Impact of intestinal helminthes infections on malnutrition and haematological indices of school-age children in Gondar town, Ethiopia.

Abstract

Introduction

Soil-transmitted helminthiases(STHs) and schistosomiasis are among the most common parasitic infections worldwide. They affect more than one billion of the world’s poorest people [1]. Globally in 2010, 819 millionroundworm (Ascarislumbricoides), 464.6 million whipworm (Trichuristrichiura), and  438.9hookworm (Necatoramericanus, Ancylostomaduodenale)were reported in Asia (70%), sub-Saharan Africa (16%) and other part of the world [2]. Globally, there are around 200 million schistosoma infected people with 160 million living inSub-Seharan Africa(SSA)[3].According to recent estimates, over 267 million pre-school-age and 568 million school-age STH infected children are found in the world with more than 1.5 billion people infected by these infections [4].According to food and agriculture (FAO) estimates, around 1.02 billion people are undernourished worldwide [5].In spite of the efforts targeted at reducing malnutrition and hunger by Millennium Development Goals, there has not been any significant improvement [6, 7]. bout 50% of all deaths among global children is related to malnutrition[8].Malnutrition related with intestinal nematodes and other parasites was reported as a major factor in reducing physical, cognitive, and educational development in children while causing decreased fitness, cognition and work productivities in adults, outside the mortalities related to this complication[9-11].The mechanisms by which these intestinal helminth infectionsaggravate malnutrition (protein energy malnutrition, anaemia and other nutrient deficiencies)include anorexia (loss of appetite), malabsorption and decreased food intakedue to nutrient loss through bleeding, vomiting or diarrhea[11-13]. Decreased barriers protection and impaired immune function during malnutrition can,in turn, aggravate the incidence of intestinal parasite infections[13].

In Ethiopia, intestinal parasitic infections have been reported as the cause of malnutrition, anemia and growth retardation among children under 5 years of age [14,15]. Factors that increase the risk of intestinal parasitic infections such as swimming, bar foot walking (lack of shoes), bad hand washing habits and low education status of parents were also reports to be associated with malnutrition [16-18]. Researches describing the role of helminth infections and their intensity on malnutrition, anemia and normal hematologicalindices in primary school children are rare. The aims of this research was to evaluate the impact of intestinal helminthes (infections and intensities) on the malnutrition, anaemia and haematological indices of school children in Azezo primary school in Gondar town..

Materials and Methods

Study area

Gondar town is located in Northwest Ethiopia with 12°35′Nlatitude and 37°28′E longitudeat an altitude of around 2200 meters above sea level. It hasa total population of 207,044 (98,120 men and 108,924 women) based on 2007 national population census[19].The ranges of mean maximum and minimum temperature in 2015 were 22.7⁰C - 29.7⁰C and 17.6⁰C - 22 ⁰C respectively with the annual rainfall of 1151 mm. The highest  rainfall was  registered in July (328 mm) and August (307 mm) compared to the driest January (4 mm) and February (6 mm) in the same year[20]. This study was conducted in the Azezo-Teda sub-city. The Azezo-Teda sub-city or zone administration is located south of the central part of Gondar. Compared to the other sub-cities, the Azezo-Teda sub-citycontains urban and rural populations.The Gondar town is expanding mostly inAzezo-Teda area. The rivers that cross Azezo-Teda sub-city are Angereb, Kaha, Shenta,Demazaand Magechrivers. The Gondar Town Administration Health Bureau regularly conducts deworming in school children in around May and June. The last date for the last deworming was June, 2017. Almost 1 year has passed since the last deworming program in the study school. Infrastructure such as piped borne water, good roads and electricity are lacking in the rural areas. Inhabitants, therefore, obtain ground water for drinking and domestic purposes. By occupation, majority are farmers, with few other working in thread mills or as laborers in central Gondar town.

Study design

This study was a cross-sectional survey, involving systematic random sampling of school aged children in Azezo-Teda sub-city, Gondar town between February and May, 2018. Azezo elementary school was sected due to high prevalence rate of intestinal helminth infections in school age children during previous study in the Azezo area. Grade level stratified random sampling by rotary mthod was used to select the study participants. Pre-testedquestionnaires were used to obtain demographic data and data about food shortage in the family and other possible risk factors which could be the source of malnutrition or intestinal helminthinfections. Using anthropometric measures, school children were grouped as stunted, wasted, obese and Normal. Kato-Katz technique was used to determine intestinal helminth infections while automated hematological and biochemical machines were used to measure haematological and biochemical values. Appropriate statistics were used to see if intestinal helminth infections is associated with malnutrition.

Sample size determination technique

 The prevalence of intestinal helminth and Schistosomamansoni infections(72.9%)[18]during previous study in Azezo primary school was used to calculate sample size of school-age students participated in the study. A single population proportion formula at 95% confidence interval [n = (Z α/2)²p (1-p)]/(0.05)²] was used to calculate the minimum 303 sample size  by using the probability of intestinal helminth and Schistosomamansoniinfections during previous study.  Including 30% contingency (91) for possible discarded samples and drop out study participants, a total of 404 sample size was planned to include in the study. At the end of the sampling, 20 participants were excluded due to incomplete data which made the overall  sample to be 384.

Sampling procedure

Following ethical approval by research ethical review board of University of Gondar and permission letter obtained from Azezo – Teda sub-city administration to conduct this research in Azezo government elementary school, study participants were recruited for the study. This elementaryschool was selected purposefully as it has students coming from both urban and rural areas and high prevalence rate of helminth infections.A stratified random sampling method was used to select the study participants based on their grade level. A rotary method was used to select the total number sample required for the study from all students attending from grade 1 up to grade 8. The students were participated in the study after the purpose and a benefit of the research explained to their parents or guardians and written consent was obtained. Only students willing to participate in the research were included. The students could also withdraw from the study at any time during the execution of the sampling process.

Data collection

After pre-tested structured questionnaire prepared in English was translated exactly to Amharic version(National language), one of the investigators interviewed the parents or guardians to obtain data about socio-demography and risk factors for possible malnutrition and intestinal helminth infections. Then, anthropometric measures, stool and blood sampleswere obtained from all study participants by senior laboratory science personnels. The questionnaire contained questions related to socio-demographic factors (first part) and possible risk factors for malnutrition and intestinal helminth infections.Data about 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 to address possible risk factors for malnutrition and intestinal infections. 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 grouped as family with accessible food. Mothers who could at least read and write were grouped as literate and those who could not read and write were grouped as illiterate.Based on residence, respondents living in Gondar town were grouped as urban inhabitants, and those in surrounding rural area were grouped as rural inhabitants.

Anthropometric Measurements

Body weight and height of the school aged children were measured using digital weighing balance and tape ruler respectively. The students were weighed without shoes and the instruments were calibrated to measure weight with 0.1 gram scalewhile 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 mass-index-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/)[21]. Weight for age (WAZ) was not been used as it is not recommended for age group above 10 years. The z-scores were classified based on WHO Child growth standards as stunting or wasting/thinness.Stuntingisdefined as insufficient height for age or when height-for-age Z score (HAZ) is less than - 2 SD (standard deviation). Underweight was defined as insufficient weight for age or when weight for age Z - scores (WAZ) is less than - 2 SD from standard median scores. 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[22,23].

Collection of stool samples and parasitological examination.

Sterile sample bottles were distributed to consenting school-aged children. Collected specimens were analysed using the Kato-Katz technique to determine the presence of intestinal helminths ova [30, 31]. The Kato Katz technique was performed on the same date to determine infection and intensity of infections. 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).

Collection of blood samples and haematological analysis

• Hematological analysis

About 3 ml venous blood was collected from consenting school-aged children in a sterile tube containing anti-coagulant. The tube was transported quicklyto the University of Gondarbefore the blood was analysed using  fully-automated haematological cell counter (Mindray BC-3200).Total white blood cells (WBC), red blood cells (RBC), hemoglobin concentration (Hgb), packed cell volume (PCV), hematocrit value; mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), lymphocytes and neutrophils were calculated using the automated blood cell counter. The haematological values were classified as normal, below normal and above normal according to standard normal range values[24]. In addition, 2 mm of venous blood sample were collected using sterile syringe from the arm of the consenting school-aged children. The blood samples were placed in a separator test tube and centrifuged at 3000 rpm for 5 minutes. The separated serum was analysed for glucose and total protein concentrations by using Mindray BS-200E chemistry analyser to determine their normal concentration in the blood according to standard reference values[25].

Ethical Consideration

Ethical approval was obtained from the University of Gondar ethical review committee. Written consent was obtained from study participant's parent/guardian after permissionwas obtained from Teda - Azezo district administration and school administration.Laboratory results were kept confidential and children who were infected with helminth infections and with abnormal hematological test, they  were linked to the Gondar district health centre for free treatment.

Statistical Analysis

Data about socio-demography and the possible risk factors were collected using questionnaire before the results for malnutritional indices, intestinal helminth infectionand haematological  testsobtained for each study school-age student were obtained in separate data sheet.After  getting height and weight measurments, height for age Z-scores (HAZ) and body mass  index for age   Z-scores (BAZ) indices were calculated by using WHO Anthroplus software.The data was cleaned in an Excel spread sheet before transferred to statistical package for social sciences (SPSS) version 20 for statistical analysis.Descriptive statistics was used to analyze the prevalenceof the different outcome variables. Mann whitnaytest (non-parametric analysis of variance (ANOVA)) was used to determine the existence of  genderor age grouprelated statistically significant difference for intestinal helminth infection, intensity of infection, co-infections, infecting species, anaemia, stunting,wasting andhaematologicalcategories in the school children.Binary logistic regression test was used to seeassociation between intestinal helminth infection, anemia, stunting,, wasting, age groups, malnutrition indices and haematological categories.Multiple logistic regression analysis was used to determine the  likelihood of anemiaor odd ratio (OR) in school children, when it was compared with uninfected,mono-infected, co-infected and species of intestinal helminth. For the statistical analysis,p-value< 0.05 was considered as significant different for 95% confidence interval

Results

Atotal of 384 school children (179 (46.7%) males and 205 (53.3%))  females) were recruited in this study with age ranged from 7 to 15 years. Of the total school children, 39.3% (151) were in 7-10 years age group while 60.7 % (233) were in 11-15 year age group (Table 1). Mean ± St.D (Median) (range) for height and weight for the all ages were 140.93 cm ±11.4 (140) (114-178) and 30.6 kg ± 7.3 (29.50) (15-55) respectively. Linear increase in height and weight for different age from age 7 to 15 were observed in statistically significant different manner for mean (P=0.000) and median (P=0.000) values.

Table 1. Socio-demographic characteristics of study participants in Azezo primary school, Gondar Town, Northwest  Ethiopia.

The overall prevalence of helminthes infections in the school children was 45.8% (178/384)with 56 light, 47 moderate and 75 heavy infections (Table 2). Ascaris infection (20.6% or 79/384) was the leading infection.The second and third leading infections wereSchistosomamansoni and hook worm infections with prevalence of 17.4% (67/384) and 13.3% (51/384) respectively. The prevalent of the remaining Tricuristrichura, Hymenolopisnana and Taeniaspp were 3.4%, 1% and 0.3% respectively. Of the total 178 school children found infected, 37(9.6%) were with double infections (15Ascaris – Schistosoma, 12 Ascaris – Hookworm, 4  Schistosoma – Hookworm and 6Ascaris and others)  and  5(2.8%)were with triple  infections (4 Ascaris -Schistosoma–others and 1 Ascaris - Hook worm –Tricuristrichura).  

Table 2: Prevalence and Intensity of intestinal helminth infections in Azezo primary school, Gondar town, Northwest Ethiopia. May,  2018.

Of the total school children analyzed for body mass index for age Z scores (BAZ), 37(9.6%) were very sever thin (sever wasted) (Z scores < -3SD), 66(19.5%) sever thin (wasted) (-3 SD < Z < -2SD), 147 (38.02%) thin (-2 SD < Z < -1SD), 134(34.9%) normal (−1SD < Z <1SD) and 1(0.3%) obese (> 2SD). Only 34.9 % were included with in the 25^th and 75^th percentiles of   the WHO normal distribution curve. Of all heights for age analyzed, 7(1.2%) were severely stunted (< - 3 SD), 40 (10.4%), stunted (< -2SD), 331(86.2%), normal height for age (-2SD > Z < 2SD) and 0.8% more height for age (> 2SD). Only 86.2% were included in the 15^th and 85^th normal curve range. Gender showed no statistically significant differences (p>0.05) for BAZ and HAZ scores (table 3).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.  Stunning was  statistically significantly different in 7-10 and 11-15 age group intervals (p=0.000). Of  thetotal 47 school children with stunted growth, 25(53%) were male while the 22 (47%) were female.  Statistically significant difference was not observed (p=0.7) for gender.Of total 47 stunted school children, the percentage with light, moderate, heavy and none infections were 34%, 4.3% 12.8% and 48.9% respectively.Of 337 school children who were not stunted, and the percentage with light, moderate, heavy and none infections were 11.9%, 13.4%, 20.5% and 54.3% respectively. 

Table 3: Prevalence of malnutrition indices in Azezo primary school, Gondar town, Northwest Ethiopia. 2018.

x

Of the total 384 school age children, 335(87.2%)had  normal blood glucose level (70 – 110 mg/dL). The remaining 49 (12.8%) school age children were hypoglycemic or with under normal blood glucose level. The number of the school children with normal total  protein level (6.6 – 8 g/dL) was 259(67.4%) while the remaining 125(32.6%)school children were hypoproteinaemic(under normal blood protein level). The number of the school children with normal haemoglobin (12-18g/dL) and normal pached cell volume (35-54%) were 361(94%) and 329(85.7%) respectively. The number of school age children with normal RBC (3.7-3.85 x10¹²/L) was 366(95.3%). The normal (4.5—9.9 X10⁹/L), below normal and above normal WBCs were 313(81.5%), 32(8.3%) and 39 (10.2%) respectively. The prevalence of the other haematological parameters are indicated in the table 4. 

Table 4. Prevalence of haematological parameters in Azezo primary school, Gondar town, Northwest Ethiopia. 2018.

Ascarislumbericoides, Schistosomamansoni and  Hook worm mono-infection or Ascarislubericoides – Schistosomamansoni and Ascarislumbericoides – Hook worm co-infections were statistically significantly associated (P≤0.001) with protein malnutrition (hypoproteinemia) and Anaemia  compared to non-infected school children. The overall infection was also statistically significantly associated with hypogycocemia, hypoproteinaemia and anemia (p=0.000)(Table 5). Intestinalhelminth infections, intensity, mono or co-infections between stunted and normal school children were not statistically significantly different (P>0.05). Mann – Whitney analysis of variance indicated intestinal helminth infections were not statistically significantly different (p>0.05) for different Gender.  Intestinal helminth infections were highly prevalent in rural (57.1%) compared to urban residence (30.1%) in statistically significant different (p=0.000) manner. Swimming or washing in the rivers were also more common for rural residences (p=0.04). But, Swimming or washing in the rivers were not associated with stunting, wasting and anaemia  (p>0.05).

Table 5: Association between intestinal helminth infection, malnutrition indices and haematological parameters. May 2019.

Discussion

In school children, 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[26]. 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 [27].Ascariasis cause malnutrition in addition to pathology associated the worm migration in the body. Chronic dysentery associated with trichuriasis is also a major problem in malnutrition and health of school children [28].Ethiopia, the second populous nation in Africa, has been classified as low income countries with 20% of poverty in both urban and rural areas[29].Low socio-economic status or low accessibility of food (poverty) in Ethiopia could be the main cause of malnutrition[30,31] similar to this study result. Factors that increase the risk of intestinal parasitic infections in Ethiopia  such as swimming, bar foot walking (lack of shoes), bad hand washing habits and low education status of parents were also reports to be associated with malnutrition [18]. In Ethiopia, intestinal parasitic infections in children are reported to be associated with malnutrition, anaemia, and growth retardation [32].The prevalence of stunting related to malnutrition in under 5 years in Ethiopia reduced  from 64% in 1990 to 47% in 2008 before further reduction to 40.4%  in 2014 [33-35]. The prevalence of intestinal helminthes infections in this study (45.8%) is lower than the previous report (2008) in another Azezo primary school (72.9%) [18]. The reduction in intestinal helminthinfecions  could be due to  the recent regular deworming  program in the primary schools. 

Ascarislumbericoides, Schistosomamansoni andhook worm mono-infection in addition to Ascarislubericoides – Schistosomamansoni, Ascarislumbericoides– Hook worm co-infections and triple infections were statistically significantly associated with protein malnutrition (hypo-proteinemia) and anaemia compared with none infected school children(P ≤ 0.001). This type of association in which intestinal helminthes infections aggravate the situation of malnutrition was reported to be common in children[36]. Prevalence of stunting and anaemia were reported higher in male than in female in Kenyia[37]. But, gender did not show any difference (p>0.05) for prevalence of stunting or anaemia in the school children studied (Table 4). The prevalence of anaemia was affected by thespecies and intensity of infections of intestinal helminthesin statistically significant ways (p=0.000) compared with none infected school children. 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. But in Kenya S. mansoni mono-infection was reported to associate with anaemia and the likelihood of anemia in Schistosoma infection was 3.68 times compared with non- infected children[37].

Except for one school child (0.3%), there was no problem of overweight in the current study in school age children. Only 34.9% of the school children were within WHO normal range based on BAZ scores. Majority (65%) were in state of thinness or underweight (thin, wasted or sever wasted). Thinness (underweight) were not statistically significantly associated (p>0.05) with age and sex. All these results could show malnutrition was very common in all children and might be related with the low socio-economic background of the community in Azezoareas  and high prevalence of intestinal helminth infections. But, analysis of HAZ scores showed 97.9% of the stunting in the school age children was found in 10 - 15 years age group compared to 2.1% in 7-10 years age group (p=0.000). Similar statistically significantly different (p=0.000) for high stunting prevalence (56.4%) in 10-15 age group compared to lower (33.6%) in 5-10 age group was reported in school children in Macha district in Northwest Ethiopia [38]. A total of  50.1% stunting in  12-14 age group school children  compared to 36.9% in 6-11 age group (p=0.000) was also reported in Arbaminch town (Southern Ethiopia)[39].Frequent stunting in children above 10 years compared to those under 10 was reported in Angola due to prolonged problem of food shortage during previous war time[40]. From the fact that anemia prevalence, glucose or protein malnutrition were not different between the age groups (p>0.05)(almost the same  probability) in addition to lack of difference in incidence of intestinal helminth infections between stunted and normal school children (P=0.49), most probably, stunting was the result of prolonged malnutrition related to poverty and parasitic infection and re-infection during early childhood. Stunting may not be restricted to areas with war and prolonged shortage of food as indicated by Olivera et al. [39]. Prolonged malnutrition and infection and re-infection of intestinal helminth in developing nation could be the main source of stunting. 

Probably, Swimming habits of school children in the rivers in the study areas, unhygienic and bare foot walking  habits mentioned during previous study [18] couldbe the important risk factor for high prevalence of intestinalhelminth infections and anemia in Azezo school children. High incidence of Ascarislumbercoides, Schistosomamansoni and hook worm infections were statistically significantly associated with lower MCH values (P≤0.001). Similarly, Schistosomamansoni and hook worm infections were associated with below normal MCHC level (p=.000). Below normal concentration of MCH and MCHC were statistically significantly associated with anaemia (Table 4). Similar study in Thailand has indicated statistically significantly lower (P<0.000) MCH, and MCHC levels in helminthes infected group compared to the helminth-free group [40][42].

Conclusions

Helminthes infection is associated with anaemia and hypo-proteinaemia, lower MCH and MCHC levels. The likelihood of anemia is very high in mono-and co-infected compared to uninfected school children. Stunting is the product of prolonged malnutrition and repeated reinfection of intestinal helminth infections in school children. Intestinal helminthes most probably aggravate the malnutrition in school children with low accessibility of food supply.  Regular monitoring of nutritional status of school children and screening and treating intestinal helminthes are required including school feeding, deworming, clean water supply and public health awareness.

Limitation of the study

Normal ranges of haematological and biochemicalobtained from  literatures were used as reference to classify the results of this study due to lack of  normal range values specific for school –age children in Ethiopia.

Abbreviations

BAZ – Body mass index for age Z-score

EPG – Egg per gram

HAZ – Height for age Z-score

SD – Standard Deviation

WBC - white blood cells

RBC - red blood cells

Hgb- hemoglobin concentration

PCV - packed cell volume 

MCV-mean corpuscular volume

MCH-mean corpuscular hemoglobin

MCHC-mean corpuscular haemoglobin concentration

Declarations

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.

Competing interests

The authors declare that he has no competing interests.

Funding

University of Gondar, office of research and community service,has funded this research.

Authors' contributions

 MB, DT and LW designed the research and participated in the research. DT collected the samples  and processed them. MB and WL supervised the processes.  DT and WL analuzed the data.WL prepared this manuscript.

Acknowledgements       

University of Gondar is acknowledged for funding the research and Gondar university research Ethics review board and Gondar City Health Bureau for providing ethical clearance. Our thanks also goes to Azezo district health clinic for provision of free health service and drug treatment for parasite positive school children. 

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