Study area and participants
The study was carried out in the schistosomiasis endemic foci of Ikata, Bafia and Mile 14-Likoko which are three rural localities in the Muyuka Health District. The study sites have been described in detailed by Ebai et al. . Environmental and socio-economic conditions in these areas favour the thriving of the vectors and the transmission of these parasites. A prevalence of malaria parasite (MP) of 35.5% and US of 34.3% was reported in the area in a study of a cross section of the population [7, 14]. Intervention measures in the area include but are not limited to the free distribution of LLINs to pregnant women and children and the mass distribution of mebendazole by the Ministry of Public Health in Cameroon to SAC in schools.
This study was conducted among SAC aged 4-14years of both sexes whose parents consented to their participation in the study. As an inclusion criterion, only children who had resided for at least three months in the study area were enrolled in the study and their participation in the study was voluntary.
Study design, sample size estimation and sampling
This cross-sectional study was carried out between March to June 2015 to coincide with the malaria and schistosomiasis transmission season. This was a repeated cross-sectional study following intervention studies in the previous transmission season [7, 14]. The sample size was determined using the formula n = Z2pq/d2 , where n was the sample size required; Z was 1.96, which is the standard normal deviate (for a 95% confidence interval, CI); p was 35.3% or 34.3%, the proportion of malaria parasite or US prevalence reported previously in the area [7, 14]; q was 1-p, the proportion of MP or US negative; and d was 0.05, the acceptable error willing to be committed. The optimum sample size was estimated to be 349 (359.5+346.3/2). To mitigate against possible loss of samples due to blood clotting and withdrawal from the study, the sample size was increased by 15% for a minimum of 401 SAC. A representative sample from each primary school and study site was obtained by selecting participants via random ballot from each class list.
Implementation of study
The inhabitants were educated on the importance, benefits and protocol of the study in several reconnaissance visits made to the localities prior to the commencement of the study. Children who presented signed consent forms were enrolled into the study and information on both demography and factors that may be associated with malaria and US were obtained through an interview using a simple structured questionnaire. Clinical evaluation was carried out subsequently where weight, height and temperature were measured. The study involved the collection of venous blood and urine sample for haematological analysis, and microscopic detection of S. haematobium eggs, respectively. Labelled blood and urine samples placed on ice blocks were transported to the University of Buea Malaria Research Laboratory for further analysis.
Questionnaire administration and clinical evaluation
A pre-tested questionnaire was administered to each participant with the aid of the teachers to obtain information on demography, hygienic practices, possible risk factors of Plasmodium and helminth infections as well as malnutrition and anaemia. The ages of participants were obtained from the school register.
The axillary temperature was measured using a digital thermometer and a participant with body temperature ≥37.5°C was considered febrile.
Height was measured to the nearest 0.1cm using a graduated ruler of length 2m. Body mass was measured to the nearest 0.5 Kg using a mechanical scale of capacity 120 Kg (KINLEE® model BR9310), and upper arm circumference was measured to the nearest 0.1cm using a graduated tape. These measurements were used to calculate an array of anthropometric indices used as proxies for malnutrition: weight-for-age (WA: under-weight); height-for-age (HA: stunting); weight-for height (WH: wasting). Anthropometric indices were computed as z-scores based on the WHO growth reference curves using the WHO AnthroPlus for personal computers manual . A child was identified as being malnourished if he or she scored < −2 in one of the anthropometric indices. A z-score between < - 2 and ≤ - 3 was considered as moderate wasting, moderate stunting or moderate underweight while Z scores of < − 3 indicated severe wasting, severe stunting or severe underweight .
Malaria parasite diagnosis and full blood count
From each participant, approximately 2ml of venous blood was collected in ethylenediamine tetra-acetate tubes for malaria parasite detection and haematological analysis. Thick and thin blood films were prepared in situ. The thin blood films were fixed in absolute methanol and together with the thick blood films were Giemsa stained and examined microscopically following standard procedures . Slides were considered positive when asexual forms and/or gametocytes of any Plasmodium species were observed on the blood film. All the slides were read twice by two independent microscopists. Malaria parasite per µL of blood was established by counting the number of parasites per 200 leukocytes and multiplying by the persons white blood cell (WBC) count. Parasitaemia was classified as low (≤ 500 parasite /µL of blood), moderate (501-5000 parasites/µL of blood) and high (>5000 parasites/µL of blood) .
A complete blood count analysis was done using a Beckman coulter counter (URIT 3300) which automatically gave values for red blood cell (RBC), WBC and platelet counts, haemoglobin (Hb), haematocrit (Hct), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC) and red cell distribution width-coefficient of variation (RDW-CV) following the manufacturer’s instructions. The classification of anaemia (Hb concentration below the WHO reference values for age or gender) and its severity was done in accordance with WHO standards (mild anaemia = 10-10.9 g/dL, moderate anaemia = 7– 9.9 g/dL and severe anaemia < 7 g/dL) [23, 25].
Urine analysis for haematuria and schistosome eggs
Each study participant collected approximately 25 mL of midstream urine into a screw cap vials after a brisk exercise between 10am and 2pm. Gross haematuria was determined by visual observation while micro haematuria was determined with the aid of reagent strips (combistix) following the manufacturer’s guide (CYBOWTM 11M a series of Health Mate Ref 0974). Eggs of S. haematobium were detected using the urine filtration technique. Following agitation, 10 mL of urine was drawn using a syringe and filtered through a polycarbonate membrane filter (STERLITECH corporation, USA). The filter membrane was examined microscopically for the presence of S. haematobium eggs as described by Cheesbrough . Schistosome egg density was expressed as the number of eggs in 10 mL urine (e/10 mL) and the intensity of infection was categorised as either light (< 50 e/10 mL) or heavy infection (≥ 50 e/10 mL) [26, 27].
Descriptive measures such as the mean and standard deviation (SD), geometric means, frequencies, and proportions were used to summarize data. Differences in proportions between populations were compared using Chi (χ2) test. The attributable risk (AR%) of anaemia caused by malaria, US and stunting was calculated accordingly : [(n1m0 − n0m1)/n(n0+m0)] ×100, where n0 = anaemic children without malaria/US/stunting and n1 = anaemic children with malaria/US/stunting, whereby n0 + n1 = n, m0 = non anaemic children without malaria/US/stunting, and m1 = non anaemic children with malaria/US/stunting, whereby m0 + m1 = m. Geometric means were computed for those positive only and the log transformed counts were used in the analysis. Geometric mean parasite density (GMPD) of P. falciparum and geometric mean egg density (GMED) of S. haematobium by age, sex and nutritional status and severity were compared by the Student’s t-test, and Mann Whitney U test where appropriate and the mean haematological parameters were compared by analysis of variance (ANOVA). Potential confounders of haematological values to be entered into a multiple linear regression (MLR) model were identified after exploratory analysis. Any potential confounder with a moderate (P < 0.2) relation with both the dependent variable and the confounder of interest was included in the later MLR models. The 95% confidence interval (CI) was reported and P-values < 0.05 were considered suggestive of statistical significance. All data was analysed using IBM-Statistical Package for Social Science (SPSS) version 21 (IBM-SPSS Inc., Chicago, IL, USA).
The study protocol was reviewed and approved by the Institutional Ethical Review Board hosted by the Faculty of Health Sciences, University of Buea following administrative authorisation from the Regional Delegation of Public Health and Basic Education. The ethical approval reference for the study is 2014/243/UB/FHS/IRB. The study was conducted in accordance with the World Medical Association (WMA) principles as stated in the Declaration of Helsinki. The population was sensitized in their various communities at the beginning of the study. Written informed consent was obtained from all parents/caregivers whose child/children participated in the study after explaining the purpose and benefits of their participation. Participation was totally voluntary, and a participant could decide to halt their participation in the study at any time without any penalty. Participants who had malaria and or helminths were given first line treatment as recommended by the national treatment guideline policy for uncomplicated malaria and helminths.