Introduction:

Malnutrition is still a major public health problem of staggering dimensions in developing countries including India. “Protein Energy Malnutrition” covers a wide spectrum of clinical stages ranging from the severe forms like kwashiorkor and Marasmus to the milder forms in which the main detectable manifestation is growth retardation. PEM is due to food gap between the intake and requirement (Bhatia, 2007).  This may lead to retardation in both physical growth and intellectual development in later years. In India, iron, iodine and vitamin A deficiency disorders have been major nutritional problems. Iron deficiency anaemia is a problem of serious public health significance, and has impact on psychological and physical development, behavior and work performance. In India the prevalence of anaemia in pregnant women (haemoglobin<11g%) of different parts of the country was in the range of 33 to 89% (Awasthi, 2003). Vitamin A deficiency, especially among preschool children is still a significant public health problem in certain pockets of the country (Arlappa,2011). Vitamin ‘A’ deficiency affects many tissues in the body; the most dramatic changes are seen in the eyes resulting in tragic consequences of total loss of vision in early life. Iodine is an essential micronutrient for normal growth and development in animals and humans. Its deficiency not only causes goiter but is also responsible for impaired brain development in the fetus and infant and retarded physical and psychomotor development in the child (WHO/UNICEF/ICCIDD, 1995). The deficiency of iodine also impairs children’s learning ability. Iodine deficiency is the most common cause of preventable mental retardation in the world today (Hess, 2009).

Aim of the Study:

The proposed study is aimed to carry out in order to learn about the epidemiology of Protein Energy Malnutrition (PEM) and micronutrient deficiencies especially with reference to Iodine deficiency disorders of the children of Allahabad district. Detailed analysis of salt consumption amount, pattern and storage practices would help to identify the main lacunae which are responsible for the low iodine bioavailability in amongst the household. Urinary Iodine excretion would give a detail picture of the individual’s iodine status which would be associated to anthropometric and other biochemical and clinical indices. It would also help in gauging and forecasting the future possibilities of developing thyroid related disorders and hence we could promulgate sound guidelines and promotional approaches so that on the basis of proper salt consumption and storage practices we could mitigate the problem of Iodine deficiency disorders.

OBJECTIVES

·        To assess the nutritional status of the children (4-12 years) and identify the prevalence of PEM.

·        To assess the prevalence of anaemia.

·        To find out the prevalence of clinical signs and symptoms of common micronutrient deficiencies.

·        To assess the dietary adequacy of micronutrients and correlate it with biochemical indices.

Methodology:

·         Selection of the Sample and Sample size:

Sample size would be n≥425, consisting children of age group 4 to 12 years of age of Allahabad district.  For a survey design based on a simple random sample, the sample size required can be calculated according to the following formula. (FAO 1990, UNICEF 1995)

                                          n =  t²x p (1- p)  ;  N= n x D

                                                       m²

n = required sample size

 t = confidence level at 95% (standard value of 1.96)

p = estimated prevalence of PEM in the project area (42.5% in this case as reported)

m = margin of error at 10% (10% of the Prevalence rate i.e 42.5% which would be = 4.25)

D= Design effect (taken as 1 in this case)

n=3.8416 x 42.5 x 57.5= 9388 = 519

                                                                (4.25) ²               18.06

To, N 10% Contingency has to be added, 0.10x519=51.9

Final Sample Size: N= 519+52 rounded off to 571

As two-third of the district’s population resides in the rural areas (census, 2011) thus the distribution ratio of the samples will also follow this trend. Thus, out of the total sample size two-third of the sample would be drawn from the rural and one fourth of the sample would be drawn from the urban sector.

Out of the twenty blocks(i.e Phulpur  Bahadurpur Pratappur Saidabad, Handia, Shankargarh, Chaka,  Karchhana, Uruwa , Meja, Kaurihar ,  Holagarh , Koraon,   Dhanupur,  Kaundhiyara       Manda), four blocks were selected randomly in order to collect the samples from theses blocks.

Methodology

                               I.          Diet survey (Swaminathan, 2002) will be conducted.

Survey Schedule:

·         It would be used to collect the general information, anthropometric status and dietary in take and details of the clinical and biochemical indices will also be recorded in it. The data on socio-demographic profile, educational status of mothers (of Children), family type, standard of living, religion and caste of the children was collected through a pretested structured questionnaire. The criteria for classifying the families in to different socio economic groups were based on the physical assets available in the household. The criterion utilized by National Family Health surveys conducted in the country was used.

• A 3 day dietary recall would be taken and it would be averaged out for one day.

·         Food Frequency Questionnaire (FFQ) will be utilized to find out the consumption of food items of various food groups. This method obtains retrospective information on the pattern of food consumption of a defined period in past on usual intakes of the food items categorized in major food groups, like cereals/grains (wheat and rice), pulses and legumes, green leafy vegetables, roots and tubers, other vegetables, fruits, milk, milk products, eggs, flesh foods, nuts and oil seeds, Fats and oils, Sugar/jaggery. The common food items consumed by a child were listed to mother to facilitate her to comprehend and recall the food items consumed by her child. The mother of the study subject was inquired about the frequency of food items of the specific food group consumed. The frequency of consumption was assessed under four categories (i) Number of days per week (1 to7 days) (ii) Once per 15 days, (iii) Once per month, and (iv) Never.

·         The dietary intake of micronutrients would be calculated using the Food Composition Tables (Gopalan, 2002) to finally compute the intakes of macro and micronutrients. The calculated values of Macronutrients (Energy, Protein, Fat and Water) and Micronutrients (Vitamins and Minerals – Iron and Zinc) would be compared to the RDA.

·         Type of Salt consumed; Salt Consumption amount per head and the details of salt storage And usage practices will also be recorded.

                            II.            Anthropometric measurements will be taken for the assessment of nutritional status by the health indicators for which reference would be used is (WHO child Growth Standards, 2006/ NCHS 2006) :-

·         Body weight (kg): Weight of the child will be taken with minimal clothing using a lever balance. Measurement will be taken to the nearest 100 g value.

·         Height (cms): Standing height of a child will be measured with anthropometry rod taken to nearest millimeter (mm). The child will be made to stand erect with heels touching together without any foot wear.  

·         Mid-arm circumference for age (cm.): It will be measured with a fiber non stretchable tape at the mid point of the acromian and olecranon on one side to the nearest mm.

Table No. 1: SD classification of malnutrition by WHO

The nutritional status of children will be calculated according to above mentioned  three measures is compared with the nutritional status of an international reference population recommended by the World Health Organization (WHO child Growth Standards, 2006/ NCHS 2006/ Dibley etal, 1987). The use of this reference population is based on the empirical finding that well-nourished children in all population groups for which data exist follow very similar growth patterns. A scientific report from the Nutrition

Foundation of India (Agarwal et al., 1991) has concluded that the WHO standard is generally applicable to Indian children.

Analytical and Biochemical Tests:

1. Quantitative estimation of Salt’s iodine content: (ICCIDD, 1995)

Reagent preparation: The preferred water for this method should be boiled distilled water, which requires provision of a distillation unit. As a simpler alternative, regular tap water treated with a mixed bed deionizing resin can be used, thus avoiding the need for an expensive distillation unit. 0.005 M Sodium thiosulfate (Na2 S2 03 ): Dissolve 1.24 g Na2 S2 03 5H2 0 in 1000 ml water. Store in a cool, dark place. This volume is sufficient for 100-200 samples, depending on their iodine content. The solution is stable for at least one month, if stored properly. 2 N Sulfuric acid (H₂ S0₄): Slowly add 6 ml concentrated H2 S04 to 90 ml water. Make to 100 ml with water. This volume is sufficient for 100 samples. The solution is stable indefinitely. Always add acid to water, not water to acid, to avoid excess heat formation and spitting of acid. Stir solution while adding acid. 10% Potassium iodide (KI): Dissolve 100 g KI in 1000 ml water. Store in a cool, dark place. This volume is sufficient for 200 samples. Properly stored the solution is stable for six months, provided no change occurs in the colour of the solution. Starch indicator solution: Dissolve reagent-grade sodium chloride (NaCl) in 100 ml double-distilled water. While stirring, add NaCl until no more dissolves. Heat the contents of the beaker until excess salt dissolves. While cooling, the NaCl crystals will form on the sides of the beaker. When it is completely cooled, decant the supernatant into a clean bottle. This solution is stable for six to twelve months. Dissolve 1 g chemical starch in 10 ml double-distilled water. Continue to boil until it completely dissolves. Add the saturated NaCl solution to make 100 ml starch solution. This volume is sufficient for testing 20 to 45 samples. Prepare fresh starch solution every day, since starch solution cannot be stored.

2. Quantitative estimation of hemoglobin:

Cyanmethemoglobin method. (Bhaskaran, 2003)

The (filter paper) method of choice for hemoglobin determination is the cyanmethemoglobin method (This is a type of colorimetric method).  The principle of this method is that when blood is mixed with a solution containing potassium ferricyanide and potassium cyanide, the potassium ferricyanide oxidizes iron to form methemoglobin.  The potassium cyanide then combines with methemoglobin to form cyanmethemoglobin, which is a stable color pigment read photometrically at a wave length of 540nm. 5 ml of Cyanmethemoglobin reagent will be pipetted into each tube.  20 ml of the appropriate sample would be added into each tube. Tubes then have to be allowed to stand for 10 minutes. Absorbance (A) has to be noted in the spectrophotometer at 540 nm, zeroing the spectrophotometer with the BLANK solution. A graph will be plotted Absorbance vs.  Hemoglobin concentration in grams % on linear graph paper.

3. Quantitative estimation of urinary iodine excretion: (ICCIDD, 1993)

Principle: Urine is digested with ammonium persulfate. Iodide is the catalyst in the reduction of ceric ammonium sulfate (yellow) to cerous form (colourless), and is detected by rate of colour disappearance (Sandell-Kolthoff reaction). Equipment Heating block (vented fume hood not necessary), colorimeter, thermometer, test tubes (13 x 100 mm), reagent flasks and bottles, pipettes, balance scales.

Reagents

1. Ammonium persulfate (analytical grade)

2. As₂O₃

 3. NaCl

4. H₂ SO₄

5. Ce(NH4)₄ (SO₄)₄ . 2H₂O

6. Deionized H₂ O

7. KIO₃ Solutions

1.0 M Ammonium persulfate: Dissolve 114.1 g H₂ N₂ O₈ S₂ in H₂O; make up to 500 ml with H2 O. Store away from light. Stable for at least one month. 5 N H₂ SO₄ : Slowly add 139 ml concentrated (36 N) H₂ SO₄ to about 700 ml deionized water . When cool, adjust with deionized water to a final volume of 1 litre. Arsenious acid solution: In a 2000 ml Erlenmeyer flask, place 20 g As₂ O₃ and 50 g NaCl, then slowly add 400 ml 5 N H₂SO₄ . Add water to about 1 litre, heat gently to dissolve, cool to room temperature, dilute with water to 2 litres, filter, store in a dark bottle away from light at room temperature. The solution is stable for months.

Ceric ammonium sulfate solution: Dissolve 48 g ceric ammonium sulfate in 1 litre 3.5 N H2 SO4 . (The 3.5 N H₂SO4 is made by slowly adding 97 ml concentrated (36 N) H₂ SO₄ to about 800 ml deionized water (careful - this generates heat!), and when cool, adjusting with deionized water to a final volume of 1 litre). Store in a dark bottle away from light at room temperature. The solution is stable for months. Standard iodine solution, 1 µg iodine/ml (7.9 µmol/l): Dissolve 0.168 mg KIO₃ in deionized water to a final volume of 100 ml (1.68 mg KIO₃ contains 1.0 mg iodine; KIO₃ is preferred over KI because it is more stable, but KI has been used by some laboratories without apparent problems). It may be more convenient to make a more concentrated solution, e.g., 10 or 100 mg iodine/ml, then dilute to 1 µg/ml. Store in a dark bottle. The solution is stable for months. Useful standards are 20, 50, 100, 150, 200, and 300 µg/l.

Procedure

1. Mix urine to suspend sediment.

2. Pipette 250 µl of each urine sample into a 13 x 100 mm test tube. Pipette each iodine standard into a test tube, and then add H₂O as needed to make a final volume of 250 µl. Duplicate iodine standards and a set of internal urine standards should be included in each assay.

 3. Add 1 ml 1.0 M ammonium persulfate to each tube.

4. Heat all tubes for 60 minutes at 100^o C.

5. Cool tubes to room temperature.

6. Add 2.5 ml arsenious acid solution. Mix by inversion or vortex. Let stand for 15 minutes.

7. Add 300 µl of ceric ammonium sulfate solution to each tube (quickly mixing) at 15-30 second intervals between successive tubes. A stopwatch should be used for this. With practice, a 15 second interval is convenient.

 8. Allow to sit at room temperature. Exactly 30 minutes after addition of ceric ammonium sulfate to the first tube, read its absorbance at 420 nm.

 Read successive tubes at the same interval as when adding the cerric ammonium sulfate. Calculation of results

Construct a standard curve on graph paper by plotting iodine concentration of each standard on the abscissa against its optical density at 405 µg/l (OD405) on the ordinate.

Note: All the test reports will be made available to the parents on demand, whenever required.

Quality Control:

The analysis of salt’s iodine content, haemoglobin and urinary iodine will be done by trained senior analysts at NABL accreditated Food Analysis and Research Laboratory, Centre of Food Technology, University of Allahabad.

Disposal of Bio-hazardous wastes: 

At the end of each blood collection and haemoglobin measurement, all materials used during the testing (gloves, lancets, alcohol swabs, and gauze pads have to be placed in sharps container (a wide-mouth plastic jar) and kept there until the end of the working day. The following are the steps that would be followed in disposing of bio- hazardous materials. First, a health investigator needs to determine a place where the waste disposal will be destroyed. An open field area with loose soil is preferable, since the materials need to be burnt and buried. Because of risk of fire, drought areas, as well as proximity to flammable materials, should be avoided.

1)      At the end of each working day, bring the sharps container (plastic jar) with bio- hazardous materials to the area selected for the waste disposal. Add a half liter of 4 percent sodium hypochlorite solution into the sharps container (plastic jar) with the bio- hazardous materials .After adding, close the container (jar) so it is airtight. The jar would be kept in an upright position for five minutes. After that, the plastic jar would be inverted and kept in that position for an additional five minutes. This step is necessary to ensure that all of the materials in the sharps container (plastic jar) are disinfected by complete immersion in the 4 percent sodium hypochlorite solution.

2)      The contents of the plastic jar, including the sodium hypochlorite solution would be transferred to a thick polyethylene bag.

3)      A forceps can be used if any material adheres or sticks to the walls of the plastic jar to transfer it to the polyethylene bag.

4)      With the help of Scissors, a hole will be made at the bottom of the polyethylene bag.

5)      The hypochlorite solution will be drained off from the polyethylene bag.

6)      A small hole will be dug with a spade to put the polyethylene bag containing the bio- hazardous materials in the pit.

7)      The waste paper will be placed on the polyethylene bag containing bio- hazardous.

8)      Some kerosene would be poured on the bag.

9)      Burn the polyethylene bag containing the bio hazardous materials in the pit.

10)  After all of the contents are burned the pit will be covered with soil. It is the health investigator’s responsibility to ensure proper disposal of bio hazardous waste.

 It is unacceptable that the materials used during the testing in one fieldwork cluster are carried by the team to the next cluster. Bio hazardous materials must be destroyed within 48 hours.

Note: Our department Centre of Food Technology runs a NABL accreditated Food Analysis and Research Laboratory. The Ferro (waste collector) is appointed for this purpose and takes away the waste from the centre.

   Statistical Analysis: 

The obtained data would be entered in MS-Office 2007 Excel Worksheet and statistical analysis will be done using SPSS-12.0. For calculations of dietary and nutrient intake software DIETCAL will be used addition to the food tables. After categorization of data, the descriptive statistics (frequency, distribution and percentages) will be calculated.