Malaria and helminth co-infections in outpatients of Alaba Kulito Health Center, southern Ethiopia: a cross sectional study

Background Distribution of malaria and intestinal helminths is known to overlap in developing tropical countries of the world. Co-infections with helminth and malaria parasites cause a significant and additive problem against the host. The aim of this study was to asses the prevalence of malaria/helminth co-infection and the associated problems among febrile outpatients that attended Alaba Kulito Health Center, southern Ethiopia November and December 2007. A total of 1802 acute febrile patients were diagnosed for malaria. 458 Giemsa-stained thick and thin blood films were used for identification of Plasmodium species and Stool samples prepared using Kato-Katz technique were used to examine for intestinal helminths. Haemoglobin concentration was measured using a portable spectrophotometer (Hemocue HB 201). Anthropometry-based nutritional assessment of the study participants was done by measuring body weight to the nearest 0.1 kg and height to the nearest 0.1 cm. Findings 458 of the total febrile patients were positive for malaria. Co infection with Plasmodium and helminth parasites is associated with significantly (p < 0.001) higher anaemia prevalence than single infection with Plasmodium parasites. And this difference was also significant for haemoglobin concentration (F = 10.18, p = 0.002), in which patients co infected with Plasmodium and helminth parasites showed lower mean haemoglobin concentration. More than one-third of the infected cases in both malaria infections and malaria/helminth co infections are undernourished. However the statistics for the difference is not significant. Conclusion Malaria and soil-transmitted helminthiasis obviously contribute to anaemia and low weight status and these conditions are more pronounced in individuals concurrently infected with malaria and soil-transmitted helminths. Hence, simultaneous combat against the two parasitic infections is very crucial to improve health of the affected communities.


Background
Overlapping distribution of intestinal helminths and malaria results in a high rate of co-infection [1,2], which may result both in synergism and antagonistic interaction between helminths and malaria parasites [3,4]. One of the main impacts of malaria and helminth infections is anaemia. Malaria causes anaemia, among other mechanisms through haemolysis and increased spleenic clearance of infected and uninfected red blood cells and cytokineinduced dyserythropoeisis [5,6]. Similarly, intestinal helminths are significant causes of anaemia as a result of direct blood loss, nutritional theft and impairment of the appetite due to immunological factor [7,8]. Based on the distinct mechanisms by which malaria and helminths reduce haemoglobin levels, it can be speculated that their combined presence might interact to enhance the risk of anaemia. And several reports [9][10][11][12] in Kenya, Nigeria, Thailand and some other countries of Africa showed suggestive of an additive impact of co-infection on anaemia in certain age groups. However such associations may be confounded by socio-economic, genetic, and nutritional factors and that the effects of co-infection may vary by malaria and helminth species and their intensities [10].
In addition, co-infections with helminth and malaria parasites have negative impact upon host nutrition through a number of mechanisms which may have additive or multiplicative impacts, especially in childhood [13]. Helminths cause and/or aggravate malnutrition through worm-induced gastrointestinal tract physiopathology and reduced food intake [14], chronic blood-loss [8] and intestinal inflammation [15]. Malaria may also contribute to protein-energy malnutrition through a number of mechanisms triggered by augmented levels of inflammatory cytokines, including anorexia and the induction of a catabolic response [16]. However, the contribution of intestinal helminth infections as well as malaria in the development of anaemia and low weight status, the concomitant occurrence of malaria and intestinal helminth infections and their clinical manifestations among all age groups, in malaria endemic areas like Ethiopia is largely unreported. Therefore, the aims of this study were to asses the associations between anemia/ nutrition status and helminth infections in patients with clinical malaria in communities of Alaba Kulito area, southern Ethiopia.

Study area and population
The study was conducted at Alaba Kulito Health Center in Alaba Woreda (administrative unit), located 313 km south of Addis Ababa. The Woreda is semi-arid with annual temperature ranging from 18 to 23°C and mean annual rainfall from 100 to 120 millimeter. Malaria and intestinal parasites are the most prevalent public health problems in the area. Malaria transmission in Alaba is unstable, seasonal and depends on altitude and rainfall. There are two main seasons for transmission of the disease; September to December, after the heavy summer rains, and March to May, after the light rains.
The study was conducted on acute malaria patients that attended Alaba Kulito Health Center in November and December 2007. Cases positive for Plasmodium species and older than one year, had no history of anti-malarial drug administration in the two weeks prior to screening, absence of any other serious chronic infection, had ability to give blood and stool samples were included in the study. However, pregnant women, children younger than 1 year and individuals with known concomitant chronic infection were excluded from the study, and this work has been published elsewhere [17].

Nutritional assessment
Anthropometry-based nutritional assessment of the study participants was done by measuring body weight to the nearest 0.1 kg and height to the nearest 0.1 cm. Then the body mass index (BMI) was calculated to determine the weight status of the study participants using the formula (weight in kg/[height in m] 2 ). In classifying the weight status of the study participants the standard weight status categories associated with BMI ranges were used for overweight and obesity in adult [18]. Similarly, the international BMI cut offs for child overweight and obesity which cover the age range 1-19 years were used for children and adolescents [18,19]. However, the interpretation of BMI values for age group 1-19 years was done after obtaining a percentile rank using a CDC-BM/-For age growth charts and Statistical Advanced Soft Ware [20] which convert BMI values to an ordered grade interpolating to the child's age. In addition, for underweight (thinness) three cut offs, namely grade-1 thinness (BMI < 16 Kg/m 2 ), grade-2 thinness (BMI = 16-16.9 Kg/m 2 ) and grade-3 thinness (BMI = 17-18.4 kg/m 2 ) were used [18][19][20] in case of adults. For children and adolescents similar cut off values derived based on adults BMI 16 and 16.5 at 18 years were used [21].

Clinical and laboratory diagnosis
Socio-demographic survey and clinical diagnosis were made by trained physicians of the health center. Thick and thin smears were prepared on a single slide for each acute febrile patient from capillary blood by finger pricking using sterile lancet. Each blood smear was stained with Giemsa and examined under the oil immersion microscope objective. Hundred fields were examined before negative result was reported. Thick smear was used to detect malaria infection and parasite quantification. The thin smear was used to identify the type of Plasmodium species. The number of parasites per microliter of blood was calculated from the thick blood smear [22]. For each participant only one slide was read to detect malaria infection and identify the type of Plasmodium species.
Clean stool cap and stick applicator were provided to malaria positive subjects volunteered to provide stool specimen. Kato-Katz thick method was used to prepare stool specimens for microscopic examination [23]. Similarly, for each participant only one slide was read to detect helminth infection and identify the type of species.

Determination of haemoglobin concentration
Haemoglobin concentration was determined using a portable haemoglobin spectrophotometer, Hemocue Hb 201 analyzer (HemoCue, Angelholm, Sweden) and specially designed microcuvette (the Hemocue Hb 201 Microcuvette, Hemocue, Angelholm, Sweden). Then, the haemoglobin values were used to assess the status of anaemia. For haemoglobin, the cut-off criterion levels below which indicating anemia was the WHO cut-off of 110 g/L for children 6-59 months; 115 g/L for children 5-11 years; 120 g/L for children 12-14 years; 120 g/L for non-pregnant women above 15 years of age and 130 g/L for men above 15 years of age [24].

Data analysis
Data entry and validation was performed in excel, and statistical analysis was done using Statistical Package for Social Science version 13.0. Mantel Heinzel Chi-square test including odd ratios at 95% CI and one way ANOVA were used to test for differences in proportions and means, respectively. Values were considered statistically significant when p-values were less than 0.05.

Ethical consideration
This study was part of the ongoing project on efficacy of anti-malarial drugs against malaria at the Akililu Lemma Institute of Pathobiology, Addis Ababa University. The study was conducted after ethical clearance was obtained from the Institute and the Health Bureau of South Nations Nationalities and Peoples Region (SNNPR). Patients were involved in the study after obtaining informed consent. Consent was also obtained from guardians and/or parents for children under 18 years of age. All positive subjects for Plasmodium falciparum were treated with coartem, for P.vivax with chloroquine, for mixed infection with P. falciparum and P. vivax with coartem and chloroquine and for soil-transmitted helminthiasis with albendazole.

Malaria and soil-transmitted helminth infections
Out of the 1802 acute febrile patients, 502 (27.9%) were positive for Plasmodium parasites and 458 subjects (233 males and 225 females) fulfilled the inclusion criteria and enrolled in the co-infection study. From the 458 malaria infected patients, 255 (55.7%) were positive for one or more STHs. The most prevalent helminth was hookworm (37.8%) followed by A. lumbricoides (24.7%) and T. trichiura (8.3%) [17].
The overall intensity of infection of intestinal helminthiasis expressed as geometric mean among the study subjects for children younger than 5 years of age, children 5-14 years old and adults ≥ 15 years old was, 955.33 (range; 24-7680), 2309 (range; 96-64424) and 1045.8(range; 24-30248), respectively. The intensity of infection tends to reach its peak in the age group ≥ 15 years old for hookworm, 5-14 years old for A. lumbricoides and < 5 years old for T. trichiura with a mean EPG of 542.72, 6249.9 and 699.33, respectively.

Haemoglobin measurement
Mean haemoglobin concentration of the study participants was, 13.1 g/dl (ranging from 5.2 g/dl to 21.4 g/dl) with standard deviation of 2.37. Plasmodium alone or both Plasmodium and helminth infected females were found to have significantly lower mean haemoglobin concentration than males. Children younger than 5 years were found to have significantly the lowest mean haemoglobin concentration as compared to the older age groups.
Based on the WHO cut off values of haemoglobin concentration, a total of 144 anaemic cases were found, making an overall prevalence of anaemia among the study subjects 31.4%. Severe anaemia (haemoglobin concentration ≤ 7 g/dl) was very rarely seen (1.7%) and most of these were young children.
Prevalence of anaemia was higher in females (33.3%) than in males (29.6%) and highest in children younger than 5 years (46.3%) (p = 0.003). It was observed that 18.7%, 40.7% and 50% of P. vivax, P. falciparum and mixed infected subjects were anaemic, respectively. Similarly, the prevalence of anaemia was higher in malaria and intestinal helminth co-infected patients. The prevalence of anaemia was 43.3%, 29.2%, 35.7% and 42.3% for hookworm, A. lumbricoides, T. trichiura and multiple intestinal helminth and malaria co infected subjects, respectively (Table 1). In general, data for the study patients indicate that co infection with Plasmodium and helminth parasites is associated with significantly (x 2 = 20.3, Odds Ratio = 2.58, p < 0.001) higher anaemia prevalence than single infection with Plasmodium parasites ( Table 2). And this difference was also significant for haemoglobin concentration (F = 10.18, p = 0.002), in which patients co infected with Plasmodium and helminth parasites showed lower mean haemoglobin concentration.
Co infection with Plasmodium and helminth parasites was significantly (p ≤ 0.001) associated with high prevalence of anaemia than single infection with Plasmodium parasites in children younger than 5 years and adults ≥ 15 years. And this difference was also significant for haemoglobin concentration (F = 18.17, p < 0.001), in which patients co infected with Plasmodium and helminth parasites showed lower mean haemoglobin concentration. Similarly, the prevalence of anaemia in males was higher (p < 0.001) in malaria and intestinal helminth co infected patients than those single infected with malaria. In contrast, no significance difference was observed both among females and children, 5-14 years ( Table 2).

Nutritional assessment
The BMI of the study participants calculated from their body weight and height measures revealed that 19 (Table 3).

Discussion
Haematological abnormalities are considered a hallmark of malaria, especially in Plasmodium falciparum infection [25]. The present study also showed lower haemoglobin level in P. falciparum cases than those with P. vivax. A relatively lower mean haemoglobin concentration was also observed in those having mixed infections than those with single Plasmodium infected patients. This study has demonstrated that cases with malaria/ helminth co-infections had significantly higher prevalence of anaemia and lower mean haemoglobin concentration when compared with malaria infection cases. And this is in agreement with previous report [12], which observed a significant difference in haemoglobin concentration in malaria/helminth co-infected study patients and patients with malaria infection alone. Similarly this finding is comparable to another report [11], which showed the occurrence of low haemoglobin concentration in pregnant women co-infected with Plasmodium parasites and helminths in Kenya. In addition similar to previous reports [10] this study also indicated that anaemia prevalence and haemoglobin concentration difference between malaria/helminth co-infections and malaria infection shows an age and sex related pattern. In general these data are suggestive of an additive impact of helminth and malaria co-infection on aggravating anaemia [26,27] in children younger than 5 years and adults ≥ 15 years [10]. However, it should be noted that such associations may be confounded by socio-economic, genetic, and nutritional factors and that the effects of co-infection may vary by malaria and helminth transmission intensities [10]. Consequently, randomized controlled trials of combining malaria and helminth-specific interventions aimed at establishing the contribution of co-infection on anemia should be conducted in a range of transmission settings. The increased prevalence of anaemia in co infected cases may be attributed to chronic blood and iron loss due to worm infections in addition to the loss  Thus, antihelminthic treatment could potentially be coadministered with malaria control to children younger than 5 years and adults ≥ 15 years in areas of seasonal malaria transmission. Although the statistics for underweight prevalence difference is not significant between malaria infections and malaria/helminth co infections, more than one-third of the infected cases in both groups are undernourished. Several studies have shown that malaria has a negative effect on the nutritional status of under 5 children and individuals older than 5 years [27][28][29]. Malaria related symptoms, such as diarrhea and abdominal pain may lead to malabsorption of nutrients and decreased intake, respectively. In addition, proinflammatory cytokine such as TNF-α released against malaria antigens would adversely affect the nutritional status [30]. Soil-transmitted helminthiasis is also associated with varying degrees of malnutrition, the pathogenesis of which is poorly defined [7]. Ascaris and hookworm secret potent inhibitors of pancreatic enzymes, which may block host nutrient absorption in the small intestine directly [30]. Furthermore, soil-transmitted helminthiasis contributes to malnutrition by impairing appetite [31].
The high prevalence of anaemia in malaria patients of the underweight status groups than those in the normal weight and overweight groups suggests malnutrition as a risk factor for anaemia besides malaria and helminth infections. Anaemia prevalence was highest in the grade-3 thinness grouped subjects compared to those in other groups when the co-infection was malaria/A. lumbricoides. This seems to support the effect of A. lumbricoides on aggravating preexisting anaemia by decreasing appetite and thus food and iron intake [7].
As indicated above malaria and helminth co infections have impact on anaemia prevalence and weight status in infected patients. However other factors like diet and socio economic status which may have impact on haemoglobin levels and nutrition status are not considered in this study. And also, for each blood and stool specimens from participants only one slide was read to detect malaria and/or helminth infection and identify the type of species. In addition, there were small sample sizes in each infection types of different helminth intensity and Plasmodium parasitaemia level to make comparisons valid. Hence this study did not assess association between helminth intensity and Plasmodium parasiteamia with anaemia and weight status. These are some of the limitations of the study.

Conclusion
Generally, malaria and soil-transmitted helminthiasis obviously contribute to anaemia and low weight status and these conditions are more pronounced in individuals concurrently infected with malaria and soil-transmitted helminths. Hence, simultaneous combat against the two parasitic infections is very crucial to improve health of the affected communities in economically developing countries.