Prevalence and factors associated with anaemia in children aged 6-24 months living a high malaria transmission setting in Burundi.

BACKGROUND: In very young children, anaemia has been linked to increased morbidity, mortality and poor cognitive development. Although Burundi has a high burden of anaemia, which may be worsened by the high burden of malaria, little is known about the extent of the problem in very young children who are most at risk of severe disease. We estimated the prevalence, and assessed the factors associated with anaemia in children aged 6-24 months using baseline data collected as part of an on-going study evaluating the effect of Micronutrient supplementation on anaemia and cognition among children in high malaria transmission settings in Burundi.
METHODS: Between February and March 2020, surveys were conducted in 498 households within the catchment area of Mukenke Health Center. One child aged 6-24 months was selected per household to participate in the survey. Following written informed consent, we administered a questionnaire to the child's primary caregiver to capture information on child's demographics, nutritional status, food intake, health (status, and morbidity and treatment-seeking practices), as well as the household markers of wealth. A physical exam was conducted, and a blood sample was collected to: 1) assess for presence of plasmodium infection using a rapid diagnosis test; 2) estimate the haemoglobin levels using a portable haemocue machine. A stool sample was also collected to examine for the presence of helminth infections.
RESULTS: The prevalence of anaemia was 74.3% (95% confidence interval [CI] 61.5%-84.0%), with most of the anaemic study participants classified as having moderate anaemia (59.2%). A total of 62 (12.5%) participants had positive malaria rapid diagnosis tests. Factors significantly associated with higher odds of developing anaemia included not receiving deworming medication (adjusted Odd ratio [aOR] = 3.54, 95% CI 1.79-6.99, p<0.001), the child's home location (Mukenke II: aOR = 2.22, 95% CI 1.89-2.62, p<0.001; Mukenke: aOR = 2.76, 95% CI 2.46-3.10, p<0.001 and Budahunga: aOR = 3.12, 95% CI 2. 94-3.31, p<0.001) and the child's age group (Children aged 6-11 months: aOR = 2.27, 95% CI 1.32-3.91, p<0.001). Education level was inversely associated with less odds of anaemia: child's primary care giver with a secondary (aOR = 0.67; 95% CI: 0.47-0.95, p = 0,024) and tertiary education level (aOR = 0.48; 95% CI: 0.38-0.61, p<0.001).
CONCLUSION: Anaemia is highly prevalent among young children in high malaria transmission setting. Anaemia is more prevalent among children who not dewormed and those with malaria. To prevent the long-term adverse outcomes of the anaemia in children, policy makers should focus on improving uptake of the deworming and malaria prevention programs, promote preventive interventions and improve the education of women especially in families with very young children.

Introduction

Anaemia is defined as a condition in which the number of red blood cells or the haemoglobin concentration within them is lower than normal, consequently affecting their capacity to carry sufficient oxygen to meet the body’s physiologic needs [1-3]. Anaemia has multiple and interconnected causes. immediate causes of anaemia are inadequate diet intake and recent morbidity [4]. Demographic, socioeconomic status, family structure, water/sanitation, growth, maternal health and recent illnesses have been previously reported to be significantly associated with childhood anaemia [5]. There is strong evidence that children living in low income household are at greater risk of anaemia compared to those with higher income. Tesema et al in a study on prevalence and determinants of severity levels of anaemia among children aged 6–59 months in sub-Saharan Africa found that Children from poorest, poorer, middle and richer household wealth were 1.39 times [AOR = 1.39, 95% CI: 1.33, 1.45], 1.32 times [AOR = 1.32, 95% CI: 1.26, 1.37], 1.20 times [AOR = 1.20, 95% CI: 1.15, 1.25], and 1.15 times [AOR = 1.15, 95% CI: 1.11, 1.20] higher odds of higher level of anaemia compared to children from the richest household wealth, respectively [6]. For Tesema et al children whose mother education level had 1.73 times (AOR = 1.73, 95% CI: 1.60, 1.86) at no formal education, 1.39 times (AOR = 1.39, 95% CI: 1.29, 1.50) at primary education, and 1.27 times (AOR = 1.27, 95% CI: 1.18, 1.36) at secondary education level than children whose mother had a higher level of education [6]. Malaria has also been documented as a major cause of anaemia in tropical regions through haemolysis of infected and uninfected erythrocytes and bone marrow dyserythropoiesis which compromises rapid recovery from anaemia [7].

Anaemia is a common childhood disease and public health threat in developing countries [8, 9]. The overall prevalence of anaemia in children under five is estimated at 43% worldwide, with the biggest burden in sub–Saharan Africa [8]. Anaemia in young children has been linked to a number of negative effects including; poor cognitive function, poor motor development, fatigue, and increased morbidity and mortality [10-13]. A number of causes of anaemia in children have been documented including inadequate dietary intake of iron and fortified foods [14], hookworm infections, and malaria infection [4, 7, 15–17], most of which are preventable. A study in Thailand reported that anaemic status of the hookworm positive group significantly improved by 2 months after deworming of the hookworm-infected children (Hb = 12.1, 95%CI 5.1–19.2) and to became comparable with the helminth-free control group(Hb = 12.0, 95% CI 9.2–15.7) [18].

Central Africa, where Burundi is located, carries the highest burden of anaemia with 71% of the children classified as anaemic [8]. The country is also highly endemic for malaria, and has a high burden of malnutrition [19, 20]. The 2016 Demographic Health Survey (DHS) showed that among children aged 6 to 59 months; 58% were stunted, 45% were anaemic, and 26.8% had malaria parasites [20]. Although previous reports showed a steady reduction in the overall burden of anaemia in the country between 1990 and 2010 [21], recent reports showed an increase in overall burden in 2017 from 45% in 2010 to 61% in 2017. This increase corresponded with a simultaneous increase of malaria prevalence from 17% to 27%. The estimated prevalence of anaemia in Burundi is way beyond the 40% WHO critical level considered as a public health problem [2, 22].Young children have contributed most to the anaemia burden in Burundi. The 2016/2017 Demographic Health Survey estimated the prevalence of anaemia at 84% and 78% in children aged 6–8 and 9–11 months respectively [22]. Young children are also the age-group with the highest risk of getting anaemia as a complication of malaria, increasing their risk of mortality and long-term adverse effects [7, 23]. Despite this high burden, there is not routine monitoring of anaemia in this high-risk group in Burundi.

In this study, we estimated the burden and factors associated with anaemia in children 6–24 months living in Mukenke Health District, northern Burundi, an area with high malaria transmission Our findings will provide information for policymakers to target interventions focusing on anaemia control in this vulnerable population.

Methods

Study design and setting

This was a cross-sectional study using baseline data collected as part of an on-going study evaluating the effect of micronutrient supplementation on anaemia and cognition among children in high malaria transmission setting in Burundi. The baseline data collection was conducted between February and March 2020 in Mukenke health district, Kirundo province. The health district is mainly a rural area with four administrative hills (Budahunga, Butegana, Mukenke, and Mukenke II). The district has approximately 35,882 households according to the 2018 annual health report [24], with the main source of income being agriculture and livestock farming. Approximately 27, 674 (17.9%) of the population in the Mukenke health sub-district are children under five years of age, with children between 6–24 months estimated at 9,276 [24]. Mukenke Health Center is the largest public health facility of Mukenke health district with the catchment area composed of 17 villages. The number of children aged 6–24 months within the catchment area of Mukenke Health Center is estimated at 1,116 [24].

Mukenke Health District is among the 23 districts (out of 46) with high malaria burden in Burundi, having an annual incidence estimated at 450 cases per 1000 population. Elsewhere annual malaria incidence was moderate (250–450 cases per 1000) for 12 health districts, low (100–250 cases per 1000) for 9 health districts and very low (less than100 cases per 1000) for 2 health districts [25]. The Health District also has a high burden of anaemia and malnutrition. According to the 2016/2017 Demographic Health Survey, the prevalence of anaemia was 79.2% and that of stunting was 62.9% in children under 5 years of age [22].

Study population

Using the community health worker registers, households within the catchment area of Mukenke Health Centre with children aged 6–24 months were identified and 501 of these households were selected by simple random sampling using the registered total number of children aged 6–24 months in Mukenke Health center catchment area (n = 1116) and the computed sample size of 492 participants. We calculated the sampling interval by dividing accessible population by the sample size (1116/492 = 2.27 rounded at 2). Using the community health worker registers in each village (the register lists the children and their households), we selected households to participate in the study using an interval of 2 as listed in the register. Meetings were held with the children’s primary care-giver to explain the purpose of the study. Following the discussions, written informed consent for their children to participate in the survey was sought from the care-givers. All children aged 6–24 months in the household were screened for eligibility to join the study. Children were enrolled if they were aged 6–24 months, were permanent residents of the selected household, and their parent/primary care giver had provided written informed consent. If a household had more than one child aged 6–24 months, one of the eligible children was randomly selected using inclusion and exclusion criteria for the study such that only one child per household participated in the study. If a selected household did not have a child in the age group on the day of the survey, we then selected the nearest northern household. Children were excluded if they had a known history of sickle cell anaemia.

Study procedures

For all eligible children, a detailed questionnaire was administered to the primary caretakers by the study team members. The questionnaire was used to capture information on: 1) the participant’s demographics, 2) nutrition status and food intake including who prepares and/or feeds the child, breastfeeding status, duration of breastfeeding, age of starting complementary foods, minimum acceptable diet, and consumption of fortified food in the last 2 weeks; 3) the child’s health including history of common childhood diseases (fever, diarrhoea, difficult or hard breathing, cough) in the last two weeks, number of times they have been treated for malaria since birth, accessibility to the nearest health facility, time required to reach the nearest health facility, vitamin A supplementation in the last 6 months, routine deworming in the last 6 months and immunisation status, 4) information on the characteristics of the primary caregiver (age, income, education level and employment), 5) detailed information on the proxy indicators of household wealth like the house structure, source of livelihood, and ownership of the house was collected.

Minimum acceptable diet was the consumption of at least four food groups from a list of seven food groups: grains, roots, and tubers; legumes and nuts; flesh foods (meat, fish, poultry, and liver/organ meat); eggs; vitamin A-rich fruits and vegetables; and other fruits and vegetables. Consuming at least four groups meant that the child had a high likelihood of consuming at least one animal source of food and at least one fruit or vegetable in addition to the staple food (grains, roots, or tubers) [26].

A physical examination was conducted on all enrolled children and included: a general examination (nutritional oedema screening, conjunctival and/or palmar pallor), axillary temperature, weight using a SECA® scale (Seca 813 Hamburg, Germany), length or height using a portable Stadiometer Height-Length Measuring Boards (infant/child ShorrBoard® Maryland, USA), and Mild Upper-arm Circumference (MUAC) using a standard MUAC tape (S0145620 MUAC, Child 11.5 Red/PAC-50). A finger-prick (or a heel prick in the case of children age 6–11 months) was done to obtain a blood sample to assess for Plasmodium infection using a rapid diagnostic test (mRDT), and haemoglobin estimation using a HemoCue® portable photometer [2, 27]. We applied altitude adjustment on haemoglobin measured values for Mukenke altitude [28] based on the midpoint of altitude range (1403.5 m for the 1331−1476 range), we used CDC formula published in 1989 MMWR equation [29] for Hb adjustment (g/L) = [(−0.032 × (altitude × 0.0032808) + 0.022 × (altitude × 0.0032808)2) × 10] = -3.20 g/L. The CDC formula provide a good estimation in assessing Haemoglobin up to 2150 m above the sea level [30].Children who were found to have moderate (haemoglobin values 70–99 g/L) or severe anaemia (haemoglobin values <70 g/L) were referred to the nearest health facility for follow-up or care. We provided a universal container, a plastic bag, a clean non-disinfectant impregnated disposable towel and tissue paper to all children primary caregiver in order to collect morning stool samples. All collected stool samples were examined for geohelminths within one to two hours period.

Laboratory evaluations

Malaria testing was performed in the field by the trained laboratory technicians, using an mRDT (SD Bioline Malaria Ag Pf/Pan rapid test, Standard Diagnostics, Inc., Yongin Si, Gyeonggi-do Republic of Korea). This is a rapid, qualitative test for the detection of HRP-II (Histidine rich protein II) specific to Plasmodium falciparum in human blood. Test kits were used before the expiration date, and were transported and stored according to the recommended storage conditions (temperature 4–30° C, avoid humidity). The kits were kept in their original packaging at room temperature and were prepared using approximately 5 μl of blood and read according to the manufacturer’s instructions. Haemoglobin concentration was assessed using a portable haemoglobinometer (HemoCue Ltd., B-Hemoglobin system (HemoCue AB, Angelholm, Sweden) and estimated to an accuracy of 1 g/dL. Stool samples were examined microscopically for the eggs of intestinal nematodes using the Kato-Katz technique [31, 32].

Data management and analysis

All data were double-entered using Census and Survey Processing System (CSPro) version 7.3 database, United States Census Bureau, September 2019 [33]. Consistency checks were performed, and all discrepancies and queries verified against original paper forms. Anaemia was defined using the World Health Organization (WHO) [2] age-specific thresholds cut off as altitude-adjusted haemoglobin lower than 110g/L. Plasmodium infection was defined as having positive mRDT results, and the proportion of children with malaria infections was calculated as the number of children with a positive results divided by the total number of children enrolled.

The anthropometric index z-scores for Weight-for-age, Weight-for-height and height-for-age Z-scores were generated after entering child sex, age, weight, height in Emergency Nutrition Assessment (ENA) for SMART 2011 tool based on WHO 2006 reference data [34, 35]. If they were less than two standard deviations below the reference mean in the WHO chart, children were classified as stunted (based on height for age Z score), underweight (based on weight for age Z score) or wasted (based on weight for height Z score) [35].

A household wealth index was created using multiple factor analysis from household head earning income, main type of roof and main type of wall of the house, the cooking place for the members of the household, members leaving in the household and the kind of toilet facility. Households were ranked according to their distribution along the index, which was then divided into quartiles.

All statistical analyses were carried out using Stata 14.0 software (STATA Corporation, College Station, TX). The outcome of interest was the prevalence of anaemia; which was calculated as the number of children with altitude-adjusted haemoglobin less than 110g/dL divided by the total number of children tested. Ninety-five percent confidence intervals (CI) were estimated for proportions and standard deviations presented for means after cluster adjustment for administrative hills level.

Using sample size of two proportions and fixing alpha at 5% and power at 80% and estimating the proportion of anaemia in children the lower wealth quintile at 69.3% and higher wealth quintile at 59.4% [22], we needed to enrol a minimum of 492 to answer this objective. Using logistic regression, associations between anaemia and potential associated factors were assessed at bivariate analysis. All variables showing an association at 20% significance level at bivariate analysis. Logical model building using both forward and backward elimination was used to generate minimum adequate model and a 5% significance level was considered significant. Children age and mother’s age were found as confounders and were included into the multivariable logistic regression model. Although child sex, relationship to primary care giver, stunting status were not showing an association at 20% significance level at bivariate analysis, we also included them into the multivariable logistic regression final model on basis of apriori knowledge [36-38]. Akaike’s Information Criteria (AIC) and Bayesian Information Criteria (BIC) were used to test the model and smaller values of AIC and BIC indicating better model fitting.

Ethical considerations

We obtained ethical approval from the School of Medicine Higher Degrees Research and Ethics Committee of Makerere University College of Health Sciences (REF #: 2019–079) and the Institutional Ethical Committee on Health Research of the Faculty of Medicine, University of Burundi (Ref #: FM/CE/02/02/2020). The study complied with the national guidelines and got clearances from the Ministry of Health and the Ministry of Intern affaires to be carried out as a household survey. Informed consent was signed by the primary caregivers of the children prior to their interviews and blood and stool sample collection from the children. Participant’s data was linked to a code number to ensure confidentiality. After communicating to the mothers/primary caretakers, children diagnosed with moderate/severe anaemia, clinical malaria or intestinal parasite infestation were referred to the health facility for etiological diagnosis and/or management.

Results

Characteristics of the study population

A total of 501 were screened for eligibility to join the study of which 498 (99.4%) were enrolled (Fig 1). The reason for exclusion of the 3 children was refusal of the primary care giver to provide written informed consent for the child to participate in the study. The study enrolled slightly more girls (51.2%) than boys, and the mean age at enrolment was 15.24 months (standard deviation [SD] ± 5.74). Majority of the children enrolled had an up-to date status of immunisation (82.1%). A total of 62 (12.5%) participants had positive mRDTs. None of the 329 children tested positive for geohelminth infections on stool examination. Almost all the study children’s primary care-giver was their mother (95.5%), with most care-givers having little or no education (66.1%). The details of the characteristics of the study population are presented in Table 1.

Fig 1

Participant flow chart.

Table 1

Characteristics of the study population.

Characteristic	Number (n)	Percentage (%)
Sex
Male	243	48.8
Female	255	51.2
Age category
6–11 m	169	33.9
12–24 m	329	66.1
Administrative hill (location)
Budahunga	120	24.1
Butegana	64	12.9
Mukenke	164	32.9
Mukenke II	150	30.1
Immunisation status Up-to date
No	89	17.9
Yes	409	82.1
Breastfeeding status
Never breast-fed	13	2.6
Stopped breastfeeding	81	16.3
Still breastfeeding	404	81.1
Mothers age
<20	69	14.7
20–40	382	81.3
>40	19	4.0
Relationship with primary care giver
Mother	476	95.6
Other relative	22	4.4
Education level of primary care giver
None/Primary	329	66.1
Secondary	147	29.5
Tertiary	22	4.4
Number of siblings under 5 years of age
0–1	321	64.5
More than 1	177	35.5
Social economic status of the household
Very poor	124	25.5
Poor	119	24.5
Middle	121	25.0
Rich	121	25.0
Diet includes fortified foods
No	465	93.4
Yes	33	6.6
Taken deworming medication in last 6 months
No	44	8.8
Yes	285	57.2
Not applicable	169	33.9
Wasted
No	415	83.5
Yes	82	16.5
Stunted
No	264	53.0
Yes	234	47.0
Underweighted
No	337	67.8
Yes	160	32.2
Malaria test results (RDT)
Negative	436	87.6
Positive	62	12.5

Prevalence of anaemia

The mean haemoglobin concentration was 99.65 g/l (Standard Deviation [SD] 0.69). Of the enrolled children, 370 had haemoglobin less that 110g/L giving an overall estimated prevalence of anaemia of 74.3% (95% CI 61.5–84.0). Of the anaemic study participants, 131 (35.4%) had mild anaemia, 219 (59.2%) had moderate anaemia, and 20 (5.4%) had severe anaemia.

Anaemia prevalence was higher in children aged 6-11months compared to the older children (83.4% versus 69.6%, aOR = 2.27, 95% CI:1.32–3.91; p-value = 0.003). The prevalence of anaemia was also higher in children who had a positive mRDT test than those that were negative (87.1% versus 72.5%, p-value = 0.017) (Table 2).

Table 2

Factors associated with anaemia among children aged 6–24 months.

Characteristic	n/N (% of anaemia)	Crude OR (95% CI)	p-value	Adjusted OR (95% CI)	p-value
Sex
Male	183/243 (75.72)	1		1
Female	186/255 (72.94)	0.86 (0.58–1.29)	0.478	0.72 (0.43–1.20)	0.209
Age category (months)
12–24	229/329 (69.60)	1		1
6–11	141/169 (83.43)	2.20 (1.31–3.68)	0.001	2.27 (1.32–3.91)	0.003
Administrative hill (location)
Butegana	38/64 (59.38)	1		1
Budahunga	101/120 (84.17)	3.64 (1.81–7.32)	<0.001	3.20 (3.07–3. 34)	<0.001
Mukenke	120/164 (73.17)	1.87 (1.02–3.42)	0.004	2.81 (2.51–3.16)	<0.001
Mukenke II	111/150 (74.0)	1.95 (1.05–3.61)	0.035	1.19 (1.82–2.63)	<0.001
Mother’s age
20–40	278/382 (72.77)	1		1
<20	55/69 (79.71)	1.47 (0.74–2.94)	0.276	1.23 (0.72–2.11)	0.439
>40	16/19 (84.21)	1.99 (1.11–3.60)	0.021	1. 75 (0.74–4.13)	0.095
Relationship to primary care giver
Mother	355/476 (74.58)	1		1
Other relative	22/498 (68.18)	0.73 (0.32–1.69)	0.463	2.71 (0.33–22.42)	0.356
Education level of primary care giver
None/Primary	254/329 (77.20)	1		1
Secondary	103/147 (70.7)	0.69 (0.49–0.97)	0.034	0. 67 (0.47–0.95)	0.024
Tertiary	13/22 (59.09)	0.43 (0.29–0.64)	0.000	0.48 (0.38–0.61)	<0.001
Number of siblings <5 years of age
0–1	235/321 (73.21)	1		1
More than 1	135/177 (76.27)	1.20 (0.96–1.45)	0.125	1.17 (0.91–1.51)	0.211
Social economic status
Very poor	91/124 (73.39)	1		1
Poor	88/119 (73.95)	1.02 (0.58–1.82)	0.921	0.95 (0.41–1.23)	0.910
Middle income	94/121 (77.69)	1.26 (0.70–2.27)	0.434	1.17 (0.48–2.84)	0.729
Rich	86/121 (71.07)	0.89 (0.51–1.56)	0.686	0.94 (0.54–1.62)	0. 823
Diet includes fortified foods
Yes	28/33 (84.85)	1		1
No	342/465 (73.55)	0.50 (0.14–1.82)	0.290	0. 42 (0.15–1.21)	0.108
Dewormed in last 6 months
Yes	190/285 (66.7)	1		1
No	39/44 (88.6)	3.90 (1.49–10.22)	0.006	3.54 (1.79–6.99)	<0.001
Not applicable	141/169 (83.4)	2.52 (1.57–4.05)	<0.001	-	-
Stunted
No	193/264 (73.11)	1		1
Yes	177/234 (77.64)	0.66 (0.51–0.86)	0.002	0.90 (0.60–1.37)	0.582
Under weighted
No	242/337 (71.81)	-		1
Yes	128/160 (80.0)	0.64 (0.47–086)	0.003	0.77 (0.61–1.07)	0.131
Malaria test results
Negative	316/436 (72.48)	1		1
Positive	54/62 (87.10)	2.56 (0.18–5.55)	0.017	2.49 (0.89–6.96)	0.136

Factors associated with anaemia in the study population

At multivariate analysis, factors significantly associated with anaemia included: 1) the administrative location of the child’s home; 2) status of deworming; 3) child’s age group; and 4) Education level of the child primary care giver. Our study showed that children from Mukenke II, Mukenke and Budahunga had 2 to 3 times higher odds of having anaemia than children from Butegana (aOR = 2.19; 95% CI: 1.82–2.63; aOR = 2.82; 95% CI: 2.51–3.16 and aOR = 3.20; 95% CI: 3.07–3.34 respectively). Children aged 6 to 11 months 2 times higher odds of developing anaemia compared to children aged 12 to 24 months (aOR = 2.27; 95% CI:1.32–3.91). Children who didn’t get deworming medication in the last 6 months had higher odds of having anaemia compared to those who took deworming medication (aOR = 3.54; 95% CI: 1.79–6.99). Children whose primary care giver have a secondary and tertiary education level had less odds of having anaemia (aOR = 0.68; 95% CI: 0.45–0.99 and 0.43; 95% CI: 0.33–0.58) than others.

Discussion

This study estimated the burden and factors associated with anaemia among children aged 6 to 24 months in Mukenke Health District, northern Burundi, an area with high malaria transmission. The overall prevalence of anaemia was 74.3% with the burden highest in children under one year of age. Factors associated with anaemia included the child’s deworming status, the child’s age group, location of the child’s home, and education level of the child primary care giver. These study findings highlight the urgent need to address the anaemia burden in this setting in order to reduce on the future possible effects in the population.

The prevalence of anaemia in the study is alarmingly high, with almost three quarters of the study population meeting the definition of being anaemic. Most worrying is that the children under one year of age were more likely to be anaemic than the older children, and majority of the anaemic children had moderate to severe anaemia. The observed burden in this setting is of concern given that anaemia in this age-group may lead to delayed growth and impaired neurological function [39, 40]. Although the causes of anaemia were not evaluated as part of the study, given the study setting, a number of factors may be attributed to the high burden observed. The possible causes of anaemia in the children in this setting may include nutritional origins especially iron deficiency which is the most common cause of childhood anaemia in African children [27].

Other causes of anaemia in this population could be infectious diseases such as malaria and HIV among others [36]. Indeed, malaria may be a key contributor to the observed burden given that the study was conducted in a high malaria burden setting. Malaria infection causes haemolysis of infected and uninfected erythrocytes and bone marrow dyserythropoiesis resulting in reduced haemoglobin concentration [7, 41]. Unfortunately, the greatest impact is in young children, and in particular infants [42]. Even if the p-value was not significant, our bivariate analysis showed that children with a positive malaria test have more than two times the odds of a having anaemia than children with a negative test. A number of other studies have demonstrated the relationship between malaria and anaemia for example, a study by Legason et al in North-western Uganda showed that anaemia was 1.5 times higher among malaria positive children compared to malaria negative [43]. On the other hand, helminth infections which have been documented to cause anaemia are also common parasitic infections in the tropics and subtropics [36]. Helminths interrupt the host’s acquisition of nutrients by ingestion and digestion of host blood resulting mainly into iron deficiency [44]. We found that deworming was associated with reduced odds of having anaemia, which is in agreement with the mechanism of action, given the treatment eliminates the helminth infections. Indeed, studies have widely reported the relationship between deworming and anaemia [45]. Although out of the scope of this work, inherited forms of anaemia especially sickle cell anaemia have been documented to cause anaemia, the commonest in this setting being sickle cell aneamia [46].

High prevalence of anaemia in the very young children has been documented in similar settings in Africa. In Ethiopia, the prevalence of anaemia in children 6–23 months was estimated at 71% in 2005, 61% in 2011, and 72% in 2016 [47]. In Namutumba district in Uganda, the prevalence was estimated at 58.8% [48]. In Armenia the highest prevalence of anaemia (67·9%) was detected among 6–12-month-old children [49]. According to the 2016/2017 DHS in Burundi, the prevalence of anaemia in same age-group was estimated at 79.2% showing only a slight reduction in estimates over the last 4 years [22].

The iron stores present at birth and in breast milk protect the infant from iron deficiency up to 6 months of age. But due to the rapid growth of children in the first months of life, there is increased micronutrient requirements including iron. At this age, although breast milk provides some nutrients, it is not sufficient to sustain his growth and development [50]. Above this age, it is well known that dietary sources of iron become critical to keep up with the child’s rapid rate of red blood cell synthesis [50], and infants should start receiving additional sources of iron to maintain sufficient haemoglobin concentration. Ensuring that their nutritional needs are met therefore requires to start complementary feeding at 6 months and complementary foods must be adequate, safe and properly fed. Food fortification and universal or targeted nutrient supplementation may also help to ensure that older infants and young children receive adequate amounts of iron and other micronutrients [51].

In addition to the child’s age group and taking deworming medication, location of the child’s home where the child resides and education level of primary care givers were associated with anaemia. Our study showed a statistical association between anaemia and administrative location of the child’s home. Children from Mukenke and Budahunga were almost 3 times more likely to be anaemic than children from Butegana. This could be explained by the fact that Budahunga hosts a site of a population of the Batwa ethnic (population group which has marginalized nutrition habits) and for Mukenke, the presence of single-parent families abandoned by fathers who were seasonal workers in the area artisanal mining. Khan et al. in Bangladesh revealed similar association; childhood anaemia was significantly associated with geographical location defined by division 60.4% among children under the age of 5 were anaemic in Barisal and 58.9% in Rangpur division while the lowest was 47.8% in Dhaka division [52]. This is also similar to what was observed by Sharma et al. in India; a significant spatial heterogeneity in the prevalence of anaemia among children [53]. By contrast, our findings are not consistent with other analysis where they didn’t find this association [5, 54].

Anaemia was negatively associated with education level of the child primary care giver. Children whose mothers had a secondary and tertiary education level were less likely to be anaemic. For Tesema et al children whose mother education level had 1.73 times at no formal education, 1.39 times at primary education, and 1.27 times at secondary education level than children whose mother had a higher level of education [6]. The level of education of primary care givers may positively influence health care and feeding practices of their children. Educated mothers are more aware of their children’s health and take into account the nutritional values of foods. Educated mother understand better and provide a healthy and hygienic diet for their children, improving their nutritional status [55, 56].

Our findings also showed an association between deworming and anaemia. Watthanakulpanich et al, in their study in Thailand reported that anaemic status of the hookworm positive group significantly improved by 2 months after deworming of the hookworm-infected children (Hb = 12.1) and to became comparable with the helminth-free control group (Hb = 12.0) [18]. On the other hand, a study on effect of deworming on school-aged children’s physical fitness, cognition and clinical parameters in a malaria-helminth co-endemic area of Ivory Cost revealed that deworming showed no effect on haemoglobin levels and anaemia. The mean Hb levels did not differ significantly between surveys (baseline, 120.4 g/l; follow-up, 119.9 g/l; p = 0.631) and the overall prevalence of anaemia remained unchanged (baseline, 34.3%; follow-up, 34.3%) [57]. Despite the variations in the above findings, it is known that repeated chemotherapy at regular intervals (periodic deworming) in high-risk groups can ensure that the levels of infection are kept below those associated with morbidity [58] and intervention studies have shown positive associations between mass deworming and decreased prevalence of anaemia among children from developing countries [54]. The deworming with two rounds of albendazole and praziquantel spaced by 2 months is highly efficacious against soil-transmitted helminths among children [59].

Study limitations

Our study has some limitations that should be taken into consideration. A cross sectional design of the study limits our ability to assess temporal or causal of the associated variables, and the study did not aim to identify the aetiology of anaemia. Our study did not measure other known risk factors like maternal anaemia, HIV disease, haemoglobinopathies and what are the levels and how they would affect our results.

Conclusions

Our study showed a very high burden of anaemia among children aged 6–24 months in Mukenke Health district with the prevalence much higher than the WHO cut-offs for declaring the burden a major public health problem. We found that having a primary care giver with secondary and tertiary education level were associated with reduced odds of anaemia while not taking deworming medication, very young children and a residence location were associated with higher odds. To prevent the long-term adverse outcomes of anaemia, policy makers should focus on improving uptake of deworming, malaria prevention programs, as well as promote women education and preventive interventions of families with very young children.

Multivariable logistic regression model.

(PDF)

Click here for additional data file.

Study questionnaire English.

(PDF)

Click here for additional data file.

Study questionnaire local language (Kirundi).

(PDF)

Click here for additional data file.

5 Jan 2022

Prevalence and factors associated with anaemia in children aged 6-24 months living a high malaria transmission setting in Burundi.

PLOS ONE

Dear Dr. % Jean Claude Nkurunziza %,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please respond to all the comments made

Please submit your revised manuscript by Feb 11, 2022. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mary Hamer Hodges, MBBS MRCP DSc

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

4. Your abstract cannot contain citations. Please only include citations in the body text of the manuscript, and ensure that they remain in ascending numerical order on first mention.

5. Please include a copy of Tables 1 and 2 which you refer to in your text on pages 11 and 12.

6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

12 Feb 2022

February 10, 2022

To: Academic Editor

PLOS ONE

Reference number: PONE-D-21-22573

Thank you very much for the careful consideration of our manuscript and for inviting us to re-submit our revised manuscript (based on the Reviewers' comments) entitled: “Prevalence and factors associated with anaemia in children aged 6-24 months living a high malaria transmission setting in Burundi ” Below are point by point responses to the reviewers’ comments. We are re-submitting a revised version of the manuscript that addresses the points raised during the review process. We look forward to your favourable consideration of this re-submission.

POINTS BY POINTS RESPONSES TO THE REVIEWERS’ COMMENTS

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

The revised manuscript was adapted to PLOS ONE’s style requirements

2. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.

Study questionnaire in English is available on protocols.io: dx.doi.org/10.17504/protocols.io.b4ryqv7w

Study questionnaire in local language (Kirundi) is available as attached file under materials section on protocols.io: dx.doi.org/10.17504/protocols.io.b4vxqw7n

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

Data of the study involve indirect identifiers (names initials, sex, age, location) and may risk the identification of study participants. Ethical restrictions may be applied on the data supporting this study in the interest of protecting confidential participant information. To access data, interested and qualified researchers may submit requests to the School of Medicine Ethics Committee at the College of Health Sciences, Makerere University (rresearch9@gmail.com), can also submit demands to the current chairperson of the committee, Prof. Ponsianoo Ocama (ponsiano.ocama@gmail.com) or to the Institutional Ethical Committee on Health Research of the Faculty of Medicine, University of Burundi (faculte_medecine_ub@yahoo.fr).

4. Your abstract cannot contain citations. Please only include citations in the body text of the manuscript, and ensure that they remain in ascending numerical order on first mention.

Ok, no citations in abstract.

5. Please include a copy of Tables 1 and 2 which you refer to in your text on pages 11 and 12.

Table 1 on pages 12-13 and Table 2 on pages 13

6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Reference list corrected and completed.

Major comments:

1. Provide more details of systematic sampling procedure referred to in Study Population section (Line 86)

Response:

Using the computed sample size of 492 participants, and the registered total number of children aged 6-24 months in Mukenke Health center catchment area (n=1116), we calculated the sampling interval by dividing accessible population by the sample size (1116/492=2,27 rounded at 2). Using the community health workers (CHW) registers in each village (the register lists the children and their households), we selected households to participate in the study using an interval of 2 as listed in the register. We selected only one participant (child aged 6-24 months) per household using inclusion and exclusion criteria (page 6 lines 89-99).

2. Line 168 Data Management and analysis. A little more detail about the cluster adjustment for administrative hills level.

Response:

We anticipate that clustering exists at the administrative hills level and we adjusted for this by including administrative hills as a covariate to the final model. The final adjusted model of significant covariates was

Logit (anaemia) = 1.89 + 1.08 Budahunga +1.01 Mukenke+ 1.40 No deworming +0.81 Tertiary Ed -0.81 poor SES -0.96 Middle SES- 0.86 Rich SES

[Stata command: logit anaemia i.chsex ib2.ChageGpe ib2.rezhill ib1.BreastFdSt ib2.MothAge ib1.Deworming i.CgChrelat i.Under5sibl i.CgEducGp i.SES3quintile3 ib1.csfpfood Stunting Wasting2 Underweight i.malarscrn, vce(cluster rezhill) ]

3. Line 177. Sentence appears to be incomplete

All variables showing an association at 20% significance level at bivariate analysis and potential confounders were considered for inclusion into the multivariable logistic regression model (page 10 lines 183-184).

4. Line 183 Describe what criterion was used for eliminating models. For instance, AIC, BIC etc,

Response:

For eliminating models, we used Akaike’s Information Criteria (AIC) and Bayesian Information Criteria (BIC). Smaller values of AIC and BIC indicating better model fitting (page 10 lines 192-194).

Minor comments:

5. Abstract/Conclusion. Line 25 and 26. Please revise sentence ‘

Sentence revised (page 3 line 27)

6. Introduction: Line 48. Change county to country

county changed to country (page 4 line 49)

7. Line 123 Study Procedures. Change plasmodium to Plasmodium

plasmodium changed to Plasmodium (page 8 line 129)

8. Line 158. Change underweighted to underweight

underweighted changed to underweight (page 9 line 164)

9. Results Line 221 Change table 2 to Table 2

table 2 changed to Table 2 (page 13 line 230)

10. Line 228- 229 MUKENKE, BUDAHUNGA and BUTEGANA should all be written in Sentence case

MUKENKE, BUDAHUNGA and BUTEGANA written in Sentence case: Mukenke, Budahunga and Butegana (page 15 lines 237- 238)

11. Line 304, 306 and 341. Same comments as above

Sentence case of Mukenke and Budahunga (page 18 lines 312), Butegana (page 18 line 314), Mukenke (page 20 line 349)

12. Conclusions Line 348. Delete in

in deleted (page 20 line 357)

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

22 Mar 2022

Please address each comment from the Reviewer

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mary Hamer Hodges, MBBS MRCP DSc

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: I have placed main concerns as introductory statement in the review which I have uploaded. The concerns are mainly on altitude adjustment, taking clustering of the data into account during analysis and the association between deworming and anaemia being chance effect as it can be verified from absence of any infection among the study children.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Plos_anaimia_manuscript.docx

Click here for additional data file.

5 May 2022

We are re-submitting a revised version of the manuscript that addresses the points raised during the second review. The specific file description is 'Response to Reviewers'.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

15 Jun 2022

Submitted filename: review round 2.docx

Click here for additional data file.

30 Jul 2022

Response to review comments for the manuscript entitled: “Prevalence and factors associated with anaemia in children aged 6-24 months living a high malaria transmission setting in Burundi” Ref: PONE-D-21-22573R2.

Thank you very much for the careful consideration of our manuscript and for inviting us to re-submit our revised manuscript (based on the second round of Reviewers' comments) entitled: “Prevalence and factors associated with anaemia in children aged 6-24 months living a high malaria transmission setting in Burundi” Below are point by point responses to the reviewers’ comments. We are re-submitting a revised version of the manuscript that addresses the points raised during the second review. We look forward to your favourable consideration of this re-submission.

POINTS BY POINTS RESPONSES TO THE REVIEWERS’ COMMENTS

Methods

Comment 1: what are the specific variables included to generate the wealth index (Line 161-

62)? Are the variable all numeric (i.e., quantitative)? If all variable are quantitative, go with

the PCA, qualitative only use the MCA and mixed type, it is advisable to use multiple factor

analysis. Our response: “Variables used in the SES included: household head earning income, house ownership, house wall, main source of energy for cooking, main type of floor and main source of drinking water. This detail is included in the manuscript (line 186-189). ”: Comment to response: This part of my comment is not well addressed. PCA is used for dimension reduction of continuous variables, multiple correspondence analysis (MCA) for categorical data and if the variables used to reduce dimension of the data is mixed (i.e both categorical and continuous) like the case in the current manuscript, multiple factor analysis is used. Thus, I recommend to use MFA instead of PCA in the current manuscript.

Response: Thank you for this in-put, we have now used the MFA instead of PCA to generate the wealth index. From the new analysis, socioeconomic status was no longer significant in the final model and has been dropped. The updated results are presented in Table 1 (Page 13-14) and Table 2 (Page 15) as well as the text (line 200-1203).

Comment 2: In line 165 through 167, and also in the abstract section, hemoglobin level is adjusted for altitude but the data is collected by questionnaire and double entered into a

software. How you get the altitude of the household for the children involved in the study. Our response: We applied altitude adjustment on haemoglobin measured values for Mukenke altitude based on the midpoint of altitude range (1403.5 m for the 1331−1476 range), we used 1989 MMWR equation for Hb adjustment (g/L) = [(−0.032 × (altitude × 0.0032808) + 0.022 × (altitude × 0.0032808)2) × 10] = -3.20 g/L (line 147-150). Comment to response: The response provided by the authors for this comment is not satisfactory. I recommend adjusting hemoglobin for altitude based on reference. Haemoglobin concentrations for the diagnosis of anaemia and assessment of severity published by WHO available at the following URL.

https://apps.who.int/iris/bitstream/handle/10665/85839/WHO_NMH_NHD_MNM_11.1_eng.pdf?sequence=22&isAllowed=y

Response: WHO-based cutoffs for Children 6 - 59 months of age are: Non- anaemia: 110 g/l or higher, 100-109 g/l for mild, 70-99 g/l for moderate and lower than 70 g/l for severe anaemia. While altitude adjustments to measured haemoglobin concentrations is -2g/l for 1000m metres above sea level and -5g/l for 1500 metres above sea level. In this study we didn’t not collect the altitude estimates for the homes of each child, however, we used the midpoint altitude estimates of the study area (Mukenke altitude ranges between 1331−1476 and we used 1403.5 m for the average estimate)as reported in elevation map . We used the CDC formula published in 1989 MMWR “equation for Hb adjustment (g/L) = [(−0.032 × (altitude × 0.0032808) + 0.022 × (altitude × 0.0032808)2) × 10]” = -3.20 g/L , . This detail is included in the manuscript, line 160-164.

Comment 3: Absence of any parasitic infections from the kato-katz technique indicates that

the young children were not at risk for such infections. The association between deworming

and anaemia seems chance effect than true effect due to not dewormed. Is there any causal pathway that deworming can increase the hemoglobin concentration in the absence of any parasitic infections? I think it is not the deworming drug that influenced the hemoglobin concertation, rather it may be the supplementations given to the children during the deworming campaign. If this was not the case, I think it is better to remove deworming from the analysis.

Response: The association between deworming and anaemia in very young children has previously been well documented in clinical trials . Our study showed that for the 329 stool samples tested, none was positive for geohelminth infections. Of these, 285 (86.6%) had received deworming medication during the December 2019 national deworming campaign and 169 (33.9%) were aged 6 to 11 months and at low risk for geohelminth infections. From this analysis we are confident that absence of any parasitic infections from the kato-katz technique in those at risk of infection could be also a result of the deworming campaign. The only additional supplementation received during the campaign was vitamin A which may not explain the association observed in the study.We have recoded the covariate “Deworming” and the analysis now takes into account the very young children (deworming not applicable). We still find a statistically significant relation in the bivariate and the multivariate analysis between anaemia and deworming: adjusted Odd ratio [aOR] of 3.54 and 95% CI 1.79-6.99, p<0.001 for 44 children who didn’t not receive deworming medication last six months out of 329 who were at risk of geohelminth infections (Table 2).

Comment 4: I think the discussion should not be crowded by numbers for comparison. Such

numbers on the prevalence of different study findings shall be presented in the introduction

section. And make the comparisons based on the level of significance and looking your

confidence intervals than putting the numbers. The same thing is true for effect size of the

factors. This comment is misunderstood by the authors. I recommend not to use the numbers which are indicated in the introduction and result section of the manuscript. Remove the odds ratio and its 95% CI in the discussion section.

Response: Thank you for this suggestion. We proceeded as recommended: We presented numbers on the prevalence of different study findings shall be presented in the introduction section (line 45-55, 65-68) and we removed the odds ratios and 95% CI in the discussion section (lines 305, 342-343 and 355).

References:

Elevation map. Elevation map for localities. Elevation of Mukenke, Bwambarangwe, Burundi (Latitude: 2.58 South, Longitude: 29.95 East, Altitude: 1331.00m/4599.74ft and Latitude: 2.58 South, Longitude: 30.32 East, Altitude: 1476.00m/4842.52ft). Elevation map for localities 2021.

Centers for Diseases Control and Prevention. CDC criteria for anemia in children and childbearing-aged women. MMWR Morbidity and mortality weekly report. 1989; 38(22):400-4.

Sarma H, Wangdi K, Tariqujjaman M, et al. The Effects of Deworming and Multiple Micronutrients on Anaemia in Preschool Children in Bangladesh: Analysis of Five Cross-Sectional Surveys. Nutrients 2021; 14(1).

Bauleni A, Tiruneh FN, Mwenyenkulu TE, et al. Effects of deworming medication on anaemia among children aged 6-59 months in sub-Saharan Africa. Parasit Vectors 2022; 15(1): 7.

Submitted filename: Response to reviewer2.2nd round_PlosOne_anaemia manuscript.docx

Click here for additional data file.

15 Aug 2022

Prevalence and factors associated with anaemia in children aged 6-24 months living a high malaria transmission setting in Burundi

PONE-D-21-22573R3

Dear Dr. % Nkurunziza %,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Mary Hamer Hodges, MBBS MRCP DSc

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: All my concerns are corrected. However, a language edition may be necessary to improve the overall quality of the paper.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

**********

24 Aug 2022

PONE-D-21-22573R3

Prevalence and factors associated with anaemia in children aged 6-24 months living a high malaria transmission setting in Burundi.

Dear Dr. Nkurunziza:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Mary Hamer Hodges

Academic Editor

PLOS ONE