficacy profile for treating hookworm infection as per WHO guideline (25). The efficacy status of 500 mg mebendazole showed doubtful efficacy profile (< 70 &>60%).
This discrepancy between our finding and other studies could be due to several influencing factors such as the diagnostic techniques applied, the study population, location of the study, allocated sample size, follow up period, and the strain of the parasite (16,(18)(19)(20)34,35). For instance, majority of the study utilized the Kato-Katz diagnostic techniques for the assessment of drug efficacy. As a result of the sensitivity and specificity variation of the diagnostic technique utilized might be the possible source for discrepancy of findings.

Conclusion
In this study, triple dose regimen of mebendazole showed satisfactory efficacy and has significantly higher CR and ERR than single dose regimen against hookworm infection. As a result, there is an imminent need to revise the treatment guideline and give special emphasis on the current deworming programs. Moreover, we recommend conducting further study on the efficacy of single and triple dose regimen of mebendazole against hookworm using molecular diagnostic techniques to explore and investigate the parasitic infection other than larval/oval stage of the parasite. And also better to undergo further investigation on species identification with respect to efficacy status of the drug.

Declarations
Authors' contributions TE conceptualized the study, contributed to the format of the data collection instruments, analysis, and write up of the first draft manuscript. MA contributed to the study protocol, development of the data collection tools field data collection and analysis. AJ was participated in the proposal development and analysis; revise the draft and final manuscript. All authors read and approved the final manuscript before submission.  Figure 1 The trial profile. This diagrammatical representation showed the allocation and selection procedure of eligible children for this study