The Association Between Malaria Parasitaemia, Intestinal Parasite and Anemia in Children Less Than 6 Month in Senegal: A Cross Sectional Survey

: Introduction: Although malaria is declining in many countries in Africa, malaria and anaemia remain frequent in children. This study was conducted to assess the relationship between malaria parasitaemia, intestinal worms, and anaemia, in children <6 months living in low transmission area in Senegal. Methods: A survey was carried out in Lamarame in the central part of Senegal. A cross sectional survey was used to select study participant. Children <6 months were enrolled after informed consent. For each child, blood thick and smear tests were performed, haemoglobin concentration was measured with HemoCue


Introduction
Malaria remains major public health problem worldwide particularly in sub-Saharan Africa.According to the World Health Organization, an estimated 219 million malaria cases and 434,000 malaria deaths occurred worldwide.92% of malaria cases and 93% of deaths are observed in Africa.Children under five are the most vulnerable group affected by malaria [1].
Also the morbidity related to intestinal parasitic infection caused by pathogenic protozoa and helminths is important worldwide, particularly in developing countries where children are most affected.It is estimated that 3.5 billion patients are affected and that 450 million of them are ill [2].According to World Health Organization (WHO), children living in endemic are most affected with an estimated number of 270 million preschool children and over 600 million school children [3].These pediatric Less Than 6 Month in Senegal: A Cross Sectional Survey infections can lead to adverse effects on nutrition, growth and cognition and contribute to the global burden of childhood anaemia [Moore et al 2012; Balarajan et al. 2011;Bethonyet al. 2006.Anaemia also remains a major public health problem, not least in malaria endemic areas, where it is primarily seen in young children in areas with stable malaria, but also in adults in malaria unstable areas [4][5].
In tropical regions, there are multiple aetiologies to anaemia and intestinal parasites as well as malnutrition can play a major role in anaemia occurrence [6], particularly when malaria prevalence is declining.Thus, exploring the relationship between malaria, intestinal parasites, malnutrition, and anaemia can provide useful information in order to guide public health programs aiming at reduction of child health problems in tropical regions.
This study was aiming (i) to assess the prevalence of malaria parasitaemia, intestinal parasites (IP), anaemia, among children less than 6 month and (ii) to explore the relationship between malaria, intestinal parasites, and anaemia.

Study Area and Population
The study was conducted at the Lamarame health post located in the Keur Socé, a rural community, at 17 Km from the city of Kaolack in central Senegal, and 200 Km from Dakar.The Lamarame health post is in the Ndoffane health district.It covers 73 villages located within a radius of 8 Km from the post, with a total population of about 30,000 inhabitants.
Malaria is endemic in this area with a seasonal pattern of transmission peaking during the rainy season (July-November).
Many programs aiming to reduce child health problems are being promoted in this area.These programs included two yearly vitamin A supplementation, and deworming with Mebendazole, in December and June of each year for children aged 1 to 5 years.
A Cross sectional survey household survey was carried out at the end of the malaria transmission season, in January 2010.one month after the second round of Mebendazole mass administration campaign.

Data Collection (i). Structured Questionnaire
A code was given to every child after parents' informed consent.Each eligible child was examined by a physician prior to a biological assessment which included blood and stool samples.The mother was interviewed directly concerning the child's symptoms as well as well as sociodemographic characteristics using a standard questionnaire.Data obtained from physical examination and parents interview were assigned on a case report form (CRF).

(ii). Biological Assessment
Blood samples were collected using finger prick blood.The first drop was used to prepare thick and thin smears for the diagnosis of malaria.Thick and thin smears were stained with Giemsa and read by a laboratory technician.Malaria was defined as any asexual parasitemia detected on a thick or thin blood smear.Parasitemia was numbered and expressed by number of trophozoites/µL using the following formula: numbered parasites ×8000/200 assuming a white blood cell count of 8000 cells per µL.Absence of malaria parasite in 200 high power ocular fields of the thick film was considered as negative.
The second drop of finger prick blood was drawn into a microcuvette for Hb determination (g/dL) using HemoCue machine (HemoCue Hb 301).Moderate and severe anaemia were defined as hb concentration below 11 g/dL and 8 g/dL, respectively.
Fresh stools samples were collected into wide mouth screw-cap clean containers.Fecal samples were examined for the detection of intestinal parasite using the Ritchie technique.Intestinal parasite was recorded positive by the presence of helminthes and/or protozoans in the faces.

Sample Size
Taking into account an estimated prevalence of malaria of the order of 20% and a prevalence of anemia at the study area of 35%, a confidence level of 95% (&=5%), a statistical power of 80% (beta=20%) for an accuracy of 5%, the number of children aged between 4 and 6 weeks to be recruited during this survey was estimated at 110; considering a percentage of 10% nonresponse or defective sampling, this size was increased to 125 children.

Data Analysis and Management
The data was entered on Excel software and the analysis was done with STATA IC 10 software.Quantitative variables were described in terms of average, standard gap.Inter-group comparisons were made using an ANNOVA test or the Student t test after verification of the conditions of application of these tests.Where these tests were not applicable, nonparametric tests (Man withney, Kruskall Wallis) were chosen.
For qualitative variables, a description in terms of numbers, percentage of data filled in was made.
The analysis of contingency tables was done using Pearson's Chi Square or Fisher's exact test according to the conditions of applicability.
A bivariate analysis was used to see the existence of the association between malaria, anemia and intestinal parasitosis.
The significance threshold of the tests was set at 5%.The recruitment of children took place over five months from December 2010 to May 2011 from the health centres of Keur Socé and Lamarame, all belonging to the rural community of Keur Socé.During this period, on a list of 296 eligible infants aged between 4 and 6 weeks, 162 were screened.12 infants were not selected for non-compliance with the inclusion criteria.Of these 12, 8 were not selected for medical reasons and the other 4 for screening error on age.Thus, 150 children were enrolled in our study cohort.

Positioning of the Children of the Study
Of the 150 included, there were 12 study outings including 2 deaths, 4 withdrawals of consent, 4 lost-of-sight and 2 excluded for change of residence outside our intervention area.

General Characteristics of the Survey Population
In our study population the age at inclusion varied between 28 and 48 days with an average of 36.3 days and 5.6 standard deviation.Male and female children accounted for 50% (75/150) each with a sex ratio of 1.The inclusion weight ranged from 2.2 kgs to 6.2 kgs.The average weight was 4.0+/-0.6.Children with low weight (between 2 and minus 4 kgs) accounted for 46.7% against 53.3% for those with a normal weight (between 4 and minus 7 kgs).
The size at inclusion ranged from 47 to 57 cm.The average size was 52.3 +/-2.1.
The number of children living with their mothers ranged from 1 to 8. The average number of children per mother in our study cohort was 3.4 +/-1.95.54.8% of the mothers surveyed had between 1 and 3 living children against 28, 1% and 17.1% who respectively had between 4 and 5 children and more than 5 children.
The minimum age for mothers of children in the study was 17 years and 40 years for the maximum.The average age was 25.3 +/-5.6.
The majority of children included in this study 76.7% (115/150) were from villages polarized by the Keur Socé health post compared to 23.3% from the Lamarame health post polarized area with no statistically significant difference (p=0.666).In this study, only one clinical malaria case was noted to be 59 days of life with a parasitic density of 1640 parasites per microlitre of blood.A thick drop made after three days of treatment with ACTs returned negative.The prevalence of malaria in our cohort was 0.7% (1/138).
No cases of malaria infection were noted between six months and 12 months.

Prevalence of Anemia
The average hemoglobin level in children in the inclusion study was 12.5 +/-6.The minimum hemoglobin level noted was 9.4 g/dl and the maximum was 17.2 g/dl.The majority of children (83.3%) at inclusion had a normal hemoglobin level.The incidence of moderate anemia which was 16.7% at inclusion increased significantly to 55.3% at 6 months and 51.9% at 12 months compared to severe anemia which was 0% at inclusion, 0% at 6 months and 1.5% at 12 months.(Table 2)

Prevalence of Intestinal Parasites
The prevalence of carriage for the main intestinal parasitic species found in our study was: Giardia intestinalis (15.4%),Ascaris lumbricoides (8.5%), Entamoeba coli (1.5%), Yeast (1.5%), Trichuris trichiura (0.8%).(Table 3) It was demonstrated in this study that there was an association between age and parasitic carriage.Children aged 12 months had a 10% higher risk of carrying intestinal parasites (0R=1.1 [0.3-3.4],p=0.845) than children aged 9-10 months.In children aged 11 months there was no association between age and parasitic carriage.
An association between sex and parasitic carriage was noted.Girls had more than 10% risk of carrying intestinal parasites (OR=1.1 [0.5-2.4],p=0.761) than boys.
It was shown that there was an association between the area of residence and the parasitic portage.Children from the Lamarame area had more than 10% risk of carrying intestinal parasites (OR=1.1 [0.4-2.7],p=0.773) than those from the Keur Socé area.This study showed that there was an association between parasitic carriage and anemia.Children with parasites were more than 40% at risk of anemia (OR=1.4[0.6-3.4],p=0.395) than negative children.

Factors Associated with Intestinal Parasitic Carriage
The existence of an association between sex and anemia has also been demonstrated.Girls were more than 20% at risk of anemia (OR=1.2[0.5-2.6],p=0.545) than boys.
Children living in the Lamarame area were twice as likely to be anemic (OR=2 [0.8-4.8],p=0.105) as those living in the Keur Socé area.
Children with parasitic infections at 12 months were less than 30% at risk of anemia compared to children at 9-10 months.
There was no association between anemia and malaria (p=0, 366).

Discussion
Malaria, intestinal parasitosis and anemia are one of the most common diseases affecting young children and especially infants with significant morbidity.The results of this study confirm here the very low prevalence of malaria before one year, but also and especially the still high prevalence of anemia and intestinal parasitic carriage in this age group.
The results of this study confirm the hypothesis that children during their first months of life are protected with relative immunity against malaria mainly due to a passive transfer of maternal antibodies, but also because of the protective role of Fetal hemoglobin (HbF).Indeed in our study cohort of 150 newborns a single case of malaria disease was detected after one year of follow-up.
Only one child was diagnosed with malaria with a relatively low parasite density of 1640 parasites per microlitre of blood.A very low prevalence in the study of 0.75% (1/133) was observed outside the 12 study withdrawals.
Several studies carried out in this direction support thus the results of our study.
In a retrospective review of records of children under the age of 15 admitted to hospital in Blantyre, Malawi, in 1998 and between 2005 and 2008, confirmed malaria cases among those under 6 months of age accounted for 4.8% 95% CI [2.8-6.7%][7].
A study in Tori Bossito in Benin in 2007 showed that the median age for the first malaria infection was 4.88 months (0.53-11.86) in infants born of infected placentas, while it was 6.11 months (0.53-12.15) in those born of uninfected placentas.
She also showed that malaria cases are graded by age, from 4.7% to 1 month, 20.3% to 3 months and 54.7% to 6 months in children in this study [8].
In Uruba, Colombia, in an endemic area with an annual incidence of more than 40 cases per 1000 inhabitants, had conducted a descriptive and prospective study in mothers who suffered from malaria during pregnancy and their newborns.116 newborns were included in the study and 80 umbilical cord samples were obtained.5 cases of congenital infections representing a prevalence of 4.3% were diagnosed with one at birth and the others during the 21-day follow-up on average [9].
A study between January 2002 and December 2009 on congenital and neonatal malaria at a rural hospital in Kenya showed that out of 4,790 thick drip slides (93.7%) of all neonates in the study: 3,187 (92.6%) during the first week of life and 1606 (96%) after, only 18 (0.35%) of the newborns were carriers of Plasmodium falciparum and no newborn was carrier of another species of plasmodium.11 of the 18 were admitted in the first week of life and were classified as having congenital malaria.Additional parasitic densities were very low [10].
In our study 85.33% of children slept under a treated net at the time of the survey, 99.3% were exclusively breastfeeding and 84.86% of mothers of children received at least one dose of TPI during pregnancy.
Intestinal parasitosis and anemia have always been important factors in morbidity in children under 5 years of age and especially those under 2 years of age.The results of our study confirmed this with, among other things, a carry rate of 25.4% intestinal parasites and a 55.3% and 51.9% anemia prevalence at 6 and 12 months of life respectively in our study cohort.This was confirmed by several studies carried out among children in Senegal and throughout Africa and elsewhere in the world.
In 2010, a study evaluating intestinal parasitosis and anemia in children under the age of ten in the same area of intervention as ours had similar results with an overall prevalence of 25.4% intestinal parasitoses.The species most frequently encountered were Giardia intestinalis (14.8%),Entamoeba coli (11%), Hymenolepis nana (1.6%), Ascaris lumbricoides (0.5%) [11].
Other studies in Africa and elsewhere in the world have confirmed the endemicity of intestinal parasitoses with varying frequencies for different species.
In Thailand in 2002, in a study assessing the prevalence of intestinal parasites in children between the ages of 3 and 5 years, a carry rate of 22.7% was found.Giardia lamblia was the most common species [17].
Concerning anemia, in the same study in 2010, had found a prevalence of anemia at 48.5%, with no association found between carrying intestinal parasites and anemia [11].
In a 2011 malaria prevalence survey at Keur Socé, 81.7% and 52.1% of children aged 6 to 2 years and 3 to 9 years had anemia prevalence in the dry season, respectively.
In Morocco in 2009, a study of school-aged children found a prevalence of moderate anemia (hemoglobin levels below 11g/dl) at 76.6% [18].

Conclusion
Malaria and intestinal parasitoses constitute in rural Africa in addition to nutritional deficiencies, the predominant factors or often associated with anemia.According to the World Health Organization, infants between six and 24 months of age are among the most vulnerable to anemia.In Senegal, the national malaria survey in 2008-2009 showed a higher prevalence among rural children at 81%.
Given the importance of malaria, intestinal parasitosis and anemia in young children, we proposed to conduct this survey to better study them.
The limits of our study: 1) The low prevalence of malaria at the time of our study in the rural community of Keur Socé; 2) The lack of evaluation of protective factors against malaria, including the determination of fetal hemoglobin, could have produced a lot of information; 3) Non-analysis of maternal antibody kinetics; 4) Not using the stool concentration method for a better diagnostic tool for stool exams; 5) Failure to use the Kato method to determine the pest load of each species.

Figure 1 .
Figure 1.Positioning of the children of the study.

Table 1 .
General characteristics of the survey population.

Table 2 .
Prevalence of anemia in the survey population.

Table 3 .
Prevalence of intestinal parasite carriage at the survey population level.

Table 5 .
Factors associated with anemia.