This randomized, open-label clinical trial was conducted at Burie and Debre Elias towns’ primary schools, Northwest Ethiopia, from March to May, 2019. This study included school-aged children aged 6 to 14 years. The study was approved by the Ethical and Review Committee of School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar, Ethiopia. This trial was retrospectively registered in www.pactr.org, number PACTR201911466695052 on November 26, 2019.
Prior to participant enrolment, all parents/legal guardians’ of the children were informed about the objective, purpose, study procedures, and the potential risk and benefits of participating in the study. Parents/legal guardians who agreed that their child should be enrolled in the study were asked to sign a written informed consent. Verbal assent was also sought from each participant. Parents/legal guardians who were unable to read and write, were asked to give thumbprint after having been read the full informed consent form by a data collector.
Intervention, trial medication, and outcome measures
This randomized clinical trial had two treatment arms: (i) a single dose of mebendazole (500mg) and (ii) a multiple dose of mebendazole (100 mg twice a day for three consecutive days). The multiple dose of mebendazole (WORMIN tab) was commercially obtained from a private pharmacy in the local market, while the single dose of mebendazole (Vermox®) was provided by the local coordinator office of the deworming program. The primary outcome considered was CR against hookworm, while ERR for determining the changes in infection intensity served as a secondary outcome measure 14-21 days of post treatment.
Eligibility criteria and Sample size
Eligible for inclusion were all hookworm-positive children with a signed informed consent who did not have additional health problems (based on medical history, physical examination, vital signs). The following exclusion criteria were also applied: children who received any form of anthelminthic treatment within the past 30 days, were unable to chew the drug (for the single), had diarrhea at the time of the first sampling, had a hemoglobin level <8g/dl, experienced a severe concurrent medical condition or had any known history of allergic reaction to mebendazole, and infected with other parasitic infection. All remaining children were randomized and allocated to one of the two treatment arms.
The desired sample size was determined by using WHO guidelines(20). The local prevalence of hookworm infections was not exactly known, and hence, it was assumed to be 50%. Fifty from each of the two treatment arms would be enough to detect differences in the CR with 80% power using a 2-sided statistical test with alpha-level of 0.05. Moreover, considering a potential loss to follow-up, 20% as a non-response rate was added. Finally, 300 school-aged children were screened for hookworm infections to get the minimum required sample size.
Data collection and Laboratory procedures
Study participants responded to a short questionnaire investigating demographic and other health-related issues using the WHO drug efficacy assessment from. A unique identification number was given to each participant. Then, each participant received a sterile stool container labeled with his/her unique identification number and was asked to provide approximately 10mg of fresh stool. All children were well informed on how to avoid any contamination of the sample. Samples were immediately transported to the nearby health center laboratory in Debre Elias and Burie hospital laboratory.
The 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(20,21). Laboratory quality control was performed by an expert microscopist by re-reading 10% of slides of each laboratory technician. Only one stool sample was collected from each participant at baseline and follow-up.
Upon completion of all the baseline parasitological and participant information survey, hookworm-positive children were subjected to a physical and clinical examination by a senior health officer. 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). Children who were found positive for Ascaris lumbericoide and Trichuris trichiura were treated with albendazole (400mg) during baseline screening.
Using simple randomization lottery techniques, eligible hookworm-positive children were randomly assigned either to the single (500mg) or multiple dose regimen of mebendazole (100 mg twice a day for three consecutive days) arm with a 1:1 ratio. Eligible children were randomly assigned and allocated to each treatment arm by the researcher. The drug administrator and children were not masked for drug treatment. Only laboratory technicians were blinded to the dose allocation, hypothesis and objective of the study.
A slice of biscuit was given to each eligible child before drug administration. The single dose and the first dose of the multiple dose of mebendazole regimens were administered to each randomized child in front of their parents by the research team and public health officer at school. After administering the drug, children in the single dose were monitored for 3 to 4 hours to observe if any vomiting and other adverse events were occurred following treatment. In the case of children who were randomized into multiple dose arm; parents/guardians were convinced to take home the remaining tablets in a sealed envelope and were instructed on how to administer the drugs. They were instructed to give the drug twice a day (every morning and evening for three days), avoid skipping/doubling any dose, follow strictly their child up to the end of treatment and study participants had been reminded not to drink alcohol on treatment days. Participants/parents/guardians were informed to report any medical discomfort following treatment to the investigators or the nearby health extension worker.
Follow-up data collection
Each treated child was revisited 14-21 days after drug administration and asked to provide one stool sample for the second time. At this time point, children were also asked about the occurrence of vomiting and diarrhea following drug administration.
A participant who vomited within 4 hours after drug administration or a participant with diarrhea was excluded for the final analysis. The same laboratory procedures took place at the follow-up. Children who remained infected with hookworm or other STH were treated with albendazole (400mg) at the end of the study.
Data was entered to Epi-data software to check data completeness and clearance, and then
transferred to SPSS version-23 for statistical analysis. All analyses were performed on a per-protocol basis. Only children with complete data sets were included in the analysis to determine the treatment efficacy. The baseline characteristics of the study participants are summarized using frequencies, mean and standard deviation (table 1). Infection intensity with hookworm was grouped in to light, moderate and heavy infections, according to WHO guidelines (22). Cure and egg reduction rates were used to assess the efficacy of the drug. Cure rate was assumed to be the proportion of individual hosts positive for hookworm who become parasitologically negative after treatment (23). Egg reduction rate was defined as the relative reduction in the mean egg output after treatment compared to pre-treatment value (24). ERR was expressed using both the arithmetic mean (AM) and the geometric mean (GM) (25).
Confidence intervals for ERR were calculated using bootstrap re-sampling method with 5000 iterations.
An 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.