|Year : 2018 | Volume
| Issue : 3 | Page : 131-136
Serum zinc levels amongst under-five children with acute diarrhoea and bacterial pathogens
Oyetundun Fausat Afolabi1, Aishat Oluwatoyin Saka2, Ayodele Ojuawo2, Sikiru Abayomi Biliaminu3
1 Department of Paediatrics, Bwari General Hospital, Abuja, Nigeria
2 Department of Paediatrics, University of Ilorin Teaching Hospital, Ilorin, Nigeria
3 Department of Chemical Pathology and Immunology, University of Ilorin Teaching Hospital, Ilorin, Nigeria
|Date of Web Publication||26-Sep-2018|
Oyetundun Fausat Afolabi
Department of Paediatrics, Bwari General Hospital, PMB 72, Bwari, Abuja
Source of Support: None, Conflict of Interest: None
Background and Aim: Acute diarrhoea contributes significantly to morbidity and mortality in under-five children globally with conflicting reports regarding the therapeutic benefit of zinc across the different causative pathogens. This study aimed to determine the prevalence of bacterial aetiology of children with acute diarrhoea and compare their serum zinc levels. Methods: One hundred children aged 2–59 months with acute diarrhoea and 100 apparently healthy matched controls were recruited in Ilorin, North Central Nigeria. Stool specimens were investigated for bacterial pathogens using conventional culture techniques, while serum zinc levels were determined by colorimetric method. Results: Bacteria were isolated in 73 (73.0%) patients and 6 (6.0%) controls. Escherichia coli was isolated in 39 (39.0%) of the patients, while Klebsiella spp., Proteus spp. and Pseudomonas aeruginosa were isolated in 28 (28.0%), 4 (4.0%) and 2 (2.0%) patients, respectively.E. coli and Klebsiella spp. were detected in 4 (4.0%) and 2 (2.0%) controls, respectively. The mean serum zinc level of 65.3 ± 7.4 μg/dl in the patients was significantly lower than 69.0 ± 6.5 μg/dl in the controls (P < 0.001). Zinc deficiency (serum zinc levels < 65 μg/dl) was detected in 47 (47.0%) patients which was significantly higher than 32 (32.0%) controls (P = 0.030). The mean serum zinc levels significantly differed amongst the bacteria isolated in the patients (P < 0.001). Conclusions: Bacterial pathogens constitute a significant burden to aetiology of acute diarrhoea in under-five Nigerian children. The prevalence of zinc deficiency was high in the study population. The serum zinc levels also differed across the bacteria isolated.
Keywords: Acute diarrhoea, bacterial pathogen, children, zinc
|How to cite this article:|
Afolabi OF, Saka AO, Ojuawo A, Biliaminu SA. Serum zinc levels amongst under-five children with acute diarrhoea and bacterial pathogens. Niger Postgrad Med J 2018;25:131-6
|How to cite this URL:|
Afolabi OF, Saka AO, Ojuawo A, Biliaminu SA. Serum zinc levels amongst under-five children with acute diarrhoea and bacterial pathogens. Niger Postgrad Med J [serial online] 2018 [cited 2019 Feb 20];25:131-6. Available from: http://www.npmj.org/text.asp?2018/25/3/131/242207
| Introduction|| |
Acute diarrhoea constitutes a major health challenge in children <5 years globally, accounting for 8.6% of the 5.8 million deaths in 2015. It is currently the fourth leading cause of morbidity and mortality in under-five children worldwide with a global prevalence of 10.0%.,, Developing countries bear the greatest burden of the disease as it contributes 10.0% each to deaths in Nigeria, India and Pakistan which together accounted for 40.0% of global under-five mortality., Bacterial pathogens contribute a huge burden to the disease accounting for 38.0% of mortalities due to childhood diarrhoea. Studies have reported Escherichia More Details coli as the most implicated bacterial pathogen associated with infectious diarrhoea in childhood, with prevalence as high as 41.4% documented in Southeast Nigeria.,,,, Other implicating bacterial organisms reported with varying prevalence depending on region include Salmonella More Details spp., Shigella spp., Campylobacter jejuni and Vibrio cholera.,,,
These bacteria disrupt intestinal function through enterotoxin production, mucosal invasion and damage resulting in net secretion of fluid and electrolytes with subsequent dehydration, electrolyte imbalance and shock. The contribution of these bacteria to childhood diarrhoea largely depends on geographic and socioeconomic conditions as they are spread through faecal–oral route potentiated by poor hygiene and sanitation as well as limited access to potable water. Identifying the spectrum of bacteria associated with childhood diarrhoea will, therefore, promote the use of specific interventions as well as preventive strategies.
Zinc is an important micronutrient necessary for protein synthesis, cell growth and differentiation, immune function and intestinal transport of water and electrolytes., Globally, it is estimated that 17.3% of the population has inadequate zinc intakes, with the highest estimates in Sub-Saharan Africa and South Asia., Zinc deficiency contributes substantially to the morbidity and mortality of young children worldwide as evidenced by high estimated prevalence and association with diarrhoea, pneumonia and malaria., Studies have documented the beneficial effect of zinc supplementation on the duration and severity of diarrhoea, thereby reducing mortality., This has led to a recommendation by the World Health Organization and United Nations Children's Fund (UNICEF) for the use of zinc supplementation for all children with diarrhoea. However, some studies have reported that the therapeutic benefit of zinc may not cut across all pathogens causing acute diarrhoea.,, Against this background, this study was conducted to determine the spectrum of bacterial aetiology amongst children with acute diarrhoea and compare their serum zinc levels.
| Methods|| |
Ethical approval for the study was obtained from the Ethical Review Board (Protocol No.: ERC PIN/2014/10/0253). All parents/caregivers of eligible children were informed of the purpose of the study, expected procedures and potential risks and benefits following which a written consent was obtained prior sample collection. It was a comparative, cross-sectional study conducted at the Emergency Paediatric Unit (EPU) of the University of Ilorin Teaching Hospital (UITH), and Oke-ose community both located in Ilorin, Kwara State, North Central Nigeria, between December 2015 and August 2016.
One hundred children aged 2–59 months with acute diarrhoea presenting at the EPU were recruited. Acute diarrhoea was defined as passage of loose or watery stools at least three times within a 24 h period, developing over a few hours or days and lasting fewer than 14 days. All the patients received standard treatment for their condition, irrespective of their decision to participate in the study. An equal number of apparently healthy age- and sex-matched controls were recruited from Oke-ose community where the hospital is located. The exclusion criteria were children with persistent diarrhoea (>14 days), severe malnutrition, pneumonia, sickle cell disease, presence of gastrointestinal anomalies, zinc supplementation and/or blood transfusion in the preceding 3 months and previously recruited children. Socio-demographic and clinical details were obtained using a structured questionnaire that was pre-tested with appropriate modifications made prior commencement of the study.
History of diarrhoea was obtained from the parent/caregiver of each patient, and a physical examination of all the study participants including weight and length/height was carried out. The weights and lengths of children <2 years of age and/or 12 kg were assessed using a bassinet weighing scale (Waymaster, England) and an infantometer, while a beam balance weighing scale with a stadiometer attached (Marsdens, England) was used for older children. Each of the weighing scales has a degree of accuracy of 0.05 kg (50 g) and was re-calibrated using standard weight periodically.
The minimum sample size required for this study was determined using the formula:
where n is the desired sample size; p1 is 69.1%; p2 is 44.0%; and Cp.power is 13 which was determined by P = 0.05 and 95% power.
N = 88 and allowing for minimum 10% attrition, 100 was the calculated minimum sample size. A purposive sampling was used in which study participants who fulfilled the inclusion criteria were recruited consecutively until the desired sample size was attained.
Fresh stool samples were collected from each study participant into clear, wide-mouth sterile universal bottles with proper identification labelled on each bottle and immediately processed or kept in a refrigerator at 4°C until processing (within 6–8 h of collection). Each specimen was examined macroscopically for consistency, colour and presence/absence of mucous and blood. The samples were then cultured on differential, selective and enrichment media, namely, MacConkey bile salt agar, Xylose Lysine Deoxycholate agar and Selenite F broth. Each of the agar plates was labelled with numbers allocated to patients after which a sterile wire loop was used to inoculate each faecal sample using streaking technique. Each plate was incubated at 37°C for 18–24 h and examined for possible bacteria growth. Agar plates with positive cultures had their representative colonies Gram stained. Biochemical tests such as citrate, indole, urease, oxidase and triple sugar iron (TSI) tests were then carried out to identify bacterial isolates using standard microbiological techniques.
Blood samples were collected for estimating serum zinc, total protein, albumin and C-reactive protein (CRP). A total of 5 ml of venous blood was drawn from the peripheral veins of all recruited children. Serum samples were obtained by centrifugation at 3000 revolutions per minute for 5 min in a benchtop centrifuge. The sera were then transferred into acid-washed plain bottles and immediately stored at −20°C till time of analysis. Serum zinc was analysed with a Jenway™ spectrophotometer 6300 model (Jenway Limited, Dunmow, Essex, United Kingdom) for measuring optical density at 560 nm, after preparation with the zinc fluid monoreagent (5-Br-PAPS) kit (Centronic GmbH, Wartenberg, Germany), a quantitative colorimetric assay of Zn2+. Children with levels of <65 μg/dl were considered zinc deficient. Furthermore, since zinc is transported in plasma bound to albumin, the concomitant serum total protein and albumin levels were determined using total protein assay and albumin assay (Agappe Diagnostics Limited, Kerala, India). C-reactive protein (CRP) levels were determined using CRP assay kits (Monobind Inc., Lake Forest, CA, USA). The CRP estimation was done to provide an objective laboratory evidence for the presence or otherwise of ongoing inflammation at the time of the recruitment. Serum zinc concentrations are reduced during acute infections and inflammation, as evidenced by elevated concentrations of CRP or other markers of the acute phase response which mainly induces zinc redistribution to the liver, and should be considered in the interpretation of results.
Data were analysed using the Statistical Package for the Social Sciences version 20.0 software (IBM Corp., Armonk, NY, USA). Measures of central tendency, dispersion of quantitative variables and proportion for the qualitative variables were presented. Chi-square test was used to test for significance of the difference between categorical variables. The independent t-test (t) and analysis of variance test were used to identify significance when comparing less than three and three or more continuous variables, respectively. The least significant difference (LSD) post hoc test was used when appropriate. Probability values P < 0.05 were accepted as statistically significant.
| Results|| |
A total of 100 children with acute diarrhoea were compared with 100 apparently healthy controls. There were 63 males (63.0%) and 37 females (37.0%) amongst the patients, while the controls comprised 60 males (60.0%) and 40 females (40.0%). The mean age of the patients was 8.7 ± 1.8 months while that of the controls was 8.8 ± 2.0 months. There was no significant difference between the two groups in terms of age, sex and height (P > 0.05). However, underweight malnutrition shown by mean weight-for-age Z-score (WAZ) was significantly higher in the patients when compared with the controls (P < 0.001) [Table 1].
Seventy-three (73.0%) of the patients had bacterial aetiology compared with 6 (6.0%) of the control [Table 2]. E. coli was isolated in 39 (39.0%), Klebsiella spp. 28 (28.0%), Proteus spp. 4 (4.0%) and Pseudomonas aeruginosa 2 (2.0%) of the patients. E. coli and Klebsiella spp. were the only bacterial pathogens detected in 4 (4.0%) and 2 (2.0%) of the controls, respectively. No isolates of Salmonella spp. and Shigella spp. were detected. There was a significant difference in the occurrence of E. coli and Klebsiella spp. between the patients and controls (P = 0.001) [Table 2]. Age group distribution of infection in the patients showed that infants had the highest infection rate constituting 55 (75.3%) of the population. There was a significant difference in the occurrence of bacterial pathogens in the patients < 24 months (P < 0.05) [Table 3].
Serum zinc and other biochemical parameters
The mean total serum zinc levels in the patient and control groups were 65.3 ± 7.4 μg/dl and 69.0 ± 6.5 μg/dl, respectively (P < 0.001) [Table 4]. Zinc deficiency was observed in 47 (47.0%) of the patients and 32 (32.0%) of the controls (P = 0.030). Furthermore, the patients with zinc deficiency had significantly lower mean serum zinc levels of 56.9 ± 7.1 μg/dl than the corresponding value of 62.7 ± 6.9 μg/dl in the controls (P < 0.001). There was also a significant difference in the mean serum total protein, albumin and CRP levels between the patient and control groups (P < 0.001) [Table 4].
|Table 4: Serum zinc and other biochemical parameters in the study population|
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Serum zinc and bacterial aetiology of acute diarrhoea
Among the patients, those with diarrhoea due to Proteus spp. had the lowest mean zinc level of 51.7 ± 2.2 μg/dl. A significant difference was observed across the mean serum zinc levels of the bacteria isolated in the patients studied (P < 0.001) [Table 5].
|Table 5: Serum zinc levels according to bacterial pathogens of acute diarrhoea|
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| Discussion|| |
This study underscores the importance of bacterial pathogens as a cause of childhood diarrhoea as demonstrated by the high prevalence (73.0%) as against the 6.0% found in children without diarrhoea. Although a similar report documented a prevalence of 85.0% in North Central Nigeria, other studies have reported lower prevalence between 40.0% and 69.0% in Africa.,,, These varying prevalence may be ascribed to differences in infrastructural and socioeconomic indices amongst the cities where the studies were conducted. Factors such as poverty, lack of potable water, poor sanitation as well as malnutrition and poor hygiene in the developing world promote the faecal–oral spread of bacterial infection.
E. coli was the leading cause of acute diarrhoea amongst the isolates in this study, which is similar to other reports from Nigeria ,,, and Africa., The prevalence of 39.0% obtained for E. coli in this study is comparable to 41.4% and 43.1% reported by workers in Nigeria , although lower than findings elsewhere., In contrast, lower prevalence rates of 19.4% and 23.0% were reported by other studies in Nigeria,, 23.7% in Burkina Faso, 11.1% in Central Africa Republic  and 4.7% in China.
In the present study, Klebsiella spp. was isolated in 28.0% of the patients. This is in contrast to lower rates detected in earlier reports., A prevalence of 34.5% was, however, found in an Indian study. Two per cent of cases of P. aeruginosa was reported in the patients, which is consistent with the prevalence of 2.7% detected by a similar study also conducted within the same geographical region (North Central Nigeria). This previous study reported an 8.4% prevalence rate of Proteus spp. which was slightly higher than the 4.0% found in this present study.
There were no isolates of Salmonella spp. and Shigella spp. cultured in this study, a finding in concordance with an earlier study conducted in Enugu, Nigeria. This is in contrast to other reports across Africa which detected these enteric pathogens at rates ranging from 2.2% to11.3% and 2.5% to 8.0%, respectively.,,,, The growth of these pathogens as well as other bacteria may have been suppressed by premedication with antibiotics prior hospital presentation as this could have been responsible for false-negative results.
In this study, the comparatively lower serum zinc levels in children with acute diarrhoea as against healthy controls are in congruence with earlier studies from Bangladesh and Iran., This finding further substantiates the well-documented interrelationship between serum zinc and diarrhoea.,, Zinc is known to play a pivotal role in the function of the immune system through antimicrobial, antioxidant and anti-inflammatory roles.,, Furthermore, its positive influence on the gut mucosa, both on a molecular and cellular level, enhances gastrointestinal barrier function., Therefore, zinc deficiency increases the susceptibility to and severity of gastrointestinal infections which then causes damage to the structure and function of the gastrointestinal tract and subsequently diarrhoea. Moreover, one of the main sources of zinc loss from the human body is through the intestine, and diarrhoea potentiates excess intestinal zinc losses. Furthermore, serum zinc is reduced during acute infections/inflammation, due to the acute phase reactant-induced redistribution of zinc to the liver, as well as decreased plasma proteins such as albumin which is the main transport protein for zinc.
The high prevalence of zinc deficiency found in the study population as well as the magnitude of the relative differences in serum zinc levels (1.5 times) between the children with acute diarrhoea and the controls in this study is in accordance with findings in Bangladeshi children by a similar study. This underscores the global burden of zinc deficiency in earlier reports with the highest prevalence documented in Africa and South Asia where the studies were conducted., On the contrary, a report emanating from Iran did not find any zinc deficiency in the healthy controls despite a high prevalence of 62.5% in the patients.
The disparity of population status of zinc as shown by the aforementioned studies may be partly explained by the quality of the local staples in the local population where these studies were conducted. The common staple food in Ilorin where the present study was done comprise rice, yam, maize and cassava, all of which are high in myoinositol hexaphosphate (phytates) which irreversibly binds zinc within the intestinal lumen, thereby inhibiting the absorption and utilisation of zinc., Furthermore, animal-rich sources of zinc such as meat, fish and poultry are consumed in limited quantity because of cost-related non-affordability of these foodstuffs by the local population.
It is noteworthy that this current study found a significant difference in mean serum zinc levels amongst the isolated bacterial pathogens of acute diarrhoea. This is in conformity with a similar study conducted amongst Iranian children. The findings from this current study would, therefore, suggest that the differing effect of zinc supplementation on isolated enteropathogens observed in some earlier reports , may be partly attributed to the causal effect of these pathogens on baseline zinc levels as well as the function of the influence of supplemented zinc on the ion channels of the gut mucosal cells where these pathogens act to induce diarrhoea., The paucity of published studies has not enabled ample comparison and/or substantiation of this finding; therefore, replication studies will be desirable in a bid to maximise the beneficial effects from the current strategy of zinc supplementation regarding isolated enteropathogens of diarrhoea.
The primary limitation of this study was that the isolated bacterial pathogens were not serotyped.
| Conclusions|| |
The results of this study showed a high burden of childhood diarrhoea due to bacterial pathogens in this clime. This study also observed a high prevalence of zinc deficiency in under-five Nigerian children, particularly those with acute diarrhoea. The serum zinc levels also differed across the bacteria isolated.
There is the need for adoption of preventive and control strategies aimed at reducing the burden of infectious diarrhoea. Furthermore, zinc supplementation and food fortification schemes should be reinforced in the population. This study, however, suggests that more research is needed to understand the differing serum zinc levels based on the isolated bacteria of acute diarrhoea.
The authors would like to appreciate the participants and their caregivers for their cooperation in this study. We also appreciate Mr Dele Amadu (Department of microbiology, UITH) for the laboratory bench space and his assistance during the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]