Consequences of polyparasitism on anaemia among primary school children in Zimbabwe

Acta Tropica 115 (2010) 103–111

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Consequences of polyparasitism on anaemia among primary school children in Zimbabwe N. Midzi a,b,1 , S. Mtapuri-Zinyowera b , M.P. Mapingure c , D. Sangweme d , M.T. Chirehwa e , K.C. Brouwer f , J. Mudzori g , G. Hlerema a , F. Mutapi h , N. Kumar d , T. Mduluza c,∗ a

National Institute of Health Research, Box CY 573, Causeway, Harare, Zimbabwe College of Health Sciences, Department of Medical Microbiology, P.O. Box A178, Avondale, Harare, Zimbabwe University of Zimbabwe, Department of Biochemistry, P.O. Box MP167, Mount Pleasant, Harare, Zimbabwe d Johns Hopkins Bloomberg School of Public Health, Department of Molecular Microbiology and Immunology, Baltimore, MD, USA e Biomedical Research and Training Institute, P.O. Box CY 1753, Causeway, Harare, Zimbabwe f University of California, San Diego, Division of Global Public Health, Department of Medicine, San Diego, CA, USA g National Microbiology Reference Laboratory, P.O. Box ST749, Southerton, Zimbabwe h University of Edinburgh, Institute for Immunology and Infection Research, Edinburgh, UK b c

a r t i c l e

i n f o

Article history: Available online 20 February 2010 Keywords: Polyparasitism P. falciparum Schistosomiasis Soil transmitted helminths Anaemia Iron deficiency anaemia

a b s t r a c t The effect of concomitant infection with schistosomes, Plasmodium falciparum and soil transmitted helminths (STHs) on anaemia was determined in 609 Zimbabwean primary school children. P. falciparum, haemoglobin levels and serum ferritin were determined from venous blood. Kato Katz, formal ether concentration and urine filtration techniques were used to assess prevalence of Schistosoma mansoni, STHs and Schistosoma haematobium infections. The prevalence of S. haematobium, S. mansoni, P. falciparum, hookworm, Trichuris trichiura and Ascaris lumbricoides were 52.3%, 22.7%, 27.9%, 23.7%, 2.3% and 2.1%, respectively. The overall prevalence of anaemia and iron deficiency anaemia (IDA) were 48.4% (277/572) and 38.1% (181/475). Haemoglobin levels among children who had P. falciparum, S. haematobium and hookworm were lower than negative individuals, p < 0.001, p < 0.001 and p = 0.030, respectively. The prevalence of anaemia and IDA in co-infections was almost double that in single infection. Children with P. falciparum/STHs/schistosome and schistosomes/P. falciparum co-infections recorded higher prevalence of anaemia and IDA (80.8% and 57.4%, respectively) than other combinations, p < 0.001. Logistic regression revealed that, age group ≥ 14years, P. falciparum, S. haematobium light and heavy infections, and S. mansoni moderate and heavy infection, hookworm light infection were predictors of anaemia. This study suggests that integrated school based de-worming and malaria control have the potential to reduce the burden of anaemia. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Concomitant infection with Plasmodium falciparum, STH and schistosomes is common in tropical and sub-tropical countries due to geographic overlap of climatic and socio-economic conditions that support survival of STHs, reproduction of malaria parasites’ vectors and schistosomiasis intermediate host snails (Tshikuka et al., 1996; Brooker et al., 2007; Midzi et al., 2008). In Africa alone, about a quarter of school children may be at risk of co-infection with P. falciparum and helminths (Brooker et al., 2006, 2007). They are also at risk of anaemia that reduces their cognitive potential,

∗ Corresponding author. Tel.: +263 4 334052; fax: +263 4 338046. E-mail addresses: [email protected] (N. Midzi), [email protected], [email protected] (T. Mduluza). 1 Tel.: +263 4 700457/795307; fax: +263 4 253579. 0001-706X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2010.02.010

retard growth and predisposes them to other diseases (WHO, 2001). Although the etiologic factors of anaemia are complex including dietary insufficiency of iron, haemoglobinopathies, other micronutrient deficiencies (vitamin A, B12 , folate and riboflavin), several studies have demonstrated the contribution of parasitic infections to anaemia. Results from meta-analysis and community based studies on malaria provide compelling evidence that malaria contributes substantially to anaemia (Geerligs et al., 2003; Korenromp et al., 2004; Ronald et al., 2006; Clarke et al., 2008). Hookworm has been shown to be an important risk factor for anaemia (Stoltzfus et al., 1997; Brooker et al., 1999). Olsen et al observed that hookworm egg intensity as low as 300 eggs/g stool was negatively related to levels of haemoglobin and serum ferritin (Olsen et al., 1998). The mechanisms by which schistosomiasis cause anaemia have been described by Freidman et al. (2005). Whilst some extensive field studies have failed to demonstrate any significant effect of schistosomiasis infection on haemoglobin (Hb) levels (Befidi-Mengue

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et al., 1993), several other studies have highlighted a significant effect of schistosomiasis on Hb levels and anaemia (Stephenson et al., 1985; Prual et al., 1992; Koukounari et al., 2007). Although schistosomes, STHs and P. falciparum are separately known etiological factors of anaemia with distinct mechanisms which reduce haemoglobin levels in human hosts (Menendez et al., 2000; Hotez et al., 2004; Freidman et al., 2005), data are still lacking on the effect of their coincidental infection on the level and severity of anaemia (Hotez et al., 2006; Midzi et al., 2008). Several studies have only explored the effect of single parasitic infections (Flemming et al., 2006; Koukounari et al., 2006), mixed infections with schistosomiasis and STH (Guyatt et al., 2001), STHs and P. falciparum (Koukounari et al., 2006) or multiple STHs infection on anaemia (Ezeamama et al., 2005). Studies on the impact of schistosomes, STHs and P. falciparum co-infection on anaemia, particularly in primary school children are relevant for the design and implementation of integrated control programmes aimed at reducing anaemia and other associated morbidities. Zimbabwe is endemic for schistosomiasis, soil transmitted helminthiasis and malaria that pose a public health problem (Taylor and Makura, 1985; Chandiwana et al., 1989; Mutapi et al., 2000). This study was therefore conducted to determine the effect of malaria, schistosomiasis and STHs co-infection on the level of anaemia and also to identify predictors of anaemia among primary school children living in an area where P. falciparum, schistosome and STHs are co-endemic (Brooker et al., 2007). 2. Methodology 2.1. Study area and population The study was conducted in July 2004 among 609 primary school children (age range 5–15 years) living in Burma Valley commercial farming areas in Mutare district that is situated to the eastern part of Zimbabwe. The geographic position, climatic conditions and demographic information of the people living in the study area have been described elsewhere. The study participants were drawn from children who attended three primary schools (Valhalla, Msapa and Kaswa). The community from which the study population was drawn has a homogeneous socio-economic status and diet composed of mainly maize meal served with green vegetables and at times dried fish as the staple food. Bananas are the common fruit eaten all year round in the area. Participants came mainly from families of farm labourers. 2.2. Study design The study used cross sectional, baseline data from a longitudinal intervention study investigating the distribution of polyparasitism with schistosomiasis, STHs and P. falciparum among primary school children in rural and farming areas in Zimbabwe: impact on anaemia and the effectiveness of regular school based deworming and prompt malaria treatment. 2.2.1. Sampling scheme Manicaland Province was conveniently chosen for the study due to its geographical location (Eastern Highlands) characterised by high annual rainfalls, wet soils and malaria endemicity (Ministry of Health Report, 2002). Multistage sampling technique was used to select the district, ward and schools in which the study was conducted. Using rotary method, Mutare District was randomly selected from 7 districts in the Province, ward 7 was selected from 36 wards in Mutare district and three primary schools among 5 primary schools in ward 7 were also selected using the same method for the study. Every child at each school was eligible except for grade 7 children. Demographic data that include age, gender and

the village where participants lived was recorded onto a questionnaire. Ages of participants were obtained from the class registers provided by teachers and from participants in cases were no information was found in registers. 2.2.2. Sample size determination The sample size was calculated using the previous prevalence of hookworm at 61.7%, Schistosoma haematobium at 58.7% and malaria at 23.5% observed in the same area by Chandiwana et al. (1989) and Mutapi et al., 2000). The following formula was used: n = (z/delta)2 P(1 − P), where n = the sample size required, z-statistic = 1.96, delta (margin of error) = 0.05 and P = proportion or prevalence of the disease (61.7%, 58.7%, 23.5% respectively). The optimum sample size was estimated as n = 373. This was adjusted by 30% considering possible loss due to follow up over a period of 33 months to n = 485. 2.2.3. Inclusion and exclusion criteria All children attending each primary school, except for children attending grade seven, were eligible. Grade seven children were not included in the study because they could not be followed up over 33 months as they would leave primary school the following year. However they received treatment for helminths and malaria. Children severely affected with other diseases and those not willing to give samples were not included in the study. 2.3. Parasitological techniques Urine and faecal samples were collected between 10:00 a.m. and 2:00 p.m. in separate wide mouth plastic specimen bottles correspondingly labelled with the laboratory identification numbers assigned to each individual. The samples were processed within 2 h of collection. Diagnosis of S. haematobium and intestinal helminths (Schistosoma mansoni, hookworms, Trichuris trichiura and Ascaris lumbricoides) was based on the detection of worm eggs in urine and faeces, respectively. 2.3.1. Detection of urinary schistosomiasis Infection with S. haematobium was diagnosed using the urine filtration technique as described by Mott et al. (1982). In brief, 10 ml of urine was filtered through the Nytrile filter membrane. The filter was stained with Lugol’s iodine and examined with an ×10 light microscope objective. S. haematobium egg intensity was expressed as the number of eggs detected per 10 ml of urine. The same procedure was repeated on three consecutive days. 2.3.2. Detection of S. mansoni and STHs The overall intestinal helminths infection status of participants was decided based on the combination of results from the formal ether concentration (Cheesbrough, 1998) and Kato Katz techniques in order to improve sensitivity of diagnosis (Goodman et al., 2007). Firstly, about 1 g portion of each stool specimen collected on the first day was preserved in a tube containing 10% formalin. The preserved specimens were processed using the formal ether concentration technique without modification. Secondly, a stool specimen was collected per study participant on 2 different successive days and a single faecal thick smear was prepared from each specimen using a 41.7 mg template (Katz et al., 1972). Faecal smears prepared were examined within 30–60 min using a light microscope in order to detect and quantify hookworm and other STHs eggs. The smears were re-examined after 24 h to detect S. mansoni eggs. Number of eggs detected from each Kato Katz thick smear was multiplied by 24 in order to express infection intensities as number of eggs per gram stool (WHO, 2002). S. haematobium infection intensities were stratified according to the World Health Organisation guidelines as light infection (1–49 eggs/10 ml urine) and heavy infection (≥50 eggs/10 ml of urine). S. mansoni, hookworm,

N. Midzi et al. / Acta Tropica 115 (2010) 103–111

A. lumbricoides and T. trichiura egg intensities were stratified into light, moderate and heavy infection according to WHO guidelines (WHO, 2002). 2.4. Blood collection and processing Approximately 5 ml of venous blood was drawn from each willing participant in blood collection tubes containing ethylene diamine tetra acetic acid (EDTA) as anticoagulant. This was used to prepare thick blood smears for malaria diagnosis and haemoglobin determination. The remaining blood was stored overnight at 4 ◦ C, centrifuged at 3000 rpm for 1 min, plasma separated, aliquoted into 2 ml screw cap storage vials and stored at −70 ◦ C. Another 5 ml of venous blood was drawn in 5 ml plain blood collection tubes, stored at 4 ◦ C overnight after which the blood tubes were centrifuged at 3000 rpm for 1 min, serum separated and stored at −70 ◦ C for later determination of serum ferritin. 2.4.1. Detection of P. falciparum Thick malaria blood smears were made from anticoagulated venous blood, air dried, Giemsa stained and observed under the microscope for identification and quantification of malaria parasites. Malaria parasites were counted against 200 white blood cells (Cheesbrough, 1998). 2.4.2. Determination of haemoglobin concentrations Haemoglobin (Hb) concentration was measured using the HemoCue photometer (HemoCue AB, Angelhome, Sweden). In brief, anticoagulated venous blood from each participant was mixed gently. A drop of blood was placed onto a plastic film using a pipette. The HemoCue microcuvette was filled with a drop of blood, placed in the HemoCue cuvette holder and the displayed haemoglobin value of blood was taken within 60 s. The Hb values were used to determine if participants were anaemic or nonanaemic. Anaemia was defined according to WHO guidelines on age and gender cut off limits (WHO, 2001). 2.4.3. Determination of serum ferritin concentration Serum ferritin concentration was determined using the Enzyme Linked Immunoassay kit (Spectro Ferritin, Ramco Laboratories Inc., Stafford, TX, USA) following manufacturer’s instructions. According to the manufacturer’s protocol, iron stores were considered depleted if serum ferritin concentration was <20 ng/ml, IDA was defined as serum ferritin <20 ng/ml among anaemic individuals. Serum ferritin values between 20 and 100 ng/ml in anaemic patients were suggestive of a combination of iron deficiency with some cause of anaemia. Overall, IDA was considered as a combination of IDA and iron deficiency with some cause of anaemia. Serum ferritin concentration >300 ng/ml indicated increased iron stores. 2.5. Treatment and other intervention measures Children infected with any of the schistosome species and STHs were treated with praziquantel at 40 mg/kg body weight and a single dose of 400 mg albendazole, respectively. Bread and orange juice (500 ml/child) were given as supplementary food following swallowing of tablets in order to enhance absorption and reduce the nauseating effect of praziquantel. Children diagnosed positive for P. falciparum were treated with a combination of chloroquine, sulphadoxine and pyrimethamine (SP) according to local malaria case management guidelines. Treatment for malaria was based on either clinical symptoms (in the absence of the research team) or parasitological results when the research team was in the field.

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2.6. Statistical methods Data were analyzed using Statistical Package for Social Scientists (SPSS) version 8.0 for Windows, SPSS inc. Chicago, USA. Association of helminths and P. falciparum infection with anaemia were determined using the Pearson Chi-Square test. Analysis of variance was used to determine any significant difference in haemoglobin levels between different infection intensity categories of helminths. Independent sample t-test was used to determine differences in Hb levels by gender, between individuals infected with parasites and those not infected. To determine the possible predictors of low Hb levels in the study population, multivariate regression analyses was performed using Hb concentration as the dependent variable, where as age group, sex, presence of P. falciparum, mean egg intensities of S. haematobium, S. mansoni, hookworm, A. lumbricoides and T. trichiura stratified according to the World Health Organisation guidelines were independent variables. Multivariate logistic regression was performed to calculate odds ratios for the association between parasitic infections and anaemia. Based on parasitological results, children were stratified into four categories: uninfected, those with single parasitic infection, double and triple parasitic infections. Single infection refers to infection with one parasite, schistosomes, STHs or P. falciparum. Double infection refers to infection with two parasite, P. falciparum and schistosomes, schistosomes and STHs or P. falciparum and STHs. Triple infection refers to infection with P. falciparum, schistosomes and STHs. Statistical significance for all analyses were determined at p < 0.05.

3. Results Six hundred and nine (609) children were recruited into the study and 50.4% of these were males. The overall mean (SD) age for the study population was 10.3 (2.3) years age range (6–17 years). The distribution of parasites and their egg intensities categorised according to WHO guidelines are shown in Table 1. S. haematobium was the most prevalent of all parasites investigated. Overall, 74.1% of children were infected with at least one of the parasites (P. falciparum, schistosomes or STHs). Among children with complete parasitological and Hb results 31.4% (154/491) had co-infections. Of the 154 children with co-infections the prevalence of malaria, STHs and schistosomiasis were 59.1% (91/154), 61.5% (96/156) and 95.5% (149/154), respectively.

3.1. Overall haematological results Among children who were screened for anaemia (n = 572), 51.4% were females. The mean Hb level was 11.6 g/dl (95%; CI: 11.5–11.8). Males had significantly higher mean Hb level than females (11.8 g/dl vs 11.3 g/dl, t = 2.868, p = 0.004). Overall, the prevalence of anaemia was 48.4% (277/572). Of the anaemic children 79.1% (219/277), 19.5% (54/277) and 1.4% (4/277) had mild, moderate and severe anaemia, respectively. Females were significantly more anaemic than males (2 = 4.29, p = 0.038). The overall prevalence of IDA was 38.1%. After stratifying children into different age groups: 5–7, 8–10, 11–13 and ≥14 years (n = 69, 248, 209 and 46, respectively), the youngest age group had the highest prevalence of severe anaemia (1.4%). The prevalence of anaemia and IDA were highest in the age group ≥14 years (60.9% and 69.7%, respectively). There was a significant difference in IDA between different age groups (2 = 16.54, p = 0.001). However there was no significant difference observed in the distribution of anaemia cases between age groups (2 = 3.16, p = 0.367.

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Table 1 Baseline prevalence of parasitic infection and stratification of helminths egg intensity according to WHO guidelines (WHO, 2002) among 609 primary school children living in Burma Valley farming area in Zimbabwe. Parameter

Parasites

Number examined (%) % Infecteda % Infected using Katob Katz technique only

S. haematobium

S. mansoni

Hookworm

A. lumbricoides

T. trichiura

P. falciparum

599 (98.4) 52.3 N/A

577 (94.7) 22.7 14.0

575 (94.4) 23.7 16.7

575 (94.4) 2.1 1.6

575 (94.4) 2.3 1.4

512 (84.1) 27.9 N/A

100 0.0 0.0

N/A N/A N/A

Parasitic infection intensity according to WHO guidelinesc % Light infection 67.7 % Moderate infection N/A % Heavy infection 32.3

44.4 39.5 16.0

84.4 12.5 3.1

66.7 33.3 0.0

a

Prevalence of S. mansoni, hookworm, A. lumbricoides and T. trichiura was based on the combination of results from the Kato Katz and formal ether concentration techniques. Prevalence of S. mansoni, hookworm, A. lumbricoides and T. trichiura was based on the Kato Katz technique only. c Infection intensities were categorised for those participants diagnosed positive by the urine filtration technique for S. haematobium and the Kato Katz technique only for S. mansoni, hookworm, A. lumbricoides and T. trichiura. b

3.2. Parasitic infection and anaemia Table 2 describes the distribution of Hb levels and anaemia in single parasite infections. The mean Hb levels were significantly lower among children who had P. falciparum, S. haematobium and hookworm infection compared to the mean Hb levels among corresponding negative individuals (11.0 g/dl vs 11.9 g/dl, t = −6.556, p < 0.001), (11.4 g/dl vs 11.9 g/dl, t = −3.973, p < 0.001) and (11.3 g/dl vs 11.8 g/dl, t = −2.986, p = 0.003), respectively. Analysis of variance showed a significant decrease in Hb levels with increasing egg intensities for S. haematobium categorised according to WHO guidelines, FS. haematobium = 8.824, p < 0.001 (Table 2). Bonferroni multiple analysis revealed that the group of participants that was uninfected had significantly higher mean Hb levels compared to those with light infection (mean Hb difference = 0.4 g/dl, 95% CI = 0.087–0.721, p = 0.007). Also non-infected participants had higher mean Hb levels than those with heavy infection (mean Hb difference = 0.6 g/dl, 95% CI = 0.227–1.028, p = 0.001). The same analysis showed no significant difference in Hb lev-

els between children with S. haematobium light infection and those with heavy infection (mean Hb difference = 0.2 g/dl, 95% CI = −0.192 to 0.639, p = 0.592). Although there was an apparent decrease in Hb levels with increasing hookworm egg intensities (Table 2), ANOVA showed no significant difference in Hb levels between different intensity categories (Fhookworm = 1.737, p = 0.158). The same trend was observed for the relationship between S. mansoni infection intensity categories (FS. mansoni = 1.201, p = 0.309). The prevalence of anaemia was significantly higher among children who were positive for P. falciparum compared to the corresponding P. falciparum negative individuals (Table 2). There was also a significant increase in anaemia with increasing S. haematobium and hookworm egg intensities (2 = 28.48, p < 0.001 and 2 = 8.16, p = 0.043 respectively). The prevalence of IDA was significantly high in P. falciparum infected individuals compared to the non-infected group. Iron deficiency anaemia significantly increased with increasing S. haematobium infection intensities (Table 2).

Table 2 Overall, anaemia and IDA by parasitic infection status among children living in Burma Valley farming area in Zimbabwe. Parasite

n

Hb ± S.E.

Anaemia (%)

2 p–value

P. falciparum Negative Positive

369 143

11.9 ± 0.07a 11.0 ± 0.11

38.8 76.9

313 <0.001

S. haematobium 0 eggs/10 ml 1–49 eggs/10 ml ≥50 eggs/10 ml

262 204 99

11.9 ± 0.08b 11.5 ± 0.11 11.3 ± 0.14

36.0 54.4 67.7

470 35 31 12

11.7 11.5 11.3 12.1

± ± ± ±

0.06 0.26 0.33 0.38

455 78 12 2

11.8 11.4 11.3 10.7

± ± ± ±

T. trichiura 0 epg 1–999 epg A. lumbricoides Negative Positive

IDA (%)

2 p–value

32.0 127

49.6

<0.001

197 178 <0.001

26.4 42.7 94

55.3

<0.001

47.2 60.0 51.6 33.3

390 27 26 0.344

36.2 51.9 53.8 10

40.0

0.137

0.07 0.18 0.28 2.15

44.8 60.3 66.7 50.0

372 68 11 0.043

35.5 45.6 63.6 5

50.0

0.119

539 8

11.7 ± 0.06 12.2 ± 0.28

47.9 25.0

448 0.199

37.9 5

20.0

0.410

536 11

11.7 ± 0.06 11.8 ± 0.22

47.4 54.5

447 0.638

37.4 6

66.7

0.141

n

*

S. mansoni 0 epg 1–99 epg 100–399 epg ≥400 epg *

Hookworm 0 epg 1–1999 epg 2000–3999 epg ≥4000 epg *

Only children who gave blood were considered in this analysis hence variation in sample sizes for each parasite from Table 1. a Student’s t-test of significant difference, p < 0.001. b ANOVA test of significant difference, p ≤ 0.001. * Kato Katz results used to stratify egg intensities according to WHO (2002) guidelines.

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3.3. Haematological characteristics in different co-infection combinations The relationship between indicators of iron status and different parasitic co-infection combinations among children who had haematological results are presented in Table 3. Among 491 children who had haematological and complete parasitological results, 22.6% (111), 46.0% (226) and 31.4% (154) had no parasitic infection at all, had single parasitic infection and co-infections, respectively. Of those with co-infections (n = 154), 83.1% (128) had double infections and 16.9% (26) had triple infections. There was a significant difference between mean Hb levels for groups of individuals that were uninfected and those infected with single or multiple parasitic infections (FANOVA = 6.927, p < 0.001) (Table 3). Boniferroni multiple analysis showed significant differences between individuals that had no infection with those with P. falciparum infection only and the groups of individuals with different parasite co-infections (Table 3.1). The mean Hb for individuals with single infection from schistosomiasis was significantly higher than mean Hb for those with other co-infections except for a group of individuals with P. falciparum + STHs co-infection (Table 3.1). Although there were apparent differences in Hb levels between different co-infection combinations, these were not significant (Table 3.1). The prevalence of anaemia and IDA increased from individuals who did not have any parasitic infection through children who had single infections with schistosomes, STHs and P. falciparum to children with polyparasites, respectively. Anaemia, iron deficiency and IDA prevalence were worse in malaria and helminth co-infections. Co-infections had almost a doubling effect on the corresponding prevalence of anaemia and IDA in non-infected and single infected individuals. The worst IDA occurred in schistosomiasis/P. falciparum and schistosomiasis/STHs/P. falciparum co-infections (Table 3). Prevalence of anaemia among children who did not have complete parasitological results (n = 81) was 46.9% and the mean haemoglobin level ± standard error was 11.8 ± 0.2 g/dl. 3.4. Predictors of Hb levels and anaemia Multivariate regression analysis using Hb level as the dependent variable where as age categories, sex, presence of P. falciparum and helminths infection intensities categorised according to WHO guidelines were explanatory variables showed that age groups 11–13 years, sex, P. falciparum were significantly associated with Hb levels (Table 4). Heavy S. haematobium infection was a predictor of low Hb level (Table 4). Multiple logistic regression analysis showed that children aged ≥14 years, presence of P. falciparum, light and heavy S. haematobium infection intensities, moderate and heavy S. mansoni and light hookworm infection intensities were predictors of anaemia. Sex, A. lumbricoides, T. trichiura infection intensities, light S. mansoni infection and the other age groups were not associated with anaemia (Table 5). 4. Discussion This study has shown an increase in anaemia, IDA and the corresponding decrease in Hb levels with increasing number of parasites infecting individuals. This is demonstrated in Table 3 where anaemia prevalence in co-infections is almost double the corresponding prevalence of anaemia in single infections with the highest prevalence of anaemia occurring in triple infections. The interaction of different mechanisms by which these different parasites reduce Hb levels in the host could be the cause for the enhanced anaemia. Helminths infection induce T-helper 2 cytokine response that results in a skewed anti-Plasmodium antibody responses towards the production of non-cytophilic

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immunoglobins ineffective against malaria instead of cytophilic ones (IgG1 and IgG3). This immune response results in haemolysis due to initial acute malaria followed by low level parasitaemia that cause dyserythropoiesis contributing to anaemia (WHO, 2001). Thus children with triple infection from P. falciparum, STHs and schistosomes are at greater risk for being anaemic. Results from this study confirm observations made in separate studies conducted elsewhere. (WHO, 2001; Nkuo-Akenji et al., 2006). Brito et al. (2006) found lower haemoglobin levels and higher prevalence of anaemia (50.9%) among Brazilian children who had S. mansoni associated with two or more intestinal helminths and had inadequate dietary iron consumption. Similarly Ezeamama et al. (2005) also observed that low-intensity polyparasite infections were associated with increased odds of having anaemia among 507 children in Leyte, the Philippines. The observed significant differences in mean Hb levels that existed between children not infected with any parasite and those with polyparasitism and significant mean Hb difference between children infected with schistosomiasis only and those with polyparasitism revealed when Bonferroni analysis was performed (Table 3.1) does not only show where the significant difference actually exist between these groups but further demonstrate the importance of polyparasitism in aggravation of anaemia. The same analysis failed to demonstrate any significant difference between groups of children with different co-infections (Table 3). Low levels of Hb and high prevalence of anaemia observed in this study among children who were positive for P. falciparum, S. haematobium and hookworm compared to those diagnosed negative for these parasites and the apparent increase in prevalence of anaemia with increasing helminths infections intensities (Table 2), present solid evidence for the causal relationship between helminths or P. falciparum infection and anaemia. In agreement with our findings, Ronald et al. (2006) observed significantly high prevalence of anaemia (66.2%) in Moshie Zongo village that had marked P. falciparum prevalence (37.8%) compared with the prevalence of anaemia (34.5%) in Manyia village that had low prevalence of P. falciparum (12.8%) in Ghana. Carneiro et al. (2006) demonstrated that the prevalence of anaemia depends on the prevalence of P. falciparum at population level in the Tanzanian study. In separate studies, Stoltzfus et al. (1997), Olsen et al. (1998) and Brooker et al. (1999) showed that Hb levels were negatively correlated with hookworm infection intensities. Beasley et al. (1999) demonstrated the importance of heavy hookworm infection intensities in the development of anaemia. On the other hand Koukounari et al. (2007) observed high intensities of S. haematobium infection in children with anaemia and severe microhaematuria. Following successful treatment Koukounari et al. (2007) observed a higher increase in Hb levels in children who had high egg intensities and anaemia at baseline. These results suggest a causal relationship between schistosomiasis, hookworm, P. falciparum and anaemia. An interesting observation is the discrepancy in prevalence of anaemia observed when it was estimated assuming single parasitic infections (Table 2) and after stratifying study participants into different parasitic infection combinations (non-infected, single and co-infected individuals) (Table 3). Results from Tables 2 and 3 show that estimating anaemia, assuming single parasitic infection in a community, would result in attributing single parasitic infections to high prevalence of anaemia that in reality could be due to unchecked co-infections. Thus there is a need for rigorous screening of other parasites known as risk factors before attributing any level of anaemia in the community to the presence of a single parasite. The observed impact of co-infection on Hb levels and anaemia (Tables 3 and 3.1), also demonstrate a compelling demand for the integrated approach in the control of anaemia in any community in order to effectively combat the disease and this can only be true

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Table 3 Relationships between parasitic co-infections with haematological characteristics among 491 children living in Burma Valley farming area in Zimbabwe. Parasite

No parasitic infection Schistosome positive only STH positive only Schistosomes + STHs STH + P. falciparum P. falciparum positive only Schisto + P. falciparum Schisto + STHs + P. f

Haemoglobin

Iron

n

Hb ± S.E.

Anaemia (%)

 p-value

n

111 157 29

12.1 ± 0.1 11.9 ± 0.1 11.8 ± 0.2 63

28.6 37.6 37.9 11.2 ± 0.2 11.2 ± 0.4 72.5 77.7 80.8

88 139 24 58.7 71.4 30

20.5 30.2 29.2

7 40 58 26

11.1 ± 0.2 10.9 ± 0.2 11.0 ± 0.3

2

<0.001

7 40.0 54 25

IDA (%)

2 p-value

54 28.6

53.7

56.0

57.4 <0.001

Only children screened for both anaemia and all parasites were included in this analysis. Key: P. f = P. falciparum; Schisto = schistosomes; STH = soil transmitted helminths. Table 3.1 Post hoc test with Bonferroni multiple analysis test for significant difference between groups of individuals with different parasitic infection combinations. Mean Hb Combination 1–combination 2

Mean difference (g/dl)

S.E.

p-Value

No parasitic infection–infected with schistosomes only No parasitic infection–infected with STH only No parasitic infection–infected with P. falciparum only No parasitic infection–infected with schistosomes + STHs No parasitic infection–infected with P. falciparum + schistosomes No parasitic infection–infected with P. falciparum + STHs No parasitic infection–infected with P. falciparum + schistosomes + STHs Schistosomiasis only–infected with STHs Schistosomiasis only–infected with P. falciparum only Schistosomiasis only–infected with Schistosomes + STHs Schistosomiasis only–infected with P. f + schistosomes Schistosomiasis only–infected with P. f + STHs Schistosomiasis only–infected with P. f + schistosomes + STHs Schistosomes + P. falciparum–P. falciparum Schistosomes + P. falciparum–schistosomes + STHs Schistosomes + P. falciparum–P. falciparum + STHs Schistosomes + P. falciparum–P. falciparum + schistosomes + STHs

0.20 0.29 1.06 0.88 1.18 0.88 1.16 0.01 0.86 0.68 0.98 0.68 0.97 −0.12 −0.30 −0.30 −0.01

0.17 0.29 0.25 0.22 0.22 0.54 0.30 0.28 0.24 0.21 0.21 0.53 0.29 0.28 0.25 0.55 0.33

1.000 1.000 0.001 0.002 <0.001 1.000 0.004 1.000 0.016 0.035 <0.001 1.000 0.036 1.000 1.000 1.000 1.000

Key: P. f = P. falciparum; Schisto = schistosomes; STH = soil transmitted helminths. Table 4 Multivariate regression analysis exploring the relationship between Hb with age, sex, presence of P. falciparum, intensities of S. haematobium, S. mansoni, hookworm, A. lumbricoides and T. trichiura infections. Independent variable

Coefficient

S.E.

t-Value

p-Value

Age group (years) 5–7 8–10 11–13 ≥14 Sex P. falciparum

Reference 0.271 0.440 0.404 −0.251 0.773

0.203 0.210 0.305 0.129 0.144

1.34 2.09 1.33 −1.95 5.36

0.182 0.037 0.185 0.051 <0.001

S. haematobium intensity (WHO categories) Uninfected—no eggs Light: 1–49 eggs/10 ml urine Heavy: ≥50 eggs/10 ml urine

Reference −0.257 −0.473

0.141 0.181

−1.82 2.61

0.070 0.009

Hookworm intensity Uninfected—no eggs Light: 1–1999 epg Moderate: 2000–3999 epg Heavy: ≥4000 epg

Reference −0.389 −0.241 −2.576

0.188 0.473 1.415

−2.07 −0.51 −1.82

0.039 0.611 0.069

S. mansoni intensity Uninfected—no eggs Light: 1–99 epg Moderate: 100–399 epg Heavy: ≥400 epg

Reference 0.020 −0.037 0.776

0.280 0.293 0.448

0.07 −0.13 1.71

0.942 0.899 0.088

A. lumbricoides intensitya Uninfected—no eggs Light: 1–4999 epg Moderate: 5000–49,999 epg

Reference −0.119 −0.277

0.629 1.014

−0.19 −0.27

0.850 0.785

T. trichiura intensityb Light: 1–999 epg

0.459

0.524

0.88

0.381

a b

Only light and moderate infection intensities were observed for A. lumbricoides. Only light infection intensity was observed for T. trichiura.

N. Midzi et al. / Acta Tropica 115 (2010) 103–111

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Table 5 Multivariate logistic regression analysis exploring the relationship between anaemia with age, sex, presence of P. falciparum, intensities of S. haematobium, S. mansoni, hookworm, A. lumbricoides, T. trichiura infections. Independent variable

Odds ratio

95% CI

z

p-value

Age group (years) 5–7 8–10 11–13 ≥14 Sex P. falciparum

Reference 0.837 1.049 3.150 1.336 5.347

0.442–1.582 0.542–2.030 1.161–8.545 0.887–2.013 2.269–8.744

−0.55 0.14 2.25 1.39 6.68

0.583 0.887 0.024 0.166 <0.001

S. haematobium intensity (WHO categories) Uninfected—no eggs Light: 1–49 eggs/10 ml urine Heavy: ≥50 eggs/10 ml urine

Reference 1.660 3.737

1.062–2.595 2.059–6.782

2.22 4.34

0.026 <0.001

S. mansoni intensity Uninfected—no eggs Light: 1–99 epg Moderate: 100–399 epg Heavy: ≥400 epg

Reference 1.045 0.364 0.141

0.416–2.626 0.137–0.967 0.025–0.786

0.09 −2.03 −2.23

0.925 0.043 0.025

Hookworm intensitya Uninfected—no eggs Light: 1–1999 epg Moderate: 2000–3999 epg

Reference 2.238 3.215

1.207–4.148 0.539–19.189

2.56 1.28

0.011 0.200

A. lumbricoides intensityb Uninfected—no eggs Light: 1–4999 epg

Reference 4.067

0.506–32.717

1.32

0.187

T. trichiura intensityb Uninfected—no eggs Light: 1–999 epg

Reference 0.247

0.259–2.359

−1.21

0.225

a b

Only two cases had hookworm heavy infection and they were left out in this analysis. Due low numbers and lack of moderate to heavy infection for A. lumbricoides and only light infection intensity was observed for T. trichiura.

if the contributing factors are identified and included in anaemia control strategy. The occurrence of more than 83% of children infected with at least a single parasite in the sub-sample with complete results demonstrates how vulnerable primary school children in subSaharan Africa are to single and overlapping parasitic infections resulting in co-morbidity including enhanced anaemia. Our study has shown no evidence of an association between S. mansoni infection intensity and Hb levels (Table 4). In agreement with our results, Latham et al. (1982) observed no association between Hb levels and S. mansoni infection in Kenyan male road workers. However, some studies suggest an inverse relationship between the degree of infection caused by S. mansoni and Hb levels (Freidman et al., 2005). Logistic regression analysis revealed that compared to participants who were uninfected, those with moderate and heavy S. mansoni infection were less likely to be anaemic (Table 5). This is surprising scientifically since we would expect those who are infected to be anaemic as well. Probably by chance these children ate supplementary food that had more iron content resulting in such a confounding trend. Our observations indicating S. haematobium heavy infection intensities, hookworm light infection and presence of P. falciparum as strong predictors of low Hb levels and anaemia (Tables 4 and 5) demonstrate that haemoglobin levels and prevalence of anaemia in a community are likely to depend on the intensity of helminth infections and presence of malaria. Unlike observation made in our study, Nkuo-Akenji et al. (2006) found no significant association between intensities of P. falciparum, hookworm and T. trichiura infections with packed cell volume (PCV). Our results are corroborated by Wilkins et al. (1985) who observed that anaemia was more common in heavy S. haematobium infected children than uninfected counterparts in the Gambian study. Stephenson et al. (1985) showed that decreases in S. haematobium and hookworm egg counts were important determinants of haemoglobin rise and that malaria parasites were equally

more important determinants of low Hb levels as helminths. Thus anaemia may represent a useful indicator of progress in control of schistosomiasis, STHs and P. falciparum malaria. The lowest Hb levels, high prevalence of anaemia iron deficiency and IDA among the older age group (≥14 years) observed in this study compared to other age groups could be due to increased demand for iron influenced by the growth spurt, blood loss due to menstruation in females compounded with single or co-infections. Among children in this age group 13.0% had no parasitic infection and 87.0% had either single or co-infection. The prevalence of anaemia (28.8%) among children with no parasites (Table 3) demonstrates that anaemia is generally high in Burma Valley farming area. Our observations confirm the observed prevalence of anaemia in Manicaland Province (30.7%) for reproductive women, age range 15–49 years (Zimbabwe Demographic Data, 2006). No parasites were screened in the later study hence the anaemia reported could not be associated with any parasitic infections as opposed to our study in which the knowledge of parasites infecting children allowed stratification of anaemia according to importance and the risk factors. High prevalence of anaemia (up to 87%) has been reported elsewhere in sub-Saharan Africa population (Schellenberg et al., 2004). These results may indicate the multifactorial nature in the etiology of anaemia. Thus the prevalence of anaemia observed in the non-infected children could be due to poor dietary intake of iron, Vitamin B12 and folate. HIV and other haemoglobinopathies such as sickle cell anaemia not ruled out in this study could also be contributing factors to the observed anaemia prevalence in non-infected individuals. Patients with HIV/AIDS show anaemia and leucoemia in about 60–70% of cases, and thrombocytopemia in over 40% (Zon et al., 1987). The limitation of our study is that it is cross-sectional, thus inference about causality of polyparasitism on anaemia should be taken with caution since the design makes it impossible to retrospectively determine if polyparasitism proceeds or follows anaemia. Causality

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can best be determined by prospective studies. Another limitation in our methodology could be the interpretation of the Kato Katz results based on examination of a single Kato Katz thick smear prepared per stool specimen per day on two successive days instead of the most acceptable duplicate thick smears prepared per specimen per day on at least two different days (Engels et al., 1996; Yu et al., 1998). Some light to moderate infections with intestinal helminths that could also be contributing to anaemia may have been missed out (Yu et al., 1998). 5. Conclusions The results from our study have fundamental practical implications for control of anaemia and parasitic diseases of public health importance. Thus integrated helminths de-worming and control of malaria through prompt diagnosis, treatment of children and provision of insecticide treated bed nets in schools would reduce the resultant deleterious co-morbidity such as anaemia in school children living in co-endemic communities. However as this is still an understudied area, further studies including prospective designs are recommended in other settings. Authorship contributions TM, NM, KCB, FM, NK contributed to the concept and design of the study protocol; NM, TM, SZ, DS, GH, JM carried out clinical assessment and parasitology; TM, NM, CMT, MPM, NK, FM carried out the analysis and interpretation of the data; NM, TM, CMT, MM, SZ drafted the manuscript. All authors read and approved the manuscript. TM and NM are guarantors of the paper. Funding The Ministry of Health and Child Welfare of Zimbabwe provided funds through the Essential National Health Research vote for the field data collection. The UNICEF/UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR) provided financial support for laboratory data analysis. International Foundation for Science W/4231 to TM for parasitology and the Wellcome Trust (Grant no WT082028MA). Schistosomiasis Control Initiative (SCI), Imperial College, UK supported the program with antihelminthic drugs (Praziquantel and Albendazole) for deworming. Conflict of interest None declared. Ethical consideration The Medical Research Council of Zimbabwe approved the study. In addition, Provincial Medical and Education Directors and teachers granted permission. Inclusion of children into the study took place after free individual and parental informed consent. Children joined the study voluntarily and were free to drop out at any time they wished without prejudice. Acknowledgements We acknowledge the Secretary for Health and Child Welfare of Zimbabwe for providing financial support for field data collection activities, the Acting director, National Institute of Health Research for all the support, all school children from Burma Valley farming areas who participated in the study, the Provincial Medical,

Education Directors for Manicaland and parents for granting us permission to conduct the study. We are also grateful to the technical staff from the National Institute of Health Research, Mr. Mugomba (Nurse in Charge for Mazonwe clinic, Burma Valley in Mutare district) who took part during field data collection. References Beasley, N.M.R., Tomkins, A.M., Hall, A., Kihamia, C.M., Lorri, W., Nduma, B., Issae, W., Nokes, C., Bundy, D.A.P., 1999. The impact of population level deworming on the haemoglobin levels of schoolchildren in Tanga, Tanzania. Trop. Med. Int. Health 4 (11), 744–750. Befidi-Mengue, R.N., Ratard, R.C., Beltran, G., D’alessandro, A., Rice, J., Befidi-Mengue, R., Kouemeni, L.E., Cline, B.L., 1993. Impact of Schistosoma haematobium infection and of Praziquantel treatment on anaemia of primary school children in Bertoua, Cameroon. J. Trop. Med. Hyg. 96, 225–230. Brito, L.L., Barreto, M., Silva, R.C.R., Assis, A.M.O., Reis, M.G., Parraga, I.M., Blanton, R.E., 2006. 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