Soil-transmitted helminth infections, associated factors and nutritional status in an estate community in Sri Lanka

Objectives: To determine the prevalence, intensity, and risk factors associated with soil transmitted helminth (STH) infections and to explore the association between STH infections and nutritional status of an estate community. Methods: A cross-sectional study was conducted in Hanthana Tea Estate (HTE) in Kandy, Sri Lanka, from September 2013 to November 2014. Demographic data were obtained using an interviewer-administrated structured questionnaire. Haemoglobin and serum albumin concentrations were measured in children. Faecal samples were analyzed by direct smears in saline and iodine and Kato-Katz technique using single-stool samples. Anthropometric measurements were obtained to calculate weight-for-age (WAZ), height-for-age (HAZ), and body-mass-index-for-age (BAZ) to evaluate underweight, stunting and wasting, respectively. Results: A total of 233 children (50% female, aged between 1 and 12 years, mean age 6.2±3.4) and 98 parents (93% female, aged between 20 and 52 years, mean age 33±6.2 years) participated in this study. The prevalence of STH infections in children and adults were 27.4% and 14.3% respectively. Ascaris lumbricoides was found in children and adult populations predominantly (26.6% and 14.3% respectively) followed by Trichuris trichiura (0.8% and 1%). Of the infected group, 57.8% of children and 92.8% of adults had a light infection. Moderate infection was found in 40.6% of the children, and 7.2% of the adults. Only one child had a heavy egg count (≥ 50000). The prevalence of STH infections was significantly higher among children than adults (p = 0.014). Not hand washing before a meal (p = 0.002) and after defecation (p < 0.001), greater de-worming period (p < 0.001), use of shared latrine facilities (p = 0.023) and lower levels of mother`s education (p= 0.035) were significantly associated with STH infections. Children with and without STH infection had comparable levels of nutritional indicators. However, 17.6% (n=41) of stunted, 19.3% (n=45) of wasted and 39.5% (n=92) of underweight


Introduction
Soil-transmitted helminthiasis (STH) is one of the most common neglected tropical diseases (NTDs) in the world and the poorest communities are most affected. 1,2 Approximately one third of the world's population is infected with STH. 3,4,5 The current level of infection for ascariasis, trichuriasis and hookworm in South Asia is 29%, 24% and 23%, respectively. 2 STH infections still remain endemic in many parts of the world including Sri Lanka, despite several comprehensive efforts made by respective governments and non-governmental organizations, to control these neglected tropical diseases. 2 Unfortunately, STH infections have become a normal and inevitable part of one's life in low-income communities of countries where it is endemic. 6,7 However, Salam et al. (2014) 8 suggested that "community based interventions are effective in reducing the prevalence and intensity of STH". 8 The most common STH are A. lumbricoides, T. trichiura, Necator americanus and Ancylostoma duodenale. 3,4 A. lumbricoides and T. trichiura are mainly transmitted by the ingestion of viable eggs in contaminated food and/or water. 9,10,11 The infective larval stages of hookworms are found freely in the soil and enter the human body by skin penetration. 12 Limited access to clean water, poverty, a lack of education, poor sanitary facilities and personal hygiene are major risk factors associated with the STH infections. 13 Previous studies have shown that hookworm infections are common among agricultural workers in China, Vietnam and tea estate workers in Sri Lanka, Bangladesh and India. 14,15,16,17 STH infections may contribute to social marginalization, malnutrition and economic instability in societies, particularly in developing nations, and a large number of children are more susceptible to STH infections than adults. 18,19 The most common clinical features of STH infections in children are anaemia, stunted growth, fatigue, proteincalorie malnutrition (PCM), and poor cognitive development. 6,7 The plantation sector in Sri Lanka, with a population of 939,000, represents significantly lowincome families in the country, with a high prevalence of child malnutrition and mortality in mothers. 20 According to a survey conducted in 1992, 90% of children living in these areas were at a risk of acquiring STH infections. 21 Poor household hygiene and sanitation and poor level of maternal education were identified as risk factors associated with STH infections of the plantation sector in Sri Lanka. 22,23 A mass deworming campaign commenced in 1994, which included children aged 3-18 years. The campaign continued for 11 years up to 2005. In 2011, a survey was conducted in the plantation sector communities to identify the prevalence of STH infection. It showed 29% prevalence despite the mass de-worming campaign conducted up to 2005. 23 The need for well planned control strategies to eradicate STH infection was emphasized.
A national survey conducted in school children in 2003, representing regions other than the plantation sector, identified only 6.9% of the population as having STH infections. 24 However, in our opinion, this does not reflect the actual situation in the country at large, since several other studies have shown that STH infections are still endemic, in poor, urban and plantation sector communities in Sri Lanka. 22,25,26,27 The main focus of the present study was to determine the prevalence and intensity of STH infections in the Hanthana Tea Estate (HTE) in Kandy, Sri Lanka. In addition, the relationship between STH infections and the nutritional status of children in this estate was investigated. The findings of the present study will be helpful in implementing new strategies to control STH infections in the plantation sector in Sri Lanka.

Study population
The present study was conducted from September 2013 to November 2014 in the Hanthana Tea Estate (latitude and longitude coordinates: 7 o 20'-7 o 29'N and 80 o 61'-80 o 64'E) in Kandy, Sri Lanka. This mountainous region (1130.38 hectares) is located 600m-1100 m above sea level with an estimated population of 5511 (National Census 2012: http://www.statistics.gov.lk/). In general, the socioeconomic status of people living in this estate is very low, compared to urban communities. These families live in heavily crowded houses with limited space, resources and poor sanitary facilities. The majority of residents, including children, often go to nearby jungles for defecation due to lack of latrine facilities for the study population. Major sources of drinking water were streams and unprotected wells. Most of the residents were employed in tea estates, factories and shops as unskilled laborers.
HTE consists of 7 divisions, namely, Factory division, West division, Top division, Middle division, Urawala division, Uduwela division and Kithulmulla division. Factory and West divisions were selected using the cluster sampling method considering divisions as clusters and each division was given a number from 1 to 7. Two numbers were selected randomly using a random number generator. The sample size was determined using a formula of n = Z 2 Pd/d 2 . 5% margin of error (d) and 95% confidence interval was used for the calculation. Prevalence (P) of STH infections among children in the plantation sector in Kandy was considered as 23%. 23 The calculated sample size was 272. All children aged between 1 to 12 years (335) and their parents (140) from the selected two divisions were included in the study. Children ˂1 year and ≥ 13 years were excluded from the study.

Data collection
All parents/guardians of the West and Factory divisions were summoned to a common place prior to collection of samples and methods and implications of the research were explained to them with the help of estate administrators and medical authorities. The demographic and socioeconomic data (gender, age, family members, mother's education attainment, occupation, and household income), sanitary facilities, living conditions and behavioral characteristics (Floor type, latrine facilities, water source, hand washing, drinking unboiled water, sucking fingers and wearing shoes) and the history of deworming were obtained from children using an interviewer administrated and structured questionnaire. These variables/data relating to parents were not assessed. The questionnaire was prepared in their native language to enable correct understanding of the purpose of the study.

Evaluation of nutritional status
The weight and height of the children were measured using a calibrated digital electronic balance and a height pole, respectively. All measurements were recorded on a pre-prepared data sheet. Adults were not enrolled to take the anthropometric measurements. Weight and height were measured twice to minimize errors. Average values were used for the final calculation. These measurements (age, weight and height) were used to calculate the following parameters, Heightfor-age Z-score (HAZ), Weight-for-age Z-score (WAZ) and Body-mass-index-for-age Z-score (BAZ) to assess stunted growth, underweight and wasting, respectively. AnthroPlus version 1.0.4 (WHO, Geneva, Switzerland) and the Epi Info software 3.5.1 (CDC, USA) were used to calculate each indicator with the international reference values given by the WHO (World Health Organization: http://www.who.int/).

Collection of stool and identification of parasites
Each participant was given a clean, wide mouthed labelled plastic container with a lid and a spoon. A single stool sample was collected from each participant. Stool samples were then transported in a cool box to the Department of Parasitology, Faculty of Medicine, University of Peradeniya, for laboratory investigation. First, direct smears were observed in saline and iodine. Then, the Kato-Katz technique was performed as recommended by WHO. 28,29 After that, the slides were observed under a light microscope, and the number of eggs were counted for each parasite. Finally, the number of eggs per gram of faeces (epg) was determined. The intensities of infection were grouped as follows into light, moderate, or heavy infections, according to WHO guidelines as follows, for "A. lumbricoides, 1-4,999 epg, 5,000 -49,999 epg and ≥50,000 epg; for T. trichiura, 1-999 epg, 1,000-9,999 epg and ≥10,000 epg; and for hookworms, 1-1,999 epg, 2,000-3,999 epg and ≥4,000 epg", respectively. 30

Collection of blood and measurement of albumin and haemoglobin levels
Two to three milliliters of blood was collected into labeled EDTA tubes using disposable syringes. The blood samples were stored in a cool box until transported to the laboratory for further investigation. Haemoglobin concentrations were measured in a private medical laboratory in Kandy using an Auto Hematology Analyzer. WHO recommended haemoglobin levels in different age categories were used to identify anaemia in children. 31 Serum albumin level was measured using a commercial kit (Liquicolor, Human, Germany) according to the manufacturer`s instructions. The reference value of the serum albumin level was provided by the manufacturer (3.8-5.1 g/dl or 38-51 g/l).

Statistical analyses
SPSS (Statistical Package for the Social Sciences) version 17 (SPSS, Chicago, IL, USA) was used for the statistical analysis. Descriptive data were used to explain the distinctiveness of the study population.
Firstly, all variables were analyzed using the univariate model to determine the relationship between the dependent variable (prevalence of STH), and the independent variables (sociodemographic, health hygiene behavior, deworming treatment, living and sanitary condition characteristics).
Secondly, significantly associated variables identified by the univariate model were included in a multivariate logistic regression analysis using forward step-wise elimination model to determine the risk factors of STH infections.
In addition, multiple regression analysis was done to assess the predictive effect of the factors associated with the intensity of infection. For nutritional status, differences in proportions for categorical variables (e.g., stunting, thinness, underweight, anaemia and hypoalbunaemia) were calculated using Chi square test of independence. Differences in mean values of continuous variables (e.g., HAZ, WAZ, BAZ, Hb and albumin) for infection status (negative and infected) were assessed using the student t-test. One-way ANOVA was used to analyze differences in anthropometric mean Z-scores of the study population for the intensity of infections (negative, light, moderate and heavy). The p-value ≤0.05 was considered to be statistically significant. Odds ratio (OR) and 95% confidence interval (CI) were calculated for logistic regression analysis. (Figure 1) Of the 7 divisions, 2 divisions (Factory division, and West division) were selected for the study. 335 children and 140 parents were included in the study (180 children and 77 parents from the Factory division and 153 children and 63 parents from the West division). Written consent was obtained only from mothers and/or guardians. 102 children and 42 parents refused to provide faecal samples due to unknown reasons. The mean age of children was 6.2 years (SD=3.4) and 117 (50%) were females. Parent ages ranged from 20 to 52 years (mean age was 33 years (SD=6.2)), of whom 91 (93%) were females. 233 (69.55%) and 98 (70%) faecal samples were collected from children and from their parents and/or guardians, respectively. In addition, 233 blood samples (2-3 ml) were collected from children to measure the haemoglobin and serum albumin levels.

Prevalence of STH infections
Of the 233 children, 64 (27.47%) children were infected with STH. Of the STH infected children, the majority (26.6%) were infected with A. lumbricoides followed by T. trichiura (0.86%). None of the children had mixed infections. The prevalence in adults was 14.6% for A. lumbricoides. Mixed infection (A. lumbricoides and T. trichiura) was detected in one adult only. Among children, the highest prevalence (39.5%) was reported in the 4 to 6 year age group. In adults, 18 to 26 year age group had the highest (16.7%) prevalence. A significantly higher prevalence of STH infections was reported among children than in adults (p< 0.05) ( Table 1).   Table 2). Table 3 shows the socio demographic, personal hygiene and other factors, which may potentially be associated with STH infections. Females had a slightly higher rate of infection than their male counterparts. Children aged between 1-6 years showed a higher prevalence (29.7%) than older children (24.7%), though not statistically significant.

STH infections and associated factors in children
Univariate analysis was used to analyze the risk factors associated with STH infections in this community (   (Table 4).

Associations between STH infections and nutritional status
The relationship between STH infections and the nutritional status of children was also assessed using an independent sample t-test. It was shown that the mean values for WAZ, HAZ, BAZ, haemoglobin and serum albumin were not significantly associated with children who are either STH negative or positive (p>0.05). Therefore, we suggest that there was no significant association between nutritional status and STH infections in the study population.

Discussion
Studies conducted in the 1980's and 1990's, showed a 100%, 95% and 89.7% prevalence of STH infections in urban slum communities in Colombo (1981), Galle (1989) and plantation sector (1994), respectively. 21,32,33 The results of the present study showed a much lower prevalence of STH infections compared to that of previous studies. However, in comparison with recent studies, the prevalence of STH reported in this study was higher than in the urban and rural areas, while it was lower than that in other plantation sectors in Sri Lanka. 22,2324,34,35,36 One to 6 year children showed a slightly higher prevalence (29.7%) than 7 to 12 year old children (24.7%). This difference may be due to various reasons including a lack of awareness, increased soil related activities of younger children than adults, and the prevailing poor health and personal hygiene in the plantation sector.
A. lumbricoides is the most predominant STH found in both children and adult populations. The close environment of the inhabitants of the study area may be heavily contaminated, especially by children with infected faeces, due to defecation in open areas and close to the line houses. The Table 5 Analysis of nutritional indicators (anthropometrics, Hb and albumin levels) in children SD: standard deviation; Hb: haemoglobin; Height-for-age Z-score (HAZ), Weight-for-age Z-score (WAZ), Body-mass-index-for-age Z-score (BAZ) *Haemoglobin cut-off values (g/dl) for different age categories are as follows 6-59 months-11.0; 5-11 years-11.5; 12-14 years-12.0 (WHO, 2011 (WHO/NMH/NHD/MNM/11.1) eggs of Ascaris are more resilient and can remain infective for years in the soil. 14 Children are therefore more likely to be infected when playing on these contaminated grounds. Soil samples were not checked for the presence of parasitic eggs in the current study.
Trichuris eggs were found only in 2 children and 1 adult. Similar low prevalence was reported by Gunawardena et al. (2011) in school children in the plantation sector in Sri Lanka. 22 A low prevalence has been shown in children living in rural communities of Pakistan as well. 23,37 Eggs of T. trichiura are not resistant to cold, drought and direct sunlight compared to Ascaris eggs which may be one of the reasons for the very low prevalence of Trichuris infection. Further investigation using a larger sample size may be indicated.
In the current study, hookworm infection was not detected in children and adults. Similar results were reported in previous studies in the Kandy area. 23,26 Hookworm is not a common backyard infection, requiring shady, sandy and moist soil for its further development. 17 The area surrounding the study participants homes were not shady which could be a contributory factor for the low detection.  have previously reported a high rate of hookworm infection among tea estate workers and among their family members. 14 Periodic deworming, improved sanitation and hygiene since 2001 may also have contributed to a decline in hookworm infection in the plantation sector. 14 Table 3 shows the intensity of A. lumbricoides infection in children and adults. Both light and moderate infections were high in children. A heavy intensity was reported in one female child.
Our results indicate that the intensity of A. lumbricoides infection gradually decreased with age. This might be due to an improvement in hygienic practices. Similar results were reported in adolescent school girls in Sri Lanka. 27 Analysis of the factors associated with STH infection in children showed that age, gender, number of family members, socioeconomic status, type of drinking water, sucking fingers, walking barefooted, source of drinking water and eating unclean fruits were not significantly associated with STH infection. Although these variables may not have a direct relationship with STH infections, these findings should be further investigated using a larger sample size to get a clearer understanding of their role in these infections. Low maternal education, infrequent hand washing using soap before meals and after defecation, shared latrine facilities and longer periods between de-worming were significantly associated with STH infection. Several studies have shown that poor maternal education is a risk factor associated with STH infection. 38,39,40,41 Generally, a mother plays a significant role in the health education of their children. In this study, we found that the mother's awareness of STH was very poor. It may be a contributing factor to the high prevalence of STH in children. A shared poor quality latrine can also lead to STH infection among children. Previous studies carried out in Sri Lanka, 36 in Nepal, 41 and in Pakistan 37 have shown similar results.
Nutritional indicators (HAZ, WAZ, BAZ, and haemoglobin and serum albumin levels) of children were also analyzed in the present study. The findings were compared with the general data of the country and with the WHO reference values.
Nutrition and growth status of many children were within the normal ranges. However, there were many under-weight children in the study group (39.5%). The Sri Lankan National Nutrition and Micronutrient Survey conducted in 2012 showed that 13.1% children aged between 6 to 59 months were stunted, 19.6% wasted and 23.5% underweight. In addition, the same survey reported that 15.7%, 20.2% and 24.7% were stunted, wasted and underweight respectively in the Kandy district. 42 A comparison between the country and district data reported that the prevalence of stunting and underweight was significantly higher in our study population.
The prevalence of anaemia in children was 15.9%. This figure was slightly higher than the country`s average (15.1%) but lower than the district average (16.9%). A reasonably high prevalence of the thalassemia traits can be a reason for the high prevalence of anaemia, but we could not find any evidences to prove it. In addition, 20.2% of children showed hypoalbuminaemia. There was no significant relationship between nutritional status and STH infections in children. This is probably due to widespread poverty in the population living in the tea plantation areas in Sri Lanka.
The Kato-Katz method was used to determine the intensity of the infection which varied from light to moderate. Limitations of the study include nonuse of the concentration technique to detect STH and examining only one stool sample per participant, both of which could increase the detection of STH.
The nutritional status and socio-demographic features in this community was studied for the first time.

Conclusions and recommendations
The findings of this study contribute to the epidemiological data of STH infections which could be used to plan an effective control programme. Prevalence of STH infections was notably high in both children and their parents, indicating a high level of transmission. Providing single houses instead of line houses, health education and the improvement of sanitary facilities are essential for the effective control of STH infections in the study population. Therefore, coordinated control and prevention programmes considering the diverse socio-demographic characteristics of estate communities is needed to eliminate STH infections in the study population. Government and local authorities should pay more attention to improving not only the health, housing and sanitary facilities of these marginalized communities, but also, their household income as a priority.