Impact of intestinal helminthes infections on stunting, wasting, normal biochemical and hematological values on school children in Gondar town, Ethiopia.

Abstract Background: Ethiopia, the second populous nation in Africa, has been grouped under low income countries. Stunting, underweight and wasting in school children in Ethiopia are very common. The aim of this research was to evaluate the nutritional status compared to WHO standards and impact of intestinal helminthes on normal growth, normal hematological and biochemical values in the school children in Azezo Elementary School in Gondar Town. Methods: Demographic data and information about possible risk factors were obtained using standard pre-tested questioners for all study subjects. WHO AnthroPlus software version 1.04 (WHO, Geneva, Switzerland) were used to classify school children as normal, stunted, wasted and obese after height and body weight measurements were obtained. The Kato Katz technique was performed to screen students for intestinalhelmenthinfections and calculate eggs per gram stool (epg). Haematological and biochemical normal values were obtained from hematology fully-automated cell counter (Mindray BC-3200) and Mindray BS-200E chemistry analyzer. Results: Atotal of 384 school children with age ranged from 7 to 15 years old were recruited in this study. Of the total school children analysed for body mass index and height for age Z scores (BAZ and HAZ scores), 103 (26.8%) were wasted (Z scores < -2SD) and 47 (12.2%) stunted (< -2SD). Gender showed no statistically significantly differences (p>0.05) for BAZ and HAZ. The prevalence of overall stunting in 7-10 age group was 0.7% (1/146) compared to 19.5% (46/236) in 11-15 age group (p=0.000). Intestinal helminthes infections were statistically significantly associated (p = 0.000) with hypo-gycocemia, hypo-proteinaemia and anemia compared with non-infected school children. The likelihood of anemia in intestinal helminth infected school children, when it was compared with uninfected was 148 times higher for both Ascarislumbericoides-Schistosomamansoni co-infection, 38 times for Hook worm, 20 times for Schistosomamansoni infection. Conclusion: Malnutrition and intestinal helminthes infections are serious problem in normal growth of the school children. Poverty associated malnutrition is the most important risk factor for stunting.

Intestinal Helminth infections; Nutritional status ; Gondar town Background According to food and agriculture (FAO) estimates, around 1.02 billion people are undernourished worldwide [1]. In spite of some progress in achievements of the nutrition related Millennium Development Goals like reducing maternal mortality, problems associated with children malnutrition and hunger have not been showing significant improvement [2,3]. In 2016, approximately 5.6 million children under age five died in the world with the highest proportion in Africa [4]. According to world food program report in 2012 [5], of 66 million primary school-age children attended classes hungry in developing world, 23 million or 34.8% were found in Africa. Globally, an estimated 165 million (26%) stunted, 101 million (16%) underweight and 52 million (11%) wasted children of under-five years of age were reported in 2011 [3]. According to the 2015 Millennium development goal (MDG) report, more than 30% of the global under nutrition for under 5 years children was found in Sub-Saharan Africa (SSA) [3,6]. In Africa, 58 million stunted, 13.9 million wasted, 10.3 million overweight and 220 million calorie deficient under five children were also been reported [3]. The severity of children mortality, delayed mental development, poor school performance, reduced intellectual capacity and reduced immunity for diseases are related with severity of stunting and wasting [7]. The percentage of stunting and wasting reflect the cumulative effects of malnutrition and parasites infections [7. 8].
Helminth parasites infections are the major factors in causing malnutrition [9,10].
Globally in 2010, 819 million Ascaris, 438.9 million Hook worm and 464.6 million Trichuristrichiura infections were reported in Asia (70%), in Sub-Sharan Africa (16 %) and other part of the world (22%) including the 1.01 billion infection prevalence in school age children in Asia (70%), Sub-Sharan Africa (16%) and Latin Ameriaca and Caribbian (13%) [11]. Declined food intake and/or an increase in nutrient wastage through blood loss, vomiting or diarrhea related to helminthes infections mostly affects the nutritional status of an individual or aggravate protein energy malnutrition, anemia and other nutrient deficiencies [12]. Chronic schistosomiasis contributes to anemia and under-nutrition, which, in turn, can lead to growth stunting, poor school performance, poor work productivity, and continued poverty while blood loss due to Hook worm infection can cause iron deficiency anemia and hypo-proteinaemia [13]. Ascariasis cause malnutrition in addition to pathology associated the worm migration in the body. Chronic dysentery associated with Trichriasis is also a major problem in health of school children [14]. Heavy S. mansoni infected children in Brazil(above 400 eggs/g of stool) showed 2.74 fold higher risk of stunting compared to uninfected children [15].
Ethiopia, second populous nation in Africa, has been classified as low income countries with 20% of poverty in both urban and rural areas [16]. The prevalence of wasting in Ethiopia was 12% in under 5 years children in 2011 and stunting reduced from 64% in 1990 to 47% in 2008 [1,16,17]

Study areas
Gondar town is located in Northwest Ethiopia at an latitude and longitude range of Lack of enough land for agriculture, the rural communities also get involved in other activities such as thread, work as labourer in Gondar town and so on to get additional income.

Study design
Across sectional study design were conducted by systematic random sampling technique to select 384 study participant at Azezo school age children during

Demographic data and Risk factor analysis
A pre-tested structured questionnaire was used to collect demographic data and the possible risk factors which could expose the school children for stunting, wasted and intestinal helminth infections. Parents and guardians were interviewed to get information for demographic data and possible risk factors. Data about age, sex, grade level, residences, accessibility of food, swimming and/or washing in rivers in the study areas, possession of shoes and proper wearing habits and hand washing habits were collected from the study participants. Family without known income or no or low (<1500 birr/month) salary and very low annual harvest, no television, serious problem in getting enough food and clothing was grouped as family with low accessible food. The other families with no problem of food accessibility as family with accessible food. Mother who could at least ready and write were grouped as literate and those who could not read and write were grouped as Illiterate. Those families who lived in the part of Gondar town were grouped as Urban residence and those who live in the surrounding rural areas were grouped as rural residence.

Anthropometric Measurements
Body weight and height for each school children were measured. Digital balance was used to weigh students with the school uniform without shoes while heights were measured with vertical length measurement with 0.1 cm scales. Measurements were taken twice to use the average for each student. Height-for-age Z-score (for stunting) and body massindex-for-age Z-score (for wasting and thinness) were calculated using the WHO AnthroPlus software version 1.04 (WHO, Geneva, Switzerland) (available http://www.who.int/ //en/)[27]. Weight for age (WAZ) has not been used as it is not recommended for age group above 10 years. Most of the school children were above 10 years. The data collected from the software were used to classify school children as stunted and wasted (thin). Stuntingisdefined as insufficient height for age or when heightfor-age Z score (HAZ) is less than -2 SD (standard deviation) from the WHO child growth standards median Z -scores. Similarly, underweight is defined as insufficient weight for age or when weight for age Z -scores (WAZ) is less than -2 SD from standard median scores. On the other hand, wasting or sever thinness is defined as insufficient mass for height ( low body mass index (BMI) for age) or when body mass index for age Z -score is less than -2 SD [28, 29].

Stool Sample Collection and determination of egg per gram
Stool specimens were collected from the school children using clean dry and leak proof containers. The Kato Katz technique was performed on the same date to determine infection and intensity of infections [30, 31]. Kato-Katz slides thick stool smears prepared from 40.7 milligram stool filtrate were examined microscopically within 30 to 60 minutes after preparation to count helminthes eggs and multiply by 24 .6 to get egg per gram stool (epg).

Hematological analysis
About 3 ml venous blood was collected from the study participant in a tube containing EDTA (dipotasium ethylene diamine tetra acetic acid) and temporarily stored in refrigerator before processed in University of Gondar referral Hospital Hematological laboratory by using haematology fully-automated cell counter (Mindray BC-3200). All

Biochemical analysis
Aftertwo millilitre venous blood samples was collected using sterile syringe from the arms of the school children, blood was placed in separator test tube, before serum was separated from the whole blood by centrifuging at 3,000 revolutions per minute for 5 minutes. Then, the separated serum was analysed for glucose and total protein by using Mindray BS-200E chemistry analyser.

Ethical Consideration
The study was conducted after University of Gondar ethical review committee approved the research. Written consent was obtained from study participant's parent/guardian after permission was obtained from Teda -Azezo district administration and school administration. Laboratory results were kept confidential and children who were infected with intestinal helminth infection and with abnormal hematological, biochemical test were linked to the Gondar District health centre for free treatment.

Statistical Analysis
After data were collected from individual school child separately using data sheet, All important information from data sheet were transferred to statistical package for social sciences (SPSS) version 20 for statistical analysis. After height for age Z-scores (HAZ) and Body mass index Z-scores (BAZ) calculated by WHO Anthroplus software calculated and school children were classified as stunted(< -2 SD), normal (-2 SD< Z<2 SD) and over height using (> 2SD) using HAZ and wasting (sever thin) (< -2SD), thin (-2 SD<Z<-1SD), normal (-1SD<Z<1 SD) and obese (>1SD). The nutritional status of each student compared with their hematological, biochemical values and helmenth infection, infection intensity and mono or co-infection using different logistic regression analysis to see the impact of intestinal helminth's infections on stunting and wasting in addition to different form of malnutrition. Value < 0.05 was considered as significant different for 95% confidence interval.

Results
Stunting was statistically significantly associated with poor family status (without enough available food) compared to other factors (table 1). Intestinal helminth infections were highly prevalent in rural (57.1%) compared to urban residence (30.1%) in statistically significant different (p=0.000) manner. But, stunning or wasting were not associated with urban or rural residence (p>0.05). Swimming or washing in the rivers was more common for rural residences (p=0.04), but it was not associated with intestinal helminth infections or stunting. Almost all the community has shoes wearing and hand washing habits.

Ascarislumbericoides, Schistosomamansoni and Hook worm mono-infection or
Ascarislubericoides -Schistosomamansoni and Ascarislumbericoides -Hook worm coinfections were statistically significantly associated (P≤0.001) with protein malnutrition (hypoproteinemia) and Anaemia (Table 3) compared to non-infected school children. The overall infection was also statistically significantly associated with hypogycocemia, hypoproteinaemia and anemia (p=0.000).    (Table 1). But, stunting was associated with low accessibility of food in poor families compared with families without shortage of food availability (p=0.000).Malnutrition due to low accessibility of food could be the main source stunting in the study area. In Ethiopia low accessibility of food due to low income of parents were reported to be statistically significant associated with stunting, wasting

Ascarislumbericoides, Schistosomamansoni and Hook worm mono-infection or
Ascarislubericoides -Schistosomamansoni, Ascarislumbericoides-Hook worm co-infections and overall infections were statistically significantly associated (P ≤ 0.001) with protein malnutrition (hypo-proteinemia) and anaemia (Table 3). This type of association between severe malnutrition and infections reported to be common in children [40]. Intestinal helminthes infections could aggravate the situation of malnutrition. Prevalence of stunting and anaemia were reported higher in male than in female [41]. But, gender did not show any difference (p>0.05) for prevalence of stunting or anaemia in school children studied (Table 3). Intensity of infection and species of intestinal helminth infections were affected the prevalence of anaemia in statistically significant ways (p=0.000) compared with none infected school children ( Table 4). The likelihood of anemia in school children, when it was compared with uninfected, increased 148 times for both Ascarislumbericoides-Schistosomamansoni co-infection, 38 times for Hook worm, 20 times for Schistosomamansoni and 3 times for Ascarislumbericoidesmono-infection (Table 4)

Ethics approval and consent to participate
Ethical clearance was obtained from Gondar University after proposal was reviewed by ethical review board of the University.

Consent for publication
The author consents to Editorial Board of the journal BMC to publish the paper. The author(s) accept responsibility for publishing this material in his own name, if any.

Availability of data and materials
The data analysed is available in the corresponding author and could be available on reasonable request.