Influence of malaria, soil-transmitted helminths and malnutrition on haemoglobin level among school-aged children in Muyuka, Southwest Cameroon: A cross-sectional study on outcomes.

BACKGROUND: The health of school-aged children (SAC) is often compromised by malaria parasitaemia (MP), soil-transmitted helminths (STH), and malnutrition in the tropics. The aim of this study was to determine the prevalence and influence of MP, STH and malnutrition on haemoglobin (Hb) levels as well as identify its predictors.
METHODS: This cross-sectional study was carried out in SAC (4-14 years) in Owe, Mpundu and Meanja villages in Muyuka, Southwest Cameroon. Hb concentration was measured using a URIT-12 Hb meter while MP and STH were determined by Giemsa staining of blood films and Kato-Katz technique respectively. Anthropometric measures (weight, height and mid upper arm circumference (MUAC)) of malnutrition (z-scores of <-2 standard deviations below mean) were obtained by standard methods. Categorical and continuous variables were compared appropriately, and multiple linear regression model was used to determine predictors of Hb level.
RESULTS: The prevalence of MP, STH, anaemia and malnutrition in the 401 SAC examined were 33.9%, 2.2%, 75.3% and 24.4% respectively. The prevalence of MP varied significantly with locality (P = 0.031). Stunting occurred commonly (23.7%) and was significantly higher in males (28.6%), children 11-14 years old (38.3%) and those of Meanja locality (47.4%) than their counterparts. Significantly higher prevalence of anaemia was observed in children of Meanja (89.5%) and those both MP positive and malnourished (86.2%). Moderate anaemia occurred commonly (60.6%) and children ≤6 years old had significantly (P = 0.034) higher prevalence (75.0%). Mean Hb level varied significantly (P = 0.004) with age and those ≤6 years old infected with MP had significantly (P = 0.022) lower values. Significant predictors of Hb levels were the MUAC (P <0.001) and the MP status (P = 0.035). Based on the Hb level (>11g/dL) and the absence of MP, STH and malnutrition, 13.7% of the SAC were considered as healthy.
CONCLUSIONS: The health of a majority of SAC is compromised by malaria, helminthiasis, malnutrition and other conditions not investigated. Anaemia is of major public health concern hence, intervention programmes that integrate malaria control with improvement of educational levels especially on proper nutrition and health care practices are desirable.

Background

Health, as defined by the World Health Organization (WHO), is a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity [1]. The relative level of wellness and illness of an individual which considers the presence of biological or physiological dysfunction, symptoms and functional impairment defines the health status of an individual. The health of school-age children (SAC) is usually compromised by common diseases such as malaria (which remains a considerable public health problem in much of the tropics and subtropics) and helminthiasis [2-4]. In Cameroon, malaria and soil-transmitted helminth (STH) infections are both widespread and are accountable for increased morbidities and associated consequences in vulnerable populations, including SAC [5-8].

Plasmodium falciparum is the most prevalent malaria parasite in the WHO African Region, accounting for 99.7% of estimated malaria cases in 2017 and Cameroon is amongst the 11 high—burden countries that account for more than 70% of the global malaria cases and deaths [2]. P. falciparum is also the most pathogenic species and remains a major cause of morbidity and mortality, with children less than five years of age and pregnant women severely affected [9]. In 2017, children aged under 5 years accounted for 61% (266 000) of all malaria deaths worldwide [2]. Consequently, control measures in endemic regions have focused on the protection of these two groups at highest risk of malaria disease. On the other hand, older children who are less often symptomatic, and may play an important role in transmission, have not traditionally been the focus of intensive detection and control strategies [10].

School age children rather than preschool children or adults, are most at risk of Plasmodium helminth co-infection and thereby at greatest risk of the consequences of co-infection [3]. Co-infections with helminth and malaria parasites have negative impact upon host and synergisms between multiple parasite species infections and infection intensity are known to exacerbate anaemia [3, 8, 11]. STH infections can accelerate or exacerbate malnutrition hence infections with STH and malaria parasite could singly or combined be contributing factors of malnutrition and/or anaemia as shown by several studies [12-15].

Malnutrition is said to be the underlying cause of deaths in 48% of children below 5 years in Cameroon [16] while the burden of malnutrition in SAC is infrequently determined especially in rural areas. Common nutritional indicators of subclinical undernutrition such as underweight, wasting and stunting in children are proxy indicators of overall well-being and reflect, the burden of infectious diseases in the community [17]. The nutritional status of SAC impacts their health, cognition, and subsequently their educational achievement [18]. Manifestation of malnutrition is often observed in terms of anaemia, micronutrient deficiencies (iron, folic acid, riboflavin, vitamin A and B12) and anthropometric measurements. Hence, the assessment of nutritional status of this population segment is essential for making progress towards improving the overall health of SAC [19].

Hb concentration is the most reliable indicator of anaemia at the population level, as opposed to clinical measures which are subjective and therefore have more room for error [20]. Anaemia is an indicator of both poor nutrition (micronutrient deficiency) and poor health (chronic infections, predominantly malaria, hereditary haemoglobinopathies). In most developing countries, anaemia is a public health problem and SAC are more vulnerable due to their rapid physical and physiological development [20, 21]. The prevalence of anaemia among SAC in Africa ranges from 64.3% to 71% [20] and in the Mount Cameroon area it ranges from 19.8% to 44.2% [22, 23]. Anaemia is considered as a public health problem (prevalence ≥ 5%) when the Hb value is below the population specific Hb threshold. The prevalence of anaemia is objective and quantifiable and can be measured in the most remote areas where access to health care is a challenge. In addition, anaemia is a major complication of several neglected tropical diseases (NTD). Moreover, it changes in predictable fashions with alterations in disease burden [24].

While studies may have been carried out on single infections with malaria parasite or co-infections with STHs in different populations, there is a dearth of knowledge on the health status of SAC in Cameroon especially in rural settings. Thus, the main objectives of this study were to determine the prevalence of MP, STH and malnutrition, assess their influence on Hb levels as well as identify the predictors of Hb levels. We hypothesised that the level of haemoglobin is a valid indicator in predicting the health status of SAC in rural areas endemic for malaria and NTDs such as soil-transmitted helminths.

Methods

Study sites

The study sites comprised of Owe, Mpundu and Meanja villages of Muyuka Sub-division located at the foot of Mount Cameroon. Owe is located at an altitude of 59m, latitude 4°17ʹ23ʺ N, longitude 9° 22ʹ50ʺ E to 84m a.s.l., latitude 4°18ʹ00ʺ N and longitude 9° 22ʹ32ʺ E. Mpundu is located at an altitude of 54m, latitude 4°14ʹ14ʺ N, longitude 9°24ʹ44ʺ E, to 68m a.s.l, latitude, 4°14ʹ42ʺ N and longitude 9°23ʹ40ʺ E. Meanja is located at 62m above sea level (a.s.l.), latitude 4° 14ʹ53ʺN, longitude 9°23ʹ48ʺ E to 69m a.s.l., latitude 4° 15ʹ53ʺN and longitude 9°24ʹ48ʺ E as shown in Fig 1. Owe, Mpundu and Meanja villages are 7km, 12km and 5.6 km away from Muyuka town respectively. The rainy season in the area, runs from March to October while the dry season runs from November to mid-March. Annual temperature ranges from 18–35°C. The natives of these villages are known as the Balongs [25]. Subsistence and small-scale cash crop farming constitute the mainstay of the villages. Other nearby landmarks include: River Mungo, a Cameroon Development Corporation (CDC) workers’ camp and CDC palm and rubber plantations. Owe has no closed potable water source but has streams which serve as a source of drinking water and other household activities while Meanja and Mpundu have closed potable water source. They lack health facilities such as clinics/hospitals and residents tend to visit pro-pharmacies more often when sick than hospitals for drugs.

Fig 1

Map showing the location of study areas in Muyuka, Southwest Cameroon.

Study design

The cross-sectional study was carried out simultaneously in three localities between the months of March and June 2015.

Study population

The study population constituted SAC (4–14 years old) of both sexes whose parents or legal representatives had signed a written consent/assent form. Primary schools were selected at random from a list of schools operating in these villages and children selected at random from each class.

Sampling size

The sample size was determined using the formula n = Z2pq/d2 [26], where n represented the sample size required; Z was 1.96, which is the standard normal deviate (for a 95% confidence interval, CI); p was 35.5%, the prevalence of P. falciparum [8] or 14.0%, the proportion of helminth infection [27]; q was 1-p, the proportion of malaria parasite negatives or helminth negative; and d was 0.05, the acceptable error willing to be committed. A minimum sample size of 268 was obtained from the average of the calculated sample size for the prevalence of P. falciparum (351) and that of helminth (185).

Sampling procedure

Five (05) schools (Government school (GS) Owe, Catholic school (CS) Owe, GS Mpundu, CS Mpundu and CS Meanja) selected by random sampling from a list of nine schools operating in these villages agreed to take part in the study. A representative number of children were selected at random by balloting from each class. First, the children, head teachers, and village chiefs were sensitized on the protocol and benefits of the exercise to the children and to the community. Informed consent forms were sent to parents/guardians through the pupils explaining the purpose and benefits of the study as well as the precautions taken to minimize risk. Children who returned with signed consent forms were enrolled in the study.

The samples collected comprised of finger prick blood for MP detection and speciation, and Hb measurement for assessment of anaemia as well as stool sample for the detection of egg or larva of STHs. The investigative methods included the use of questionnaires, clinical evaluation and laboratory methods such as Giemsa-stained microscopy and Kato-Katz technique.

Questionnaire

A simple pre-tested structured questionnaire (S1 File) was administered to each pupil with the assistance of schoolteachers and investigators. Information on important demographic data such as age, sex, area of residence was obtained as well as information relating to malaria and STH infection epidemiology. In brief, the questionnaire sought to obtain information on insecticide treated net (ITN) possession and use, house type, bushes around the residence, pre-fever history, history of anti-malarials, toilet availability and type, hand washing, shoe wearing, and farming frequency.

Clinical evaluation

The axial temperature of each child was measured using a mercury thermometer to determine the presence of fever. Children who had temperatures ≥ 37.5°C were reported to have fever.

The age, height, weight and mid upper arm circumference (MUAC) of participants were measured in order to determine anthropometric indices. Height and weight measurements were recorded to the nearest 0.1cm and 0.5kg respectively. Weight was measured using a mechanical weighing scale and heights and MUAC were measured using a measuring tape. Ages of children were obtained from school records with the permission of the head teacher. The z scores height-for-age, weight-for-age, weight-for-height were computed based on the WHO 2006 growth reference curves [28]. Stunting was defined as height-for-age z (HAZ) score of < -2; underweight as weight-for-age z (WAZ) score of < -2 and wasting as weight-for-height z (WHZ) score < -2. A child was considered malnourished if he or she scored < -2 in one of the z scores (HAZ, WAZ, WHZ). Z score of < -3 indicated severe stunting, underweight and wasting.

Collection of blood and stool samples

Finger prick blood was collected from each pupil with signed assent form. The first drop of blood was immediately wiped off and the next drop was placed on the Hb test strip already inserted into the URIT-12 haemoglobin meter (URIT Medical Electronic co., Ltd, London, United Kingdom) for Hb determination. The Hb value displayed was recorded to the nearest 0.1 g/dL. A participant was considered anaemic when Hb concentration fell below the WHO reference values for age or gender [29]. Anaemia was further classified as either mild (between 10.0 g/dL and the level in the WHO reference values for age), moderate (7.0–9.9 g/dL based on WHO reference for age) or severe (<7 g/dL and WHO reference for age) [29]. Two drops of whole blood were placed on a labelled, grease-free glass slide 1mm apart for the preparation of thin and thick films for the detection and speciation of malaria parasites, as described by Cheesbrough [30].

Each child was provided with a labelled screw-capped plastic container to return with a fresh midday stool sample. A single stool sample was collected from each participant. The fresh samples were preserved with 10% formalin to maintain the morphology of the egg. Samples were transported in a specimen box to the Malaria Research Laboratory of the University of Buea for diagnosis by the Kato-Katz technique.

Laboratory procedures

Both thin and thick blood films prepared in the field were air-dried. The thin blood film was fixed in 75% methanol, and both thick and thin blood films were stained using 10% Giemsa solution for 15 minutes [30]. Slides were then microscopically examined for the presence of malaria parasites by two independent parasitologists, and in the case of any disparity they were read by a third parasitologist. Slides were considered positive when asexual forms and/or gametocytes of any Plasmodium species were observed on the blood film. Parasite density per μL of blood was determined based on the number of parasites per 200 leukocytes on thick blood film assuming a white blood cell count (WBC) of 8000 leucocytes/uL of blood [30]. Asymptomatic malaria parasitaemia was defined as the presence of Plasmodium parasites with an axillary temperature of < 37.5°C, while clinical malaria parasitaemia was defined as the presence of any species of Plasmodium together with an axillary temperature of ≥ 37.5°C or reported fever in the previous 48 hours, or headache or joint pain. Parasitaemia was classified as low (≤ 500 parasite /μL of blood), moderate (501–5000 parasites/μL of blood) and high (>5000 parasites/μL of blood).

Stool smears were prepared and examined using the Kato-Katz thick smear method, as described by Cheesbrough [30]. Duplicate smears were prepared for each specimen. Each slide was allowed to clear for 30 min, and then examined at 100× total magnification within one hour of preparation to avoid missing hookworm eggs. Morphological identification of eggs of A. lumbricoides, T. trichiura and hookworm were based on identification aids [30]. All the eggs in the 41.7 mg of stool were counted and multiplied by 24 to compute the number of eggs per gram of faeces (epg). As a quality control measure, all positive slides and 20% of randomly selected negative smears were re-examined within 24 hours by a third experienced parasitologist who had no knowledge of the previous results. An average of the counts was utilised.

Statistical analysis

All data collected was entered into Microsoft Excel and cleaned for entry errors (MS Excel 2016). Data was further analysed with the IBM-statistical package for the social sciences (IBM-SPSS) version 19 (IBM—SPSS, Inc, Chicago, IL, USA). Data was summarized into means and standard deviations (SD), and proportions used in the evaluation of descriptive statistics. Prevalence of malaria, STH infections, anaemia and malnutrition were determined and compared using the Chi-square (χ2) test. The mean (SD) Hb levels were compared using non-parametric tests [(Mann Whitney U and Kruskal Wallis tests) and parametric tests (t-test and analysis of variance (ANOVA)] where appropriate. Malaria parasite counts were log transformed before analysis. The Pearson correlation coefficient (r) was used to evaluate the linear correlation between age, sex, MUAC, nutritional status, MP status, level of education, type of school, locality and Hb level. The attributable risk (AR%) of anaemia caused by malaria and malnutrition was calculated accordingly [31]: [(𝑛1𝑚0 − 𝑛0𝑚1)/𝑛(𝑛0+𝑚0)] ×100, where 𝑛0 = anaemic children without malaria and 𝑛1 = anaemic children with malaria, whereby 𝑛0 + 𝑛1 = 𝑛, 𝑚0 = non anaemic children without malaria, and 𝑚1 = non anaemic children with malaria, whereby 𝑚0 + 𝑚1 = 𝑚. The multiple linear regression (MLR) model (enter) with Hb as the dependent variable was run to examine the influence of the following independent variables; age, sex, MUAC, nutritional status, MP status, level of education, type of school and locality. All 401 participants were included in the model. Significant levels were measured at 95% confidence interval (CI) with significant differences set at P < 0.05.

Ethics statement

Administrative clearances were obtained from the Regional Delegation of Basic Education as well as from the Catholic Education Board. The institutional review board hosted by the Faculty of Health Sciences, University of Buea issued the ethical clearance document after reviewing the study protocol, participant’s information sheet and assent forms. Authorization to proceed with the study in these villages within the selected school was obtained from village chiefs and school head teachers. Children participated in the study if a parent or guardian signed the informed consent form. The parents or guardian and their children were informed that their participation in the study was voluntary and they could withdraw at any time without any explanation.

Results

Socio-demographic characteristics of the study participants

A total of 401 pupils with a mean (SD) age of 8.94 (2.9) years (range = 4–14) of both sexes (212 females and 189 males) were examined. Majority of the participants were pupils from Owe locality (47.6%), Catholic schools (58.1%) and between the age range of 7–10 years (58.4%). A greater proportion of the participants used bed nets (75.1%) and lived in plank houses (85%) as shown in Table 1. The proportion of children whose house floors were cemented (93.0%) was higher compared to their counterparts with earthen house floors (0.7%).

Table 1

Characteristics of the study participants.

Characteristic		Number examined	%
Sex	Male	189	47.1
	Female	212	52.9
Age group (years)	≤ 6	60	15.0
	7–10	234	58.3
	11–14	107	26.7
Locality	Owe	191	47.6
	Meanja	38	9.5
	Mpundu	172	42.9
Schools	Government school	168	41.9
	Catholic school	233	58.1
ITN use	Yes	301	75.1
	No	100	24.9
House type	Plank	341	85.0
	Block	60	15.0

Clinical profile of participants

The mean (SD) HAZ, WAZ, and WHZ scores were -1.17 (1.2), -0.34 (1.2) and 2.91 (4.0) respectively. The prevalence of fever, MP, STH infections, anaemia and malnutrition were 15% (95% CI = 11.7–18.7%), 33.9% (95% CI = 29.5–38.7%), 2.2% (95% CI = 1.8–2.7%), 75.3% (95% CI = 70.9–79.3%) and 24.4% (95% CI = 20.5–28.9%) respectively (Table 2).

Table 2

Clinical characteristics of the 401 SAC.

Characteristics	Number	%
Prevalence
Prevalence of fever (≥ 37.5°C)	60	15.0
Overall prevalence of MP	136	33.9
Overall prevalence of STH	9	2.2
Prevalence of anaemia	302	75.3
Prevalence of malnutrition	98	24.4
Means	Mean (SD)	Range
Mean age in years	8.9 (0.1)	4–14
Mean weight in kg	124.6 (0.6)	98–157
Mean height in cm	27.29 (0.3)	15–53
Mean MUAC in cm	18.6 (2.08)	13.6–33.0
Mean HAZ	-1.20 (1.2)	-5.50–2.40
Mean WAZ	- 0.35 (1.2)	-2.76–5.51
Mean WHZ	0.93 (1.6)	-2.79–8.28
Mean temperature in°C	36.85 (0.1)	33–39.5
Mean haemoglobin in g/dL	10.6 (1.4)	4.4–14.3
Mean MP density	1326.2 (3939)	40–34000

HAZ = Height-for-age z score; MP = Malaria parasite; MUAC = Mid upper arm circumference; STH = Soil-transmitted helminths; WAZ = Weight-for-age z score; WHZ = Weight-for-height z score.

All MP detected were Plasmodium falciparum. Out of the 9 children with STH infection, 5 (55.6%) were infected with Trichuris and 4 (44.4%) with Ascaris, no mixed infections, hookworm, and Strongyloides were observed. Out of the 60 (15%, 95% CI = 11.7–18.7%) pupils with fever, 22 (36.7%, 95% CI = 25.2–49.4%) of them were positive for MP. ITN usage was highest in children ≤ 6 years old (72.1%), followed by 7–10 years (64.8%) and least in 11–14 years old (53.3%).

MP, STH and malnutrition prevalence

Out of the 136 children with malaria parasitaemia 114 (83.8%, 95% CI = 76.7–89.1%) of the infections were asymptomatic. MP prevalence varied significantly (P = 0.031) among the localities only. The children of Meanja had the highest prevalence of MP (50.0%) when compared with their Owe and Mpundu equivalent (Table 3).

Table 3

Prevalence of MP, STH and malnutrition by sex, age, locality and type of school.

Parameter	Category	N	MP % (n)	STH % (n)	Stunting % (n)	Underweight % (n)	Wasting % (n)	Malnutrition % (n)
Sex	Male	189	34.9 (66)	2.6 (5)	28.6 (54)	4.8 (9)	0.0 (0)	29.6 (56)
	Female	212	33.0 (70)	1.9 (4)	19.3 (41)	4.2 (9)	0.7 (1)	19.8 (42)
	P- value		0.688	0.619	0.03	0.803	0.304	0.022
Age group in years	≤ 6	60	36.7 (22)	1.6 (1)	6.7 (4)	1.7 (1)	1.7 (1)	10.0 (6)
	7–10	234	29.5 (69)	1.3 (3)	21.4 (234)	3.4 (8)	0.0 (0)	21.8 (51)
	11–14	107	42.1 (45)	4.7 (5)	38.3 (41)	8.4 (9)	0.0 (0)	38.3 (41)
	P–value		0.067	0.139	< 0.001	0.061	0.117	< 0.001
Locality of School	Owe	191	28.8 (55)	2.1 (4)	25.1 (48)	6.8 (13)	0.7 (1)	26.7 (51)
	Meanja	38	50.0 (19)	5.1 (2)	47.4 (18)	2.6 (1)	0.0 (0)	47.4 (18)
	Mpundu	172	36.0 (62)	1.8 (3)	16.9 (29)	2.3 (4)	0.0 (0)	16.9 (29)
	P–value		0.031	0.431	<0.001	0.101	0.560	<0.001
School Type	Government	168	30.4 (51)	3.0 (5)	21.4 (36)	6.0 (10)	0 0 (0)	22.0 (37)
	Catholic	233	36.5 (85)	1.7 (4)	25.3 (59)	3.4 (8)	0.6 (1)	26.2 (61)
	P- value		0.201	0.500	0.366	0.229	0.367	0.339

P- value obtained using χ2. P -values in bold are statistically significant.

STH was prevalent in 2.2% (95% CI = 1.8–2.7%) of the children with no significant differences observed with respect to gender (P = 0.619), age (P = 0.139), locality (P = 0.431), and the type of school (P = 0.500) as shown in Table 3.

Of the 98 children with malnutrition, stunting the most common form occurred in 95 (96.9%), while underweight and wasting occurred in 18 (18.4%) and 1 (1.0%) of them, respectively. Overall, the prevalence of stunting was 23.7% (95% CI = 19.8–28.1%), underweight was 4.5% (95% CI = 2.9–7.0%) while wasting was 0.3% (95% CI = (0.06–1.8%). The prevalence of malnutrition, more specifically stunting was significantly higher in males (28.6%), children of the 11–14 years age group (38.3%) and those of Meanja locality (47.4%) than their respective counterparts as shown in Table 3.

Anaemia prevalence and its severity

Overall, anaemia occurred in 75.3% (95% CI = 90.9–79.3%) of the pupils with similar prevalence in males (74.6%) and females (75.9%). Although not statistically significant, the prevalence of anaemia was higher in the ≤ 6 years age group (81.7%), children from GS (78.6%) and those positive for MP (80.9%) than their contemporaries. However, significantly higher (P = 0.035) prevalence of anaemia was observed in children of the Meanja locality (89.5%) when compared with those of Owe (70.7%) and Mpundu (77.3%) as revealed in Table 4.

Table 4

Prevalence and severity of anaemia with respect to sex, age, locality, type of school and malaria parasite.

Characteristic		N	Anaemia prevalence % (n)	χ2 P value	Mild anaemia prevalence % (n)	Moderate anaemia prevalence % (n)	Severe anaemia prevalence % (n)	χ2 P value
Sex	Male	189	74.6 (141)		13.7 (29)	61.3 (130)	0.9 (2)
	Female	212	75.9 (161)	0.756	12.2 (23)	59.8 (113)	2.6 (5)	0.584
Age group in years	≤ 6	60	81.7 (49)		6.7 (4)	75.0 (45)	0.0 (0)
	7–10	234	74.8 (175)	0.433	11.1 (26)	61.5 (144)	2.1 (5)	0.034
	11–14	107	72.9 (78)		20.6 (22)	50.5 (54)	1.9 (2)
Locality	Owe	191	70.7 (135)		12.6 (24)	55.5 (106)	2.6 (5)
	Meanja	38	89.5 (34)	0.035	13.2 (5)	76.3 (29)	0.0 (0)	0.132
	Mpundu	172	77.3 (133)		13.4 (23)	62.8 (108)	1.2 (2)
School Type	Catholic	233	73.0 (170)	0.199	14.2 (33)	57.5 (134)	1.3 (3)	0.334
	Government	168	78.6 (131)		11.3 (19)	64.9 (109)	2.4 (4)
MP status	Positive	136	80.9 (110)	0.064	13.2 (18)	66.2 (90)	1.5 (2)	0.290
	Negative	265	72.5 (192)		12.8 (34)	57.7 (153)	1.9 (5)
MP density category	Low	85	81.6 (71)		17.2 (15)	63.2 (55)	1.1 (1)
	Moderate	40	80.0 (40)	0.948	5.0 (2)	72.5 (29)	2.5 (1)	0.669
	High	9	77.8 (9)		11.1 (1)	66.7 (6)	0.0 (0)

P—values in bold are statistically significant.

Mild anaemia = Hb between 10.0 g/dL and the level in the WHO reference values for age.

Moderate anaemia = Hb between 7.0–9.9 g/dL based on WHO reference for age.

Severe anaemia = Hb < 7 g/dL and WHO reference for age.

Low MP density = ≤ 500 parasite /μL of blood.

Moderate malaria parasite density = 501–5000 parasites/μL of blood.

High malaria parasite density = >5000 parasites/μL of blood.

Moderate anaemia (Hb = 7.0–9.9 g/dL) occurred commonly (60.6%, 95% CI = 55.7–65.3%) in the participants than mild (13.0%, 95% CI = 10.3–16.6%) and severe anaemia (1.8%, 95% CI = 0.9–3.6%). While no significant differences in anaemia severity were observed by sex, locality, type of school, MP status and density, the prevalence of moderate anaemia was highest in children of the ≤ 6 years age group (75.0%) and the difference was statistically significant at P = 0.034 (Table 4).

Infection status, anaemia prevalence and haemoglobin levels

Of the 401 participants, 48.6% (195) had no MP, STH or malnutrition. Correspondingly, 25.7% (103) had MP only, 0.7% (3) had STH only,16.2% (65) were malnourished only, 7.2% (29) had both MP and were malnourished, 0.5% (2) were infected with STH and malnourished and 1.0% (4) were co-infected with MP and STH. The prevalence of anaemia and mean (SD) Hb levels in g/dL as influenced by infection category and nutritional status is shown in Table 5. Children who were MP positive and malnourished as well, had the highest prevalence of anaemia (86.2%) followed by those infected with MP only (79.6%) and malnutrition only (79.6%). However, the AR of anaemia caused by MP + malnutrition, MP only and malnutrition only were 0.96%, 3.8%, and 1.1% respectively.

Table 5

Anaemia prevalence and mean (SD) Hb (g/dL) levels by sex and age as influenced by infection category and nutritional status.

Parameter	n	Prevalence of anaemia % (n)	Overall mean (SD) Hb (g/dL) level	Mean (SD) Hb (g/dL) level by sex		P valuea	Mean (SD) Hb (g/dL) level by age group in years			P- Valueb
				Male	Female		≤ 6	7–10	11–14
STH Only	3	66.7 (2)	10.3 (1.8)	10.0 (2.4)	11.0 (-)	-	8.3	11.4 (0.5)	-	-
MP Only	103	79.6 (82)	10.5 (1.2)	10.4 (1.2)	10.5 (1.1)	0.706	10.0 (0.9)c	10.4 (1.2)	10.9 (1.3)c	0.022
Malnutrition only	65	73.8 (48)	10.5 (1.6)	10.2 (1.7)	10.8 (1.4)	0.296	10.3 (0.7)	10.4 (1.4)	10.6 (20)	0.753
MP + Malnutrition	29	86.2 (25)	10.4 (1.2)	10.5 (1.3)	10.2 (1.1)	0.326	9.0 (0.3)	10.3 (1.1)	10.6 (1.2)	0.143
Negative	195	71.8 (140)	10.8 (1.3)	10.9 (1.3)	10.7 (1.4)	0.338	10.4 (1.3)	10.8 (1.3)	11.2 (1.4)	0.058
Total	401	75.3 (302)	10.6 (1.4)	10.6 (1.3)	10.6 (1.4)	0.889	10.2 (1.2)d	10.6 (1.3)	10.9 (1.5)d	0.004

Abbreviations: STH Soil transmitted helminth, MP Malaria parasite

MP Only = Infection with malaria parasite only.

Malnutrition only = Participants with malnutrition only (Absence of MP and STH).

MP + Malnutrition = Participants with malaria parasite and malnutrition.

Negative = Negative for MP and STH as well as absence of malnutrition.

P–valuea obtained by t- test.

P–valueb obtained by ANOVA.

Figures in bold are statistically significant.

c Mean (SD) Hb (g/dL) significantly different (Post Hoc Turkey HSD test: P = 0.017).

d Mean (SD) Hb (g/dL) significantly different (Post Hoc Turkey HSD test: P = 0.003).

Overall, the mean (SD) Hb varied significantly (P = 0.004) with age with the highest level occurring in children 11–14 years old [10.9 (1.5) g/dL] and the lowest in the ≤ 6 years old [10.2 (1.2) g/dL]. As shown in Table 5, the mean (SD) Hb (g/dL) levels in all conditions were lower than the normal Hb levels for age and sex. Statistically significant (P = 0.022) only, was the difference in mean Hb levels with age in children infected with MP where, children ≤ 6 years old had the lowest mean Hb [10.0(0.9) g/dL] when compared with the 7–10 years [10.4 (1.2) g/dL] and the 11–14 years [10.9 (1.2) g/dL] age groups. The mean Hb level was lowest in those with parasitaemia of 15000 parasites/μL when compared with the aparasitaemic and the other quartiles of malaria parasite densities as shown in Fig 2.

Fig 2

Haemoglobin level by malaria parasitaemia in the study population.

Predictors of haemoglobin levels

The tolerance statistics of the multiple linear regression (MLR) model were all below 1 and all the variance inflation factors (VIF) were less than 2. Bivariate correlations with Hb level revealed a significant positive relationship with age (r = 0.150, P = 0.001), MUAC (r = 0.263, P < 0.001) and level of education (r = 0.145, P = 0.002) while a significant negative relationship was observed with MP status (r = -0.085, P = 0.045), type of school (r = -0.099, P = 0.024) and locality (r = -0.089, P = 0.038). In the MLR model, the only factors identified as significant predictors of Hb levels were the MUAC (P < 0.001) and the MP status (P = 0.035) as shown in Table 6.

Table 6

MLR model showing influence of independent variables on haemoglobin level.

Independent variable	B	Standard error	95% CI	P- value
Age	-0.007	0.041	-0.088–0.075	0.873
Sex	0.032	0.134	-0.231–0.295	0.813
MUAC	0.157	0.042	-0.074–0.240	<0.001***
Nutritional status	-0.113	0.166	-0.440–0.214	0.498
MP status	-0.294	0.139	-0.567 - -0.021	0.035*
Level of education	0.148	0.086	-0.021–0.316	0.085
Type of School	-0.204	0.135	-0.468–0.061	0.131
Locality	-0.066	0.070	-0.206–0.072	0.347

Abbreviations: MUAC Mid upper arm circumference, MP Malaria parasite.

Dependent variable: Hb (g/dl)

* P is significant at the 0.05 level.

*** P is significant at the 0.001 level. Model summary: R = 0.311, R2 = 0. 097, Adjusted R2 = 0.078, F = 5.251, P < 0.01, N = 401.

Health status in the population

Based on the Hb level (Hb > 11g/dL) and the absence of fever, MP, STH and malnutrition, 13.7% (95% CI = 10.7–17.4%) of the SAC were considered as healthy in the study population. The proportion of healthy children was comparable among the age groups (≤ 6 years = 13.3%, 7–10 years = 15% and 11–14 years = 11.2%; P = 0.645) and sex (female = 13.7%, male = 13.8%; P = 0.982). Among the 195 negative for MP, STH, and malnutrition, 55 (28.2%, 95% CI = 22.4–34.9%) were classified as healthy. Even though more males (32.1%) and children of the 11–14 years age group (33.3%) were healthier than females (25.4%) and those ≤ 6 years (24.2%) and 7–10 years (27.8%) old the differences were not statistically significant (P = 0.308, P = 0.693), respectively.

Among the 346 (86.3%) unhealthy children, anaemia occurred most frequently (40.5%), followed by MP (29.8%) and malnutrition (18.8%) as shown in Fig 3. The occurrence of the various conditions varied significantly (χ2 = 34.88, P < 0.001) with the age group. Children in the ≤ 6 years group had the highest prevalence of anaemia (48.1%) and MP (38.5%) while those of the 11–14 years age group had the highest prevalence of malnutrition (25.3%), MP + malnutrition (15.8%), malnutrition + STH (2.1%) and MP + STH (3.2%) as revealed in Fig 3.

Fig 3

Prevalence (%) of the different conditions in unhealthy participants as influenced by age.

Discussion

Understanding the burden of malaria, malnutrition and helminth infection in SAC is essential to finding delivery mechanisms to help implement control measures in this at-risk population. This study assessed the health status of SAC with respect to malaria, malnutrition and STH infections using the level of Hb as an indicator.

The prevalence of STH infections was very low (2.2%) when compared with that (33.7%) of a similar study carried out in the same region two years earlier [32]. However, it is comparable to the 1% obtained in SAC in Tiko Health District [33] and 2.5% in selected rural, semi-urban and urban communities [22] all in the Mount Cameroon area. While a high burden of infection is not very common in regions targeted for elimination, the low prevalence could be as a result of the mass chemotherapy with mebendazole in school children initiated in Cameroon through the Ministry of Public Health since 2004 [5]. Furthermore, a combination of auto-medication, history of chemotherapy, changes in environmental and behavioural factors by individuals may have led to the sustained low prevalence of STH infection in the area.

The low occurrence of STH infection in SAC in the area limits the expression of its influence on the Hb level. However, out of the 3 children who were infected with STH only, 2 (66.7%) were anaemic. Moreover, those with STH infection had lower mean Hb levels when compared with their negative counterparts. Although this observation is limiting, it corroborates the negative effect of STH infection on the Hb values reported in abundant STH (Ascaris lumbricoides, Trichuris trichiura) infections among school children of Kashmir Valley India [34].

The overall MP prevalence (33.9%) is comparable to the 33.0% obtained in school children in Bomaka and Molyko in the Mount Cameroon Area [35] while, it is lower when compared with the 44.26% obtained by Kimbi et al. [36] in SAC children in Muea in the Mount Cameroon area and the 50.7% by Makoge et al. [37] in primary school pupils in Mbonge Sub-Division, Cameroon. Plasmodium falciparum infection prevalence as high as 60% in school children has been reported in Malawi [38] and a comparable prevalence of 30% in similar transmission settings in Uganda [39]. It is worth noting that majority (83.8%) of the MP infections were asymptomatic as most SAC do not have any symptoms because they have acquired some immunity. While acknowledging the decline in malaria prevalence due to sustained malaria control interventions implemented by the Cameroon Government through the National Malaria Control Programme [40] this burden highlights the need to embark upon malaria control in SAC in Cameroon.

Although all three localities are rural, children of Meanja had the highest significant prevalence of MP. More specifically the prevalence was highest in SAC of the 11–14 years age group when compared with the other age groups. As expected, the heterogeneity in prevalence of malaria parasite is demonstrated in the area of study. Even though the distribution of malaria cases with age is influenced by the transmission intensity [41], it is likely that other ecological factors and behaviours favoured the occurrence and age-related distribution of the infection in the area. Inhabitants of Meanja live near the CDC rubber and palm plantations which together with a lot of bushes around, creates an ecosystem appropriate for Anopheles mosquito development in the locality. In addition, older children in rural areas are more involved in outdoor activities such as farming and household activities like fetching water from far water sources early in the morning exposing them to the mosquito vector.

The high prevalence of anaemia (75.3%) in SAC in this rural area with a significant majority (60.6%) being moderate anaemia is an indication that anaemia is a major public health problem. This prevalence was higher than those obtained by other authors in the region and elsewhere [22, 37, 38, 42]. This probably reflects the poor state of health of children in the area. The aetiology of anaemia in SAC is multifactorial and the relative importance of each cause varies from place to place. Albeit it is difficult to differentiate the influence of malaria parasite on anaemia in SAC, the attributable risk of anaemia due to malaria parasite (3.8%), malnutrition only (1.1%) and MP and malnutrition (0.96%) is very low. However, it is very evident from the findings that SAC with these conditions especially those with MP and malnutrition had the highest prevalence of anaemia and lowest Hb levels when compared with their negative counterparts suggesting their contributions to the burden of anaemia in the area. The highest occurrence of anaemia in SAC of Meanja locality (89.5%) that also had the highest prevalence of malaria parasite lends more support to this assertion.

The observed prevalence of malnutrition (24.4%) in SAC in this area shows it is a public health issue. The very low mean HA (-1.17) and WA (-0.33) z scores culminating in the presence of stunting (23.7%) and underweight (4.5%) observed in the children highlights the degree of growth failure in height and weight in SAC in this rural area. The prevalence of stunting is similar to that observed in primary school children in Nairobi-Kenya [43] but higher than the 11.3% observed in SAC in North-Eastern Ethiopia [44]. Male children like their counterparts in Kenya and Ethiopia [44, 45] had a significant tendency of being stunted than females while a study in Southern Ethiopia [46] showed no major differences in prevalence of stunting in males and females. The growth and development of male children is influenced by environmental and nutritional stress more than the female [47]. Hence, it is likely that the mean energy intake for boys in this rural area did not meet the energy requirements as boys are more hyperactive in this age range than females who spend more time in food preparation and may thus have increasingly access to excess food.

In line with the other studies stunting was found to be significantly higher in the older age group [44, 46]. Stunting which is a marker of chronic malnutrition is more likely to be apparent with increase in age. Hence, its increased presence in older children which in addition are in a transition to the adolescent stage in life that has its own nutritional needs [48] which may not be adequate in this rural environment.

Findings from the study revealed the mean Hb level of SAC in these rural settings are comparatively lower than the WHO level for age and sex even though an ideal Hb level is not yet established. While the mean Hb level in all conditions were lower, that of children in the 6 years age group and below with malaria parasite infection was statistically significant. Furthermore, the model identified MP status as a significant predictor of Hb level with a negative relationship. Several other studies have associated Plasmodium infection to lower Hb level [42, 49, 50]. This highlights the insidious effect asymptomatic malaria parasitaemia may have on the Hb level of younger children who have not developed anti disease immunity.

On the other hand, the MUAC was identified as a significant predictor of Hb level with a positive association as shown in the MLR model. While further investigation is necessary to assert this association, the MUAC which measures only acute malnutrition could be an alternative of great value especially in resource poor settings were Hb measurement is unaffordable. Blood Hb measurement, a common indicator for diagnosing anaemia, requires trained personnel, expensive equipment or well-developed laboratory facilities which is often unavailable in rural areas. Measuring the MUAC is much cheaper and easier than measuring weight and height. In addition, it is less affected by acute dehydration than weight-based indices [51].

Furthermore, keeping an update information on the level of education of parent or caregiver is of utmost value as findings in a bivariate analysis demonstrated a significant positive association of parent/caregiver level of education with the Hb level. While acknowledging the limitations of self-reported levels of education and its potential of introducing bias, this finding is not surprising as low educational level can lead to low income and socio-economic status and thus inability to provide for proper feeding and affordable health care. Basic causes of anaemia reported in other studies include, maternal level of education and household wealth rank [52].

The magnitude of unhealthy SAC (86.3%) in the rural setting surpasses the occurrence of healthy children and that is a cause for concern. Although the presence of these conditions contributed to the unhealthy status of SAC, the fact that anaemia occurred in 71.8% of the negatives demonstrates their health is compromised by other ailments that were not investigated in the study. One of the limitations of this study is that the number of infections investigated are fewer albeit those evaluated are reported to be of common occurrence in the Mount Cameroon area [8, 37]. More research involving a wider range of morbidities (micro-nutrient deficiency, bacterial and viral infections) other than the conditions investigated that may be responsible for the unhealthy status of the children needs to be carried out. However, the lowered haemoglobin level observed in measurable and unmeasurable ailments lends support to the fact that haemoglobin level could be used as an indicator of the health status of children.

Conclusions

It is evident from this study that the health of majority of SAC in these rural settings in the Mount Cameroon area is compromised by malaria, helminthiasis, malnutrition and other conditions not investigated. Anaemia is of major public health concern and the Hb level could serve as a prognostic marker of the health status of SAC. Children with MP and malnutrition had the highest prevalence of anaemia and lowest Hb levels indicating their contributions to the burden of anaemia even though the attributable risk to it was insignificant. Malaria parasite is a significant negative predictor of Hb level hence, there is a need for intervention programmes targeting SAC in rural areas that integrates proper malaria control measures with improvement of educational level of parent/caregiver especially on proper nutrition and health care practices that will ensure health and well-being of the children.

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27 Nov 2019

PONE-D-19-31286

Haemoglobin level as an indicator of health status of school-aged children in Muyuka, Southwest Cameroon: Influence of malaria parasites, soil-transmitted helminths and malnutrition

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Reviewer #1: I read the manuscript with great interest. The manuscript was very interesting and provided good information on the Hemoglobin level as an indicator of health status of school-aged children. However, it has some points that need to be addressed to improve its quality and impact. Here are some of my observations:

The map for the target area should be included in the manuscript.

The references were mostly from more than 5 years ago. If possible, add more recent references.

Reviewer #2: Dear Editor

I have thoroughly read the manuscript, although it represents an important public health problem in School Aged Children it has a number of issues in literature review, statistical analysis and discussion. The jauthors should revise the manuscript by addressing a number of typographical, data presentation and discussion before it is considered for publication. I have highlighted a number of issues in the attached revised doc

Reviewer #3: Review for Manuscript Entitled “Haemoglobin level as an indicator of health status of school-aged children in Muyuka, Southwest Cameroon: Influence of malaria parasites, soil-transmitted helminths and malnutrition”

Abstract

Background

• Line 60- You mention an abbreviation SAC without prior statement of its long form. Much as it is appearing for the first time in the background section it has to be in its long form, and then can subsequently be mentioned as a acronym.

• Line 98- Put a percent symbol after 64.3

• Line 99-Put a percent symbol after 19.8

• Line 100-Indicate the specific threshold at which anaemia is considered a public health problem

Methodology

Study site

The description of the study site need to have a reference, no reference is provided for the description of the study site, kindly provide the reference.

Study design

• After stating the study design, for the purpose of making the paper easy to follow by the reader, this section should have the following subsections:

• Study population

• Sample size

• Sampling procedure

• In this paper, the primary objective was to determine the prevalence of Malaria parasite, STH and Malnutrition. But in calculating the sample size you used 14.0% which was the prevalence of STH, but you have not indicated that this is what is going to give enough sample size to estimate the population prevalence of Malaria parasites and Malnutrition as well.

• Line 146-148: You state that “The minimum sample size was calculated using the prevalence of P. falciparum malaria and helminth infections of previous studies: 35.5 and 14.0%, respectively, in the Mount Cameroon area”. But the formular for sample size calculation that you have provided allows for using only one proportion, how did you use these two proportions?

• But after you have calculated the sample size, you have not indicated in this section what was the calculated minimum sample size.

• Line 152-154: “Samples collected comprised of finger prick blood and stool for MP detection and speciation, Hb 153 measurement for assessment of anaemia and detection of egg or larva of STHs respectively” , rephrase this statement to make it clear as to which sample was used for whic purpose.

Clinical evaluation

• Line 172: You have written o.1cm instead of 0.1, kindly change accordingly.

Collection of blood and stool samples

• Line 192: Samples were fixed in 10% formalin and then examined using the Kato-katz technique, can the author explain, how did they perform kato-katz on a formalin fixed stool, Kato-katz performs well when done on fresh stool. Can you explain this?

• Line 194: You have written Kato-katz concentration technique. Kato-katz is not one of the concentration techniques applied on stool samples.

Statistical analysis

Line 228: SPSS is no longer Statistical Package for Social Sciences, please supply the correct long form.

Results

• In this subsection you were supposed to provide only socio-demographic and economic profile of the study participants. But you have also provided findings from laboratory analysis and other measurements, these should fall under a separate subheading and a separate table.

• Line 260-261: Out of 9 children with STH, 55.5% were infected with Trichuris trichiura, please indicate both the number and percentages.

Anaemia prevalence and its severity

• When writing confidence interval, you must indicate that it is 95% CI, do not just write “CI” as it appears in line 293, 301, 302 etc.

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Reviewer #1: Yes: Associate Professor Dr. Talal Alharazi

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31 Dec 2019

Response to Comments and Concerns of the Editor

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The corrections in the naming of the files and author affiliations have been effected.

2. Please address the following:

- Please refer to any post-hoc corrections to correct for multiple comparisons during your statistical analyses. If these were not performed please justify the reasons. Please refer to our statistical reporting guidelines for assistance (https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting).

The Post Hoc Turkey HSD test has been included where significant differences in means were observed as indicated. The correction is highlighted in Turquoise.

- Please ensure you have thoroughly discussed any potential limitations of this study within the Discussion section, for example the potential bias introduced by using self-reported data.

The factors discussed where measurable parameters that limited the potential of introducing a bias. However, limitations in the study have been reported in lines 501-503 and the addition of potential limitations highlighted as indicated.

- 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. In addition, please include any details of the pre-testing of this questionnaire.

The simple pre-tested questionnaire used has been included as supporting information 1.

Thank you for your attention to our queries.

3. Your ethics statement must appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please also ensure that your ethics statement is included in your manuscript, as the ethics section of your online submission will not be published alongside your manuscript.

The ethics statement has been moved to the methods section as requested

Reviewer comments and responses

The changes made in the manuscript based on reviewers’ comments and concerns have been highlighted in yellow and green in the manuscript with tract changes for easy appraisal.

Reviewer #1: I read the manuscript with great interest. The manuscript was very interesting and provided good information on the Hemoglobin level as an indicator of health status of school-aged children. However, it has some points that need to be addressed to improve its quality and impact. Here are some of my observations:

The map for the target area should be included in the manuscript.

The map of the study area has been included in the manuscript as requested see (Fig 1).

The references were mostly from more than 5 years ago. If possible, add more recent references.

While a single reference or two has been added, the authors believe they did an appropriate citation of relevant and recent articles in the field. At least 20 of the articles cited were published between 2014 and 2019. The articles beyond that cited are very relevant to the background methods and discussion of the results.

Reviewer #2: Dear Editor

I have thoroughly read the manuscript, although it represents an important public health problem in School Aged Children it has a number of issues in literature review, statistical analysis and discussion. The jauthors should revise the manuscript by addressing a number of typographical, data presentation and discussion before it is considered for publication. I have highlighted a number of issues in the attached revised doc

The corrections effected as requested by the reviewer are highlighted in yellow.

• Line 60: The correction has been effected.

• Line 62: The correction has been effected.

• Line 92: Literature has been reviewed and a sentence added thus “Manifestation of malnutrition is often observed in terms of anaemia, micronutrient deficiencies (iron, folic acid, vitamin B12) and anthropometric measurements.”

• Line 96: The aetiology of anaemia has been included as requested.

• Line 98: The sentence has been rephrased for clarity.

• Line 99: The prevalence of anaemia stated in line 103, specifically for SAC is the most recent. The updates given by WHO in 2011 does not include this specific age group. The recent updates are for children 6 months -5 years, pregnant women and women between the ages of 14 and 49 of various categories.

• The sentences in line 99-104 have been revised for clarity as requested.

• Line 140: The number of schools from which selection was made had been included and the method of selection as follows “Out of a list of 9 schools operating officially. Random selection by balloting was used to obtain a representative sample”.

• Line 151: The authors believe using the prevalence of malaria parasite and STH to estimate the population size to be sampled is appropriate. The main aim of this paper is to assess the influence of these infections on the haemoglobin level of SAC. Anaemia is one of the outcomes of these infections. Infections may not necessarily lead to anaemia in every circumstance.

• Line 153: Note taken. Correction effected.

• Line 153: The laboratory method has been included.

• Line 162: The authors believe the ensuing statement clarifies the previous sentence. However, the questionnaire has been attached as a supporting information for clarity (S1 Questionnaire).

• Line 172: Correction effected.

• Line 185: Haemoglobin measurement and anaemia classification has been combined as requested.

• Line 223: The time (When?)“within 24 hours” has been included.

• Line 230: Percentages has been replaced with proportions as requested.

• Line 249: The correction has been effected as indicated.

• Table 1: It is actually ITN Use and not possession. The correction has been effected based on the question in the questionnaire and the responses of the participants.

• Lines 271-273: The sentence has been rephrased for clarity as requested.

• Table 2: The phrase “as influenced by” has been deleted as requested.

• Lines 294-296 has been rephrased for clarity as requested.

• Line 300: The Hb range of moderate anaemia has been included as requested.

• Line 303. The “with” has been replaced with “by” as requested.

• Table 3: The definitions of the various categories of anaemia has been provided again beneath the table as end note even though it has been clearly defined in the methods.

• Line 314: The title has been rephrased as requested.

• Line 315: The correction has been effected.

• Line 380: Correction has been effected.

• Lines 392-394: The sentence has been rephrased for clarity. Secondly the sentence does not need any reference because the possible explanation given for the low prevalence of STH observed in the area is based on the responses obtained from the questionnaire.

• Line 398: The sentence has been rephrased for clarity.

• Conclusion: The statement has been revised.

Reviewer #3: Review for Manuscript Entitled “Haemoglobin level as an indicator of health status of school-aged children in Muyuka, Southwest Cameroon: Influence of malaria parasites, soil-transmitted helminths and malnutrition”

Corrections of Reviewer 3 are highlighted in green

Abstract

Background

• Line 60- You mention an abbreviation SAC without prior statement of its long form. Much as it is appearing for the first time in the background section it has to be in its long form, and then can subsequently be mentioned as a acronym.

The abbreviation has been written in full as indicated

• Line 98- Put a percent symbol after 64.3

The symbol has been added.

• Line 99-Put a percent symbol after 19.8

The symbol has been included.

• Line 100-Indicate the specific threshold at which anaemia is considered a public health problem

Anaemia is considered a public health problem when the prevalence is ≥ 5%. That has been included.

Methodology

Study site

The description of the study site need to have a reference, no reference is provided for the description of the study site, kindly provide the reference.

The reference has been included.

Study design

• After stating the study design, for the purpose of making the paper easy to follow by the reader, this section should have the following subsections:

• Study population

• Sample size

• Sampling procedure

The study design has been organized in the different subsections as requested

• In this paper, the primary objective was to determine the prevalence of Malaria parasite, STH and Malnutrition. But in calculating the sample size you used 14.0% which was the prevalence of STH, but you have not indicated that this is what is going to give enough sample size to estimate the population prevalence of Malaria parasites and Malnutrition as well.

The prevalence of both MP and helminth were used in determining the minimum sample size. The correction has been effected as indicated in the highlight.

• Line 146-148: You state that “The minimum sample size was calculated using the prevalence of P. falciparum malaria and helminth infections of previous studies: 35.5 and 14.0%, respectively, in the Mount Cameroon area”. But the formular for sample size calculation that you have provided allows for using only one proportion, how did you use these two proportions?

Yes the formula indicates just one prevalence. Hence, the sample size was calculated twice using both prevalence and an average was obtained. That has been indicated in the corrected version of the manuscript.

• But after you have calculated the sample size, you have not indicated in this section what was the calculated minimum sample size.

A minimum sample size of 268 samples obtained has been included.

• Line 152-154: “Samples collected comprised of finger prick blood and stool for MP detection and speciation, Hb 153 measurement for assessment of anaemia and detection of egg or larva of STHs respectively” , rephrase this statement to make it clear as to which sample was used for whic purpose.

The sentence has been rephrased for clarity as requested.

Clinical evaluation

• Line 172: You have written o.1cm instead of 0.1, kindly change accordingly.

The correction has been effected.

Collection of blood and stool samples

• Line 192: Samples were fixed in 10% formalin and then examined using the Kato-katz technique, can the author explain, how did they perform kato-katz on a formalin fixed stool, Kato-katz performs well when done on fresh stool. Can you explain this?

The statement has been rephrased for clarity. The stool samples were not fixed but in order to maintain the morphology of the egg during the period of transportation before examination in the laboratory, the samples were preserved in 10% formalin. This has been reported to preserve the morphology of Hookworm for up to 15 days and Trichuris for up to 30 days. We actually went ahead to compare our observations with that of formol ether concentration techniques even though that was not reported here because that is not the subject of this paper.

• Line 194: You have written Kato-katz concentration technique. Kato-katz is not one of the concentration techniques applied on stool samples.

The word concentration has been deleted from the sentence. The authors are sorry for the mix up. It is due to the fact that we carried out both a concentration method (formol ether) which we didn’t report here and the Kato-katz technique.

Statistical analysis

Line 228: SPSS is no longer Statistical Package for Social Sciences, please supply the correct long form.

The name has been corrected to IBM-SPSS.

Results

• In this subsection you were supposed to provide only socio-demographic and economic profile of the study participants. But you have also provided findings from laboratory analysis and other measurements, these should fall under a separate subheading and a separate table.

The laboratory analysis and other measurements have been separated from socio-demographic characteristic as requested.

• Line 260-261: Out of 9 children with STH, 55.5% were infected with Trichuris trichiura, please indicate both the number and percentages.

The correction has been effected.

Anaemia prevalence and its severity

• When writing confidence interval, you must indicate that it is 95% CI, do not just write “CI” as it appears in line 293, 301, 302 etc.

The corrections have been effected as indicated.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

29 Jan 2020

PONE-D-19-31286R1

Haemoglobin level as an indicator of health status of school-aged children in Muyuka, Southwest Cameroon: Influence of malaria parasites, soil-transmitted helminths and malnutrition

PLOS ONE

Dear Dr. Sumbele,

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Hesham

Hesham M. Al-Mekhlafi, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Academic Editor’s Comments:

Dear authors,

Thank you for submitting your revised manuscript to PLoS One. This version has addressed the reviewers’ comments; however, corrections remain and additional comments should be addressed before this manuscript can be accepted. Please consider the following:

1- In laboratory procedures section, it is mentioned that malaria parasite density per μL of blood was determined; however, information on this variable was not provided in the manuscript. Using the term “malaria parasitaemia” in the current manuscript without providing these results is unclear and can be misleading. It can be replaced with malaria infection. Moreover, it is interesting to examine the association between the level of parasitaemia (parasite density) and Hb level and anaemia prevalence as well, and this should be incorporated into the results & discussion of this manuscript.

2- The attributable risk (AR%) was mentioned in statistical analysis section (please remove details on formula used) and in discussion and conclusion; however, results were not provided in results section.!

3- I suggest change the title. Presentation of your results and your conclusion are not compatible with the title “Haemoglobin level as an indicator of health status of ….”. Basically, the title can be “Influence of malaria, soil-transmitted helminth infections and malnutrition on haemoglobin level and anaemia prevalence among school-aged children in Muyuka, Southwest Cameroon”. You may think about alternative titles.

4- Tables format should be improved and edited for consistency and English. For instance:

4.1. Table 5: change columns of total to “overall mean (SD) Hb level” and this column can be placed after prevalence o anaemia column. P-value for sex can be placed after the columns of mean Hb for sex and p-value for age groups will be accordingly after the columns of age. Indicate the use of t-test and ANOVA in the footnotes.

4.2. Table 3: the data are % (n) but it is indicated as (n) only.

4.3. Table 6: please report important necessary results only for the output of MLR analysis; collinearity statistics and correlation coefficient columns can be removed. Please read about reporting such results in scientific papers or refer to previous literature. For instance, provide B value, standard error, 95% CI and p value for the included variables.

5- Figure 2 can be removed. Renumber the figures.

6- What is URIT-12 haemoglobin meter? Company, City, Country, and cite reference for its validity.

7- Follow first appearance rule; for instance, spell out the MUAC in abstract and then add the abbreviation between brackets. Follow this in the entire text and tables. In tables, define the abbreviations in the footnotes.

8- Moreover, the manuscript needs extensive editing by a native English ‎speaker. Please ‎note that poor English may ‎ultimately be a reason to reject the ‎manuscript.‎ ‎

[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: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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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

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: Yes

**********

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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: No

Reviewer #3: Yes

**********

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Reviewer #2: The authors have adequately addressed all my comments. However, proof reading of the manuscript is needed before publication

Reviewer #3: (No Response)

**********

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Reviewer #2: No

Reviewer #3: No

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12 Feb 2020

Response to Comments and Concerns of the Editor

Academic Editor’s Comments:

Dear authors,

Thank you for submitting your revised manuscript to PLoS One. This version has addressed the reviewers’ comments; however, corrections remain and additional comments should be addressed before this manuscript can be accepted. Please consider the following:

1- In laboratory procedures section, it is mentioned that malaria parasite density per μL of blood was determined; however, information on this variable was not provided in the manuscript. Using the term “malaria parasitaemia” in the current manuscript without providing these results is unclear and can be misleading. It can be replaced with malaria infection. Moreover, it is interesting to examine the association between the level of parasitaemia (parasite density) and Hb level and anaemia prevalence as well, and this should be incorporated into the results & discussion of this manuscript.

The authors apologize for not detailing the report of the malaria parasite (MP) density in the manuscript. The relationship between MP density and anaemia was reported in Table 4 earlier but the categories were not clearly defined as shown in the highlight. We have included the definition of low, moderate and high parasite densities in the method and as a foot note in the Table for clarity. As concerns malaria parasitaemia, the mean MP density and range has been included in Table 2 and the relationship between MP and Hb level reported in Fig 2. No statistically significant association was observed between MP density and anemia likewise between Hb level and MP even though a seemingly decreasing trend was revealed in Figure 2. This was taken into consideration during the discussion earlier therefore, the discussion was based on the prevalence of MP and AR in association with the other conditions rather than density alone. Hence, the authors don’t see the need for more discussion on malaria parasite density and Hb level in this instance. Furthermore, the relationship between Hb level and malaria parasitaemia was discussed further in Lines 508-511.

2- The attributable risk (AR%) was mentioned in statistical analysis section (please remove details on formula used) and in discussion and conclusion; however, results were not provided in results section.!

The authors wish to disagree on this point. The results on attributable risk were presented in lines 349 to 350 in the results as indicated in the highlight that is why they were discussed. In the context of the study, this calculation is necessary to show the relative contribution of each factor to the occurrence of anaemia.

3- I suggest change the title. Presentation of your results and your conclusion are not compatible with the title “Haemoglobin level as an indicator of health status of ….”. Basically, the title can be “Influence of malaria, soil-transmitted helminth infections and malnutrition on haemoglobin level and anaemia prevalence among school-aged children in Muyuka, Southwest Cameroon”. You may think about alternative titles.

The title has been revised to “Influence of malaria, soil-transmitted helminth infections and malnutrition on haemoglobin level among school-aged children in Muyuka, Southwest Cameroon: a cross sectional study on outcomes. We the authors believe this is more embracing. Placing haemoglobin level and anemia prevalence on the title is kind of odd. Anaemia is an outcome of these infections. It as well embraces the health status.

4- Tables format should be improved and edited for consistency and English. For instance:

The tables have been edited and formatted as shown in the tracked changes.

4.1. Table 5: change columns of total to “overall mean (SD) Hb level” and this column can be placed after prevalence o anaemia column. P-value for sex can be placed after the columns of mean Hb for sex and p-value for age groups will be accordingly after the columns of age. Indicate the use of t-test and ANOVA in the footnotes.

The corrections have been effected as requested. See tract changes for the corrections.

4.2. Table 3: the data are % (n) but it is indicated as (n) only.

Since the table is reporting prevalence, we the authors thought it wasn’t necessary to indicate percentage again as the unit of measurement. However, we have included it as requested. See tracked changes.

4.3. Table 6: please report important necessary results only for the output of MLR analysis; collinearity statistics and correlation coefficient columns can be removed. Please read about reporting such results in scientific papers or refer to previous literature. For instance, provide B value, standard error, 95% CI and p value for the included variables.

The Table was a composite table of two different out puts. However, the table has been revised as requested to include the B value, standard error and 95% CI for variables calculated.

5- Figure 2 can be removed. Renumber the figures.

Figure 2 has been deleted and the results written out for clarity. A new Fig 2. that shows the Hb level and malaria parasite counts in the population has been produced in response to editors request of evidence of parasitaemia in the manuscript.

6- What is URIT-12 haemoglobin meter? Company, City, Country, and cite reference for its validity.

The specification has been included “URIT Medical Electronic co., Ltd, London, United Kingdom)”.

7- Follow first appearance rule; for instance, spell out the MUAC in abstract and then add the abbreviation between brackets. Follow this in the entire text and tables. In tables, define the abbreviations in the footnotes.

The corrections have been effected as indicated in the tracked changes.

8- Moreover, the manuscript needs extensive editing by a native English ‎speaker. Please ‎note that poor English may ‎ultimately be a reason to reject the ‎manuscript.

The manuscript has been extensively edited by a native English speaker as requested. See the tracked changes for the copy editing.‎ ‎

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

Reviewers' comments

The comments in the file attached by reviewer 2 had been adequately addressed in the first round of review process as indicated by the reviewer.

Submitted filename: Response to Reviewers 2.docx

Click here for additional data file.

11 Mar 2020

Influence of malaria, soil-transmitted helminths and malnutrition on haemoglobin level among school-aged children in Muyuka, Southwest Cameroon: a cross-sectional study on outcomes

PONE-D-19-31286R2

Dear Dr. Sumbele,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Hesham

Hesham M. Al-Mekhlafi, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please note that weight and height values (mean and range) in Table 2 are in opposite order. To correct this, variable names should be exchanged. This can be corrected prior to the production stage.

Reviewers' comments:

16 Mar 2020

PONE-D-19-31286R2

Influence of malaria, soil-transmitted helminths and malnutrition on haemoglobin level among school-aged children in Muyuka, Southwest Cameroon: a cross-sectional study on outcomes

Dear Dr. Sumbele:

I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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.

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on behalf of

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Academic Editor

PLOS ONE