Prevalence and predictors of zinc deficiency among children and non-pregnant women in Nepal: analysis of Nepal micronutrients status survey 2016

1 Ministry of Health and Population, Government of Nepal, Kathmandu, Nepal Queensland Brain Institute, The University of Queensland, Australia 3 Chief, Nutrition Section, Family Welfare Division, Department of Health Services, Ministry of Health and Population, Kathmandu, Nepal 4School of Optometry and Vision Science, Faculty of Science, Kensington NSW 2033, UNSW, Sydney, Australia 5Department of Infectious Diseases and Immunology, Kathmandu Research Institute for Biological Sciences (KRIBS), Lalitpur, Nepal 6Centre for Primary Health Care and Equity, University of New South Wales, Sydney, Australia 7School of public Health and Community Medicine, UNSW, Sydney, Australia 8Centre for research policy and implementation, Biratnagar, Nepal 9Department of Public Health, Torrens University, Sydney, Australia 10Little Buddha College of Health Sciences, Min Bhawan, Kathmandu, Nepal 11Director General, Department of Health Services, Ministry of Health and Population, Kathmandu, Nepal


Introduction
Zinc deficiency is a common and long-standing public health problem of low and middleincome countries (LMICs). According to the WHO data, zinc deficiency is a leading cause of mortality and morbidity in LMICs (Organization, 2002). Zink deficiency is considered as a public health concern when the prevalence of low serum zinc concentration is >20% (Whitehead Jr et al., 2017). Nearly one-third of the South Asian population is at risk of inadequate zinc supply (Wessells & Brown, 2012) but zinc deficiency varies from 4% to 73% across the subregions. Mild to moderate level of zinc deficiency is more common throughout the world than severe zinc deficiency (Brief, 2007;Ezzati, Lopez, Rodgers, & Murray, 2004). Since the first documentation of zinc as a nutrient for human health in 1963, several studies have proven zinc as an essential micronutrient with a key role in myriad of biological functions which ranges from DNA synthesis to physical growth (Brown, Peerson, Rivera, & Allen, 2002;MacDonald, 2000;Prasad, 1991). Zinc is equally important forideal growth of the foetus and maternal tissue development (King, 2000). Zinc deficiency is mainly associated with insufficient intake and/or absorption from the foods. Additionally, the human body has no tissue reservoir for zinc, unlike iron and vitamin A so, adequate zinc supply through dietary is necessary to prevent zinc deficiency (Ackland, Michalczyk, & nutrition, 2006;Mackenzie, Iwasaki, Tsuji, & signaling, 2008). Children, especially from low-income countries such as Nepalwith poor diet and gastrointestinal infections are at higher risk of zinc insufficiency (Brown et al., 2004). Zinc deficiency can appear as a symptom of the disease which leads to detrimental effects in human health including immune anomalies as zinc involves in innate immunity, rough skin, dwarfism, poor appetite, and mental fatigue, among others (Livingstone, 2015;Report & Reducing risks, 2002). Zinc deficiency is also correlated with anaemia, cardiac diseases, and impairs neurogenesis at the early stage of development (Adamo et al., 2019;Atasoy & Bugdayci, 2018). Globally, zinc deficiency is highly associated with chronic and infectious diseases like cancer, and diabetes, and measles, HIV, tuberculosis, and pneumonia, respectively (Sigel, Sigel, & Sigel, 2013). Zinc supplementation can reduce the risk of low-birthweight infants' deaths, and it is also used as an adjuvant with rehydration treatment of diarrheal diseases (Sazawal et al., 2001; "Therapeutic effects of oral zinc in acute and persistent diarrhea in children in developing countries: pooled analysis of randomized controlled trials %J The American journal of clinical nutrition," 2000).
The Government of Nepal (GoN) has introduced zinc supplements to manage childhood diarrhoea in 2007 but, it demands regular evaluation and proper monitoring (Ghimire, Agho, Renzaho, Dibley, & Raynes-Greenow, 2018). GoN has listed Zinc tablet of 10 mg, 20 mg (scored tablets) in the national list of essential medicines to use in acute diarrheal cases as an adjunct to ORSs (Alam et al., 2017).
Micronutrient deficiencies is a hidden hunger among Nepalese community, particular women and children, and the inadequate zinc status of maternal women results in adverse consequences such as abortion, low-birthweight, and congenital malformation (Jiang, Christian, Khatry, Wu, & West Jr, 2005). In essence, the monotonous Nepali food menu consists of more cereal items but food like red meat is limited in the daily supply of kitchen of many households. Phytate rich nutrients of cereals inhibit zinc absorption, particularly when phytate:zinc molar ratio (P:Z) is >15 in the consumed diet (Ram Lonnerdal, 2000). Poor dietary intake, inappropriate food supply, food insecurity, presence of more phytate and/or fiber in diets, and improper food preparation can cause zinc deficiency (Bruno De Benoist et al., 2007). National data on zinc deficiency based on population studies and associated predictors are lacking in Nepal. To address this gap, this study aimed to assess the national level prevalence of and factors associated with the zinc deficiency among children aged 6-59 months and non-pregnant women aged 15-49 years in Nepal.

Study participants
We used cross-sectional data from a nationally representative Nepal National Micronutrient Status Survey 2016(NNMSS-2016. A detail methodology has been presented elsewhere (Ministry of Health and Population et al., 2018). In brief, the NNMSS-2016 study was conducted to provide up to date status on the basic health and associated demographic statistics. Complete details about the study population, study area, and sampling techniques are published in the NNMSS report. Stratified multistage cluster sampling without replacement approach was applied in the study. Three geographical regions (Mountain, Hill, and Terai) and five development zones (Eastern, Central, Western, Mid-western, and Far western) were included in the study considering 180 clusters (75 from the Terai and Hill each, and 30 clusters from the Mountain) and 15 strata by systemic sampling where clusters were used as the primary sampling units (PSUs) (Ford et al., 2020). A total of 24 households were selected from each cluster (n = 4,320) using a systematic random sampling where 4,309 (99.7%) households have completed the interviews. Geometric mean zinc and prevalence of zinc deficiency were determined among 1,462children aged 6-59 months and 1,923 nonpregnant women aged 15-49 years. Serum zinc was corrected for inflammation in children 6-59 months but not the women. Zinc deficiency was defined using the cut off as below (kIZiNCG, 2007): For children 6-59 months: morning, non-fasting: <65 μg/dL or afternoon, non-fasting: <57 μg/dL. For non-pregnant women: morning, non-fasting: < 66 or afternoon, non-fasting<59 μg/dL depending on the time of day: morning (until noon), non-fasting: 66 μg/dL; afternoon, non-fasting: 59 μg/dL

Biological specimen collection and laboratory processes
Blood samples were collected to measure micronutrient status and inflammation markers. Trained phlebotomists and pathologists collected blood and stool samples from the participants at their houses. Blood samples were non-fasted samples as fasting was not possible in the survey design. Stool samples were collected by the survey staffs within 24 hrs of the interview. The collected stool samples were examined for Helicobacter pylori antigen detection using an enzyme-linked immunoassay (ELISA) test kit on a Mago clinical analyzer which provides both negative and positive controls in each analytical test. For the validity of each analytical test, positive and negative controls were used where the absorbance was at least 0.8 OD units and less than 0.09 OD units, respectively. Internal quality control of laboratory was strictly followed.
Serum zinc concentration was determined by atomic absorption spectrophotometry (AAS). A BioRad serum control with three levels of control materials was used for the AAS and samples were run in duplicate. Haemoglobin was measured by photometric method using Hemocue® Hb 301 analyser at the household on small blood samples . Following criteria was used for defining anaemia(WHO, 2011):Children 6-59 months: < 11.0 g/dL; Nonpregnant women 15-49 years: <12.0 g/dL.
The other marker of anaemia such as ferritin and soluble transferrin receptor (sTfR) was measured by ELISA. Vitamin A and retinol binding protein (RBP) were measured by HPLC and ELISA, respectively. RBC folate level was determined by a gold standard microbiological method. The markers of inflammation namely AGP and CRP were measured by ELISA. Body mass index (BMI) was calculated for the anthropometric measurement of children and women. The detailed methodology has been described in detail elsewhere (Ministry of Health and Population et al., 2018). Data on socio-demographic characteristics, participation in national nutrition and other intervention, recent micronutrient supplementation (zinc, iron, folic acid, vitamin A, multiple micronutrient supplementation or powders), two week recall of fever, cough and diarrhoea and other relevant information were collected by using structured questionnaire.

Statistical analysis
All analyses were performed using Stata 15. The reported values were weighted by sample weights in order to obtain the national estimates. Logistic regression was used to assess unadjusted and adjusted odds ratio (AOR) considering the sampling design where ecological zones were used as strata and 'wards' as a cluster. Only significant association observed in bivariate logistic regression were included in the multivariate model. P<0.05 was considered to be statistically significant.

Ethics statement
The ethical approval to conduct the survey was approved by the Ethical Review Board (ERB) of Nepal Health Research Council (NHRC) (Reg. No.: 201/2015). All participants gave informed consent before they were included in the study.

Results:
This study reports the data on micronutrient deficiencies in children (6-59 months) and nonpregnant women (15-49 years) from a national micronutrient survey of Nepal 2016. A total of 1462 (N=1462) children and 1923 (N=1923) non-pregnant women were sampled for the survey representing all geographical regions and socio-demographic groups.
The prevalence of zinc deficiency among the children was 22.9% (N=335). Besides zinc deficiency, other nutritional problems such as anaemia, stunting and underweight were also significantly prevalent in these children [  Table  2]. The location of residence (rural or urban) is a non-modifiable risk factor while diarrhoea possibly can be modifiable and manageable.  Among the non-pregnant women, the mean zinc concentration was 46.94 µg/dL in the deficient group, and the overall prevalence of zinc deficiency was 24.7% (N=497). Besides, anaemia and obesity were also one of the significant health problems among women [  Haemoglobin adjusted for altitude and smoking (WHO, 2017a). c Anaemia defined as altitude-and smoking-adjusted Hb <12.0 g/ dL (WHO, 2017a).  Note. Estimates are unadjusted odds ratios and adjusted odds ratios with 95% confidence intervals from logistic regression models, accounting for weighting and complex sampling design. a Folate cut off based on the risk of megaloblastic anaemia defined as RBC folate <305.0 nmol/ L (Institute of Medicine 1998).

Discussion:
This paper details the findings from the Nepal National Micronutrient Status Survey 2016 (NNMSS2016) focusing on zinc deficiency in pre-school children aged 6-59 months and nonpregnant women (NPW) aged 15-49 years. This is the first nationally representative data on the zinc status of Nepal. Our results suggest that zinc deficiency in Nepalese children and NPW is a significant nutritional problem. A few cross-sectional studies conducted among schoolage children, women of reproductive age, and pregnant women have also shown a high prevalence of zinc deficiency in Nepal (Ram K. Nepal et al., 2014;Tamang, Yadav, Acharya, & Lamsal, 2020). Zinc deficiency is not only a public health problem in developing countries like Nepal, but recent data from some developed countries such as Japan and New Zealand also report a higher burden of zinc deficiency (Daniels et al., 2018;Yasuda & Tsutsui, 2016). We didn't find nationally representative data from India on zinc deficiency, a neighbouring country of Nepal that share similar socio-cultural and food practices(Gonmei& Toteja, 2018). However, several studies from various regions of India report that prevalence of zinc deficiency were high (43.8%) in 6-60 months children and about 53% in non-pregnant women (Kapil & Jain, 2011;Menon et al., 2011).
The National Demographic and Health Surveys (NDHS) from the Government of Nepal provide the nationally representative data on micronutrient status in the Nepalese population(Ministry of Health and Population (MOHP) [Nepal], New ERA , & Inc., 2012; Ministry of Health Nepal, New ERA, & ICF, 2017) but these measures have not measured zinc status as one of the parameters in assessing infant and maternal nutrition. The prevalence of zinc deficiency in the current study was 22.7% in children and 24.7% in NPW. Zinc deficiency is considered a public health concern when the prevalence reaches 20% (de Benoist, .A community-based study in Bhaktapur district had observed >2/3 rd of the participating non-pregnant women (13-35years )were zinc deficient(Ram K. .Since there are no clinical trials aiming role of zinc supplementation in pregnancy and infant outcomes in Nepal, some cross-sectional community-based studies(Ram K. have reported higher burden of zinc deficiency across the country. Based on our findings, zinc deficiency is a public health concern in Nepal and thus, we suggest longitudinal studies or interventional studies targeting the risk groups of different ecological zones of the country. Studies have shown that inflammation influences nutritional markers including zinc concentration (Karakochuk et al., 2017), one study has reported 31% of Nepalese children of 6-8 years of age had high levels of α-acid glycoprotein (AGP) and C-reactive protein (CRP), prominent markers of inflammation (Schulze et al., 2014). Also, an unpublished data from a baseline survey in 2012 showed a high percentage of children (6-23 months of age) with subclinical inflammation(Ministry of Health and Population et al., 2018).Therefore, the micronutrient concentration of the included participants in this study has been adjusted based on the values of AGP and CRP, which will further minimise bias (Strand, Adhikari, Chandyo, Sharma, & Sommerfelt, 2004).
Our finding showed that children residing in rural areas had higher risk of zinc deficiency compared with those from urban areas. An Ethiopian study also reported higher odds of zinc deficiency in pregnant women residing in rural areas (Kumera et al., 2015). The location of residence (rural vs urban) is a non-modifiable predictor of zinc status. The increased risk of zinc deficiency in children residing in rural areas might reflect the limited consumption of food products from animal sources compared with those living in urban areas. Meat products are quite expensive, and poor people often cannot afford to buy them. Animal products such as meat and oysters are a good source of zinc (Ma & Betts, 2000) which are very limited and often hard to get in the kitchen of rustic communities in Nepal. Plant-based diets, the typical Nepalese staple foods contain a high amount of phytates which are potent inhibitors of zinc absorption in the intestines (Lönnerdal, 2000). A study from Africa showed significant association of serum zinc status with dietary diversity in pregnant women (Kumera et al., 2015). However, this study did not assess food consumption pattern in the participants, which could have further highlighted the contribution of dietary intakeof zinc.
The occurrence of diarrhoea in children during two weeks preceding the survey was associated with increased risk of zinc deficiency. A study conducted among 6-35 months Nepalese children has reported significant association of dysentery with zinc status (Strand et al., 2004). Diarrhoea or dysentery leads to loss of body fluids and zinc can be excreted in stool (Castillo-Duran, Vial, & Uauy, 1988). Studies have suggested zinc supplementation improves the gastrointestinal mucosal integrity and promotes immune system, thereby potentially reducing severity and duration of diarrhoea (Lazzerini & Wanzira, 2016). Based on our findings, nutritional awareness, improving the dietary pattern to include meat products, provision of health/medical services and livelihood programs along with nutritional counselling for the people living in rural areas might help to improve zinc status as well as overall food habit to improve nutritional status. The body mass index is a modifiable predictor of zinc status in the NPW. As our data show that underweight increases the odds of zinc deficiency, maintaining body weight, fitness and consumption of adequate nutrients might help prevent zinc deficiency in those groups of NPW.
The infection of H. pylori increased the likelihood of zinc deficiency as did the fever occurring during two weeks preceding the survey. H. pylori infection is more commonly discussed in the context of anaemia (Cardaropoli, Rolfo, & Todros, 2014;John, Baltodano, Mehta, Mark, & Murthy, 2018) and evidence linking association of H.pylori with zinc status is scarce. A study in dyspepsia patients showed an association of H. pylori-induced gastric inflammation with reduced zinc concentration in gastric tissues. H. pylori infection can induce increased reactive oxygen isotypes resulting in oxidative stress and zinc deficiency further exacerbates the inflammation (Sempértegui et al., 2007). There are evidences that H. pylori infection can transmit in the family through personal contact and proper hygiene maintenance may prevent its transmission and spread (Salih, 2009). Relationship between fever and zinc deficiency is less clear; however, a study found that dengue diagnosed in children with zinc deficiency had slightly longer duration of fever and hospital stay as compared to children with normal zinc level(Rerksuppaphol& Rerksuppaphol, 2019). Probably zinc level drop in blood during fever due to higher hepatic synthesis of zinc binding acute phase proteins including metallothioneins (Gammoh & Rink, 2017).
Our study observed that good economic status (rich vs poor) and risk of folate deficiency were protective factors for zinc deficiency in NPW. Economic status is a non-modifiable factor. However, through proper economic support and livelihood opportunities from the government, people with low economic condition can gradually increase their living standard. The growth of income will support them to buy nutritious food that may help to improve their overall nutrition uptake. Low folate levels as a protective factor for zinc deficiency in our study is quite intriguing as many studies suggest no significant interaction between folate and zinc at the context of intestinal zinc absorption (Butterworth et al., 1988;Hambidge, Hackshaw, & Wald, 1993;Tamura et al., 1992). Evidence of the interaction between folate and zinc is less clear (Hansen et al., 2001). Thus, future studies, preferably interventional in design, should investigate the role of folate in maintaining zinc status in NPW.
Low zinc levels in high proportion of 6-59 months children and NPW of 15-49 years in our study suggest that zinc deficiency is a public health concern in Nepal. Our study identified several modifiable predictors of zinc deficiency such as body mass index, diarrhoea and fever occurrence, and appropriate intervention as discussed above could improve the zinc as well as the overall nutritional status of children and women. Interventional studies with zinc supplements, particularly in vulnerable groups are warranted to verify the relationship between zinc status and its observed risk factors.

Strengths and limitation of the study
The NNMSS 2016 is a nationally representative sample addressing all ecological regions and socio-demographic groups, which is its biggest strength. As far as our knowledge, this study is the first study from Nepal using nationally representative data in order to assess the predictors of zinc deficiencies. This study has some limitations as well, perhaps the greatest limitation of our study is its cross-sectional design; therefore, we could not ascertain the causality between the predictors and zinc deficiency. We were also unable to determine the contribution of dietary zinc on serum zinc levels because of a lack of dietary information.

Conclusion
Our study suggests that zinc deficiency is a significant public health concern among NPW and children in Nepal, especially among children and NPW from rural communities with poor economic status. Community focussed interventional programs, awareness, counselling and sustainable livelihood policies may help to improve zinc status as well as overall health of the target population.