This randomized, single-blinded clinical trial was conducted at Burie and Debre Elias towns’ primary schools, Northwest Ethiopia among school-aged children (6-14). Before the initiation of the study, ethical clearance was obtained from Ethical and Review Committee of School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar, Ethiopia. This trial is retrospectively registered with www.pactr.org, number PACTR201911466695052 on November 26, 2019.
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 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).
Upon completion of all the baseline parasitological and participant information survey, all children who found positive for hookworm infection were subjected to physical and clinical examination by senior health officer. Then height was measured with a standard meter (to the nearest 0.1 cm), and weight with an electronic balance (to the nearest 0.1 kg). Haemoglobin levels were measured in capillary blood using the finger-prick method (HemoCue®301).
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 (500mg) (Vermox®) of mebendazole. A slice of biscuit was given to each eligible child before time of drug administration.
Eligible children who were randomized in single dose mebendazole ordered to take the drug in front of their parents and public health officer at school. While children who were randomized in triple dose arm; parents/guardians were asked to took the sealed drug with envelops and instructed to give the drug for their child with proper time to avoid interruption of time for the second dose of administration. Moreover parents/guardians were instructed to follow their child up to the end of treatment and reminded not to drink alcohol.
Moreover, participants/parents/guardians were reminded to notify about any medical discomfort happen following treatment to the investigators or the nearby health extension worker. Children were revisited after 14 -21 days of drug administration and asked to provide approximately 10gm of fresh fecal specimen for the second time using sterile stool container labeled with their identification number.
At the time of second visit, children were asked about the occurrence of vomiting and diarrhea following drug administration. A participant who vomited within 4 hours after drug administration and participant with diarrhea were excluded for the final analysis. Similarly, to the baseline parasitological investigation McMaster diagnostic technique was utilized at follow up time. Laboratory quality control was performed through re-reading 10% of the slides of each laboratory technician by an expert microscopist.
Data entry and analysis
Data was entered to Epi-data software to check data completeness and clearance then
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.