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Article
Prevalence of Vitamin B12 and Folate Deficiencies in
Indian Children and Adolescents
Tattari Shalini1, Raghu Pullakhandam1, Santu Ghosh2, Bharati Kulkarni1, Hemalatha Rajkumar1,
Harshpal S. Sachdev3, Anura V Kurpad2, G Bhanuprakash Reddy1*
1National Institute of Nutrition, Hyderabad, India
2St John’s Medical College, Bangalore, India
3Sitaram Bhartia Institute of Science and Research, New Delhi, India
*To whom correspondence should be addressed: Dr. G. Bhanuprakash Reddy, National Institute of Nutrition,
Jamai-Osmania, Tarnaka, Hyderabad - 500007, India. Tel: 91-40-27197252; Email: geereddy@yahoo.com;
reddyg.bp@icmr.gov.in.
Abstract: Deficiencies of vitamin B12 (B12) and folate (FA) are of particular interest due to their
pleiotropic role in 1-carbon metabolism. In addition to adverse birth outcomes, deficiencies of B12 and
FA, or an imbalance in FA/B12 status, are linked to metabolic disorders. Indian diets that are
predominantly plant food-based could be deficient in these vitamins, but there are no national
estimates of the prevalence of B12 and FA deficiency in Indian children and adolescents, nor of their
associations with age, sex and growth indicators. The recent Comprehensive National Nutrition
Survey (CNNS-2016-18) provided estimates of the prevalence of B12 and FA deficiency at the national
and state level among preschool (1-4y: 9,976 and 11,004 children respectively), school-age children (5-
9y: 12,156 and 14,125) and adolescents (10-19y: 11,748 and 13,621). Serum B12 and erythrocyte FA were
measured by the direct chemiluminescence method and their deficiency was defined using WHO cut-
offs. The prevalence of B12 and FA deficiency was high among adolescents (31.0%, CI: 28.7-33.5 and
35.6%, CI: 33.1-8.2), compared to school-age (17.3%, CI: 15.4-19.3 and 27.6%, CI: 25.5-29.9) and
preschool children (13.8%, CI: 11.7-16.2 and 22.8%, CI: 20.5-25.2, respectively). The prevalence of both
B12 and FA deficiency was significantly higher by 8 and 5% points respectively, in adolescent boys
compared to girls. The prevalence of B12 deficiency was higher in moderately stunted school-children
(by 18.9% points) than in normal children, but no such difference was observed for FA deficiency.
There was wide regional variation in the prevalence of B12 and FA deficiency, but no rural-urban
differences were observed across all age groups. The national prevalence of B12 deficiency among
preschool or school-age children was <20% (the cut-off that indicates a public health problem).
However, FA deficiency in these age groups, and both FA and B12 deficiencies in adolescents were
>20%, which warrants further investigation.
Keywords: Vitamin B12 deficiency; Folate deficiency; School-age children; Adolescents; CNNS
1. Introduction
Vitamin B12 (B12) and folate (FA), are critical micronutrients, required in a plethora of metabolic
and biological functions [1]. One central pathway is the methyl transfer reaction in the methionine cycle,
which converts homocysteine (Hcy) to methionine. Folate is engaged in many methylation reactions
covering DNA, proteins, phospholipids and neurotransmitter metabolism [2], while B12 and FA have
overlapping biological functions in DNA synthesis and the development of red blood cells (RBC) and
the myelin sheath, that are essential for normal growth and development [3]. B12 is only found in animal
source foods such as meat, poultry, fish and dairy products, while folate is abundant in both animal
and plant foods.
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B12 and FA deficiencies occur during the lifecycle, with different outcomes. During pregnancy,
they are associated with an increased risk of adverse outcomes such as neural tube defects and low
birth weight, intrauterine growth retardation, miscarriage and pre-eclampsia [4-6]. In children, B12 and
FA deficiency can result in megaloblastic anemia, poor growth and stunting, increased risk of
infections, cognitive dysfunction, neurologic damage and brain atrophy in severe cases [1-3,7]. There is
also a high prevalence of the double burden of malnutrition, where multiple biomarkers of
cardiovascular disorders (CVD) are elevated even in undernourished children and adolescents [8]. B12
and FA deficiencies are associated with hyperhomocysteinemia, which is a CVD risk factor [9-11] and
during pregnancy, an imbalance in FA/B12 status has been associated with adverse birth outcomes [4],
and adiposity and insulin resistance in the offspring [12]. Although a high of prevalence of B12 (27% -
67.2%) and FA (12% - 42%) deficiencies have been reported in India [10,11,13-16], the studies were not
nationally representative.
The Indian Comprehensive National Nutrition Survey (CNNS) was conducted during 2016-2018,
and evaluated the anthropometry, along with serum B12 and erythrocyte FA concentrations, among 1–
19-year children and adolescents across all the Indian geographic states. This offered an opportunity to
quantify the prevalence of B12 and FA deficiency at a national and state-level in children and
adolescents, stratified by age and gender. We also evaluated the association of B12 and FA deficiency
with demographic and socioeconomic variables, as well as reported morbidity, and anemia prevalence.
2. Methodology
2.1. CNNS survey, serum B12 and erythrocyte FA analysis
The CNNS was a community-based cross-sectional survey conducted among Indian children and
adolescents in 29 states and union territory of Delhi, during February 2016 to October 2018 in
collaboration with UNICEF, India and Population Council, under the supervision of the Ministry of
Health and Family Welfare, Government of India. The methodological details of the survey are
available in the CNNS report [17]. Briefly, CNNS used a multi-stage stratified, probability proportional
to size (PPS) cluster sampling to enrol pre-school children (1–4y), school-age children (5–9y) and
adolescents (10–19y) to adequately represent the national, state, male-female and urban-rural
population. For biological sampling, 50% of all the children who completed anthropometry were
selected by systematic random sampling. Children/adolescents with physical deformity, cognitive
disabilities, chronic illness, acute febrile/infectious illness, acute injury and pregnancy were excluded.
Ethical approval was obtained from the Institutional Review Boards of Population Council, New York,
USA and the Post Graduate Institute of Medical Education and Research, Chandigarh, India [17].
Informed consent from the parent/caregiver of children under 10 years, informed consent of
parent/caregiver of adolescents (11-17y) as well as the latter’s assent, and informed consent of
adolescents above 17y were obtained. All procedures and methods were performed in accordance with
the Declaration of Helsinki.
Household socioeconomic and demographic characteristics, information on history of morbidity
in the preceding two weeks and iron-folic acid (IFA) supplementation in the previous week, and
anthropometric data of one child/adolescent per age group were collected from each household. Wealth
index based on possession of common household items and facilities was computed as described in
National Family Health Survey (NFHS)-4 [18]. Access to facilities like drinking water, hand washing
and sanitation was categorized based on WHO/UNICEF Joint Monitoring Programme for Water
Supply, Sanitation and Hygiene (WASH) guidelines [19]. Age-sex standardized height-for-age (HAZ),
weight-for-height (WHZ), and BMI-for-age Z-scores were calculated using the WHO Growth Reference
Standards [20].
The day before sample collection, parents and children were instructed to ensure overnight fasting
(8–10h) in the latter. Venous blood samples with recording of binary (yes/no) information on fasting
status and time of sample collection were obtained by trained phlebotomists. The blood samples were
transported in cool bags (3L-12H-08P, PronGo) to the nearest collection centre, where the serum/plasma
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and erythrocytes were separated and divided into aliquots, within 6h of sample collection. Biochemical
analyses were carried out by SRL Labs in Mumbai, Gurugram and Kolkata, India, and are reported in
detail elsewhere [21]. Briefly, serum B12 and erythrocyte FA levels were estimated using a
chemiluminescence based competitive immunoassay (Siemens Centaur) [17]. Hemoglobin was
estimated in whole blood by the cyanmethemoglobin method (Beckman Coulter, LH 750). Rigorous
quality control procedures were implemented for sample collection, transportation and testing using
standard internal and external quality assurance procedures [17,21].
Using WHO guidelines, B12 deficiency was defined as serum B12<203 pg/ml and FA deficiency as
erythroyte FA<151 ng/ml for all age-groups [22]. Anemia was diagnosed using WHO Hb cut-offs
(g/dL): <11.0 (1–4 y), <11.5 (5–11 y), <12.0 (12–14 y), <12.0 (15–19 y, girls), and <13.0 (15–19 y, boys) [23].
According to the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia
(BRINDA) report, B12 and FA concentrations do not require adjustment for inflammation [24], and
hence, no adjustment was performed in the present study.
A total of 1,05,243 children and adolescents (preschool: 31,058, school-age: 38,355 and adolescents:
35,830) were interviewed and anthropometric data collected, of which serum B12 and erythrocyte FA
concentrations were available for 33,880 and 38750 children and adolescents (preschool: 9,976 and
11,004, school-age: 12,156 and 14,125 and adolescents: 11,748 and 13,621, respectively) (Figure 1). The
socio-demographic characteristics were almost similar among participants in whom anthropometric
data were collected (total sample) and the study sample (B12 and FA), except that proportion of children
included in the study sample was higher in 3-4 and 7-9y compared to 1-2 and 5-6y (61% vs 39%)
respectively (Table S1). Table 1 shows the age-specific general characteristics of the study population.
Among preschool children, 35% were stunted and underweight, 16% were wasted, and about 15% had
diarrhoea two weeks prior to the survey in both B12 and FA study sample.
2.2. Statistical analyses
Statistical analyses were conducted using SPSS statistical package (version 23, SPSS Inc., Chicago,
IL, USA). The proportion of demographic characteristics of the study sample included in the present
analysis were compared with the proportion in the entire CNNS survey sample to rule out selection
bias due to nested sampling. Serum B12 and erythrocyte FA concentrations are presented as geometric
mean (GM) and geometric standard deviation (GSD), since their distributions were skewed. Relevant
sampling weights were used wherever indicated in order to ensure representativeness of the estimates
at the national/ state level as well as at the local level, such as rural, urban and urban slum areas in
metropolitan cities. The prevalence of B12 and FA deficiency, along with 95% confidence intervals (CI),
was estimated at the national as well as state level. Sub-group analyses were also performed to evaluate
urban-rural, age, gender, socio-demographic, and WASH differentials. The association between the
prevalence of B12 and FA deficiency with age groups in different states was evaluated using the
Spearman rank-order correlation.
3. Results
3.1. Serum B12 and erythrocyte FA concentration and prevalence of B12 and FA deficiency by age and sex
The GM of serum B12 (pg/mL) and erythrocyte FA (ng/mL) concentration was significantly
different among pre-schoolers, school-age children and adolescents (Table 2A & 2B). The national
prevalence of B12 and FA deficiency was higher among adolescents (31.0%, CI: 28.7-33.5 and 35.6%, CI:
33.1-8.2), compared to school-age (17.3%, CI: 15.4-19.3 and 27.6%, CI: 25.5-29.9) and preschool children
(13.8%, CI: 11.7-16.2 and 22.8%, CI: 20.5-25.2, respectively) (Table 2A & 2B). Though B12 and FA
concentrations tended to decline with age (1-19 years) in both genders, the decline was significantly
greater in adolescent boys compared to girls (Table 2A & 2B). As a consequence, the prevalence of B12
and FA deficiency increased with age (Figure 2), and adolescent boys had 8% points and 5% points
higher B12 and FA deficiency compared to girls (Table 2A & 2B).
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Table 1. Characteristics of the study population.
Characteristics
1-4 years
5-9 years
10-19 years
Vitamin B12
(n=9,976)
% (95% CI)
Folate
(n=11,004)
% (95% CI)
Vitamin B12
(n=12,156)
% (95% CI)
Folate
(n=14,125)
% (95% CI)
Vitamin B12
(n=11,748)
% (95% CI)
Folate
(n=13,621)
% (95% CI)
Age in years
Mean (95% CI)
2.78 (2.74-2.83)
2.78 (2.74-2.83)
7.02 (6.97-7.06)
7.02 (6.98-7.07)
14.3 (14.2-14.4)
14.3 (14.2-14.4)
Sex
Boys
52.9 (50.3-55.4)
52.5 (50.0-55.0)
51.6 (49.8-53.3)
51.2 (49.4-52.9)
50.6 (48.6-52.5)
50.6 (48.8-52.4)
Girls
47.1 (44.6-49.7)
47.5 (45.0-50.0)
48.4 (46.7-50.2)
48.8 (47.1-50.6)
49.4 (47.5-51.4)
49.4 (47.6-51.2)
Residence
Urban
24.9 (21.7-28.4)
25.7 (22.5-29.1)
23.6 (20.7-26.8)
25.1 (22.1-28.2)
25.2 (22.1-28.6)
25.7 (22.6-29.0)
Rural
75.1 (71.6-78.3)
74.3 (70.9-77.5)
76.4 (73.2-79.3)
74.9 (71.8-77.9)
74.8 (71.4-77.9)
74.3 (71.0-77.4)
Wealth Index
Poorest
15.9 (13.8-18.3)
15.8 (13.7-18.1)
18.1 (16.2-20.3)
18.0 (15.8-20.4)
17.8 (15.5-20.4)
18.3 (16.0-20.8)
Poor
21.2 (18.5-24.1)
20.5 (17.9-23.4)
21.3 (19.4-23.3)
20.2 (18.5-21.9)
20.5 (18.8-22.3)
20.6 (18.9-22.3)
Middle
22.3 (20.4-24.3)
21.7 (19.9-23.5)
21.5 (19.9-23.3)
21.0 (19.4-22.5)
21.4 (19.8-23.1)
20.3 (18.8-21.9)
Rich
20.8 (18.9-22.9)
21.7 (19.7-23.9)
21.0 (19.4-22.7)
21.6 (19.9-23.3)
20.9 (19.3-22.7)
21.0 (19.4-22.8)
Richest
19.8 (17.7-22.0)
20.3 (18.3-22.5)
18.0 (16.4-19.8)
19.3 (17.6-21.1)
19.3 (17.5-21.2)
19.8 (17.9-21.8)
Mother’s Schooling
Primary
34.3 (31.7-36.9)
34.6 (31.9-37.3)
47.8 (45.4-50.3)
47.9 (45.6-50.3)
16.3 (14.3-18.4)
15.9 (14.1-18.0)
Secondary
44.3 (41.8-46.8)
43.6 (41.3-46.0)
40.1 (38.0-42.3)
39.8 (37.8-41.8)
68.7 (66.2-71.1)
69.3 (67.0-71.5)
Higher Secondary 10.8 (9.3-12.5) 11.4 (9.9-13.1) 6.7 (6.0-7.6) 6.9 (6.2-7.8) 9.7 (7.7-12.1) 9.5 (7.7-11.6)
Graduation and above
10.7 (9.2-12.4)
10.4 (9.0-12.0)
5.3 (4.7-6.1)
5.4 (4.8-6.1)
5.3 (4.4-6.3)
5.3 (4.5-6.3)
Father’s Occupation
Professional
7.9 (6.8-9.2)
8.5 (7.4-9.9)
9.4 (8.1-10.8)
9.5 (8.3-10.8)
10.0 (8.5-11.7)
9.4 (8.0-10.9)
Sales and services
26.8 (24.6-29.1)
28.0 (25.7-30.4)
23.1 (21.4-25.0)
24.2 (22.3-26.1)
24.4 (22.7-26.2)
24.1 (22.5-25.8)
Manual, Agriculture 51.5 (48.8-54.1) 50.4 (47.6-53.2) 54.8 (52.2-57.4) 53.8 (51.4-56.2) 51.3 (48.8-53.7) 52.4 (50.0-54.7)
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Others
13.8 (11.8-16.0)
13.1 (11.3-15.1)
12.7 (11.1-14.5)
12.5 (11.0-14.2)
14.3 (12.5-16.4)
14.2 (12.4-16.1)
Child schooling Yes - - 92.2 (91.1-93.2) 92.1 (91.1-93.0) 80.7 (78.9-82.4) 80.9 (79.2-82.5)
No
-
-
7.8 (6.8-8.9)
7.9 (7.0-8.9)
19.3 (17.6-21.1)
19.1 (17.5-20.8)
Stunting
No Stunting (HAZ < -2SD)
64.4 (62.1-66.7)
64.5 (62.3-66.7)
79.3 (77.7-80.8)
78.6 (76.9-80.3)
73.0 (71.0-74.9)
73.4 (71.6-75.2)
Moderate (HAZ: -3 to -
2SD)
24.0 (22.2-26.0)
23.3 (21.6-25.0)
15.5 (14.3-16.8)
16.4 (15.1-17.8)
21.3 (19.6-23.1)
20.7 (19.2-22.3)
Severe (HAZ < -3SD)
11.5 (10.1-13.2)
12.2 (10.7-13.9)
5.1 (4.3-6.1)
5.0 (4.2-5.8)
5.7 (4.9-6.6)
5.9 (5.1-6.7)
Underweight
Not present (WAZ < -2SD)
64.8 (62.0-67.6)
65.1 (62.5-67.6)
-
-
-
-
Moderate (WAZ:-3 to -
2SD)
26.2 (23.7-28.8)
26.7 (24.3-29.2)
-
-
-
-
Severe (WAZ < -3SD)
9.0 (7.8-10.3)
8.2 (7.2-9.4)
-
-
-
-
Wasting/
Thinness
Not present (WHZ < -
2SD)
84.0 (82.1-85.6)
85.0 (83.5-86.5)
76.4 (74.8-78.0)
76.6 (75.2-78.0)
75.5 (73.7-77.2)
75.7 (74.0-77.2)
Moderate (WHZ:-3 to -
2SD)
12.2 (10.8-13.8)
11.6 (10.4-12.9)
18.5 (17.1-19.9)
18.4 (17.1-19.8)
18.1 (16.6-19.7)
17.9 (16.6-19.4)
Severe (WHZ < -3SD)
3.8 (3.0-4.8)
3.4 (2.8-4.1)
5.1 (4.3-6.0)
5.0 (4.3-5.8)
6.4 (5.7-7.3)
6.4 (5.6-7.3)
Drinking water source
Piped & Improved
85.0 (82.4-87.3)
85.2 (82.6-87.5)
84.9 (82.5-87.0)
85.2 (82.9-87.2)
85.6 (83.5-87.5)
86.2 (84.2-87.9)
Non-piped & Improved
8.9 (7.0-11.2)
9.2 (7.4-11.3)
8.2 (6.6-10.2)
8.1 (6.7-9.9)
8.1 (6.8-9.6)
7.7 (6.5-9.1)
Unimproved 6.1 (4.8-7.6) 5.6 (4.3-7.4) 7.0 (5.7-8.4) 6.7 (5.3-8.4) 6.3 (5.1-7.9) 6.1 (5.0-7.6)
Handwashing
Basic
50.3 (47.5-53.1)
52.4 (49.7-55.2)
46.8 (44.1-49.5)
49.3 (46.7-51.9)
47.8 (45.3-50.3)
48.7 (46.2-51.3)
Limited
36.1 (33.4-38.8)
33.8 (31.3-36.5)
39.4 (36.6-42.3)
37.2 (34.6-39.8)
35.5 (33.1-38.0)
34.7 (32.5-37.1)
No facility
13.6 (11.7-15.9)
13.7 (11.8-15.9)
13.8 (12.1-15.8)
13.5 (11.8-15.4)
16.7 (14.7-19.0)
16.5 (14.4-18.8)
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Sanitation
Improved & Not shared
43.8 (40.5-47.2)
44.1 (41.0-47.1)
39.7 (37.2-42.3)
40.5 (38.0-43.0)
47.8 (45.3-50.4)
47.1 (44.5-49.7)
Improved & Shared 12.2 (10.8-13.7) 13.0 (11.6-14.6) 12.3 (11.0-13.8) 12.3 (11.0-13.8) 8.7 (7.7-9.8) 9.0 (8.0-10.1)
Unimproved
44.0 (40.1-48.0)
43.0 (39.3-46.7)
48.0 (44.8-51.2)
47.2 (44.1-50.4)
43.5 (40.6-46.4)
43.9 (41.1-46.8)
History of diarrhoea in
the two weeks prior to
survey
Yes
15.0 (12.9-17.5)
15.3 (13.3-17.6)
9.4 (8.2-10.7)
9.0 (8.0-10.2)
-
-
No
85.0 (82.5-87.1)
84.7 (82.4-86.7)
90.6 (89.3-91.8)
91.0 (89.8-92.0)
-
-
History of fever in the
two weeks prior to
survey
Yes
30.7 (28.3-33.2)
31.5 (29.2-33.9)
21.8 (19.8-24.0)
22.2 (20.5-24.0)
-
-
No
69.3 (66.8-71.7)
68.5 (66.1-70.8)
78.2 (76.0-80.2)
77.8 (76.0-79.5)
-
-
HAZ; height-for-age, WHZ; weight-for-height, WAZ; weight-for-age.
Table 2A. Serum vitamin B12 levels and prevalence of vitamin B12 deficiency among children and adolescents stratified based on sex and age groups.
Sex
1-4 years (n=9,976)
5-9 years (n=12,156)
10-19 years (n=11,748)
Vitamin B
12
(pg/mL)
Geometric mean (95%
CI)
Vitamin B
12
deficiency % (95%
CI)
Vitamin B
12
(pg/mL)
Geometric mean (95%
CI)
Vitamin B
12
deficiency % (95%
CI)
Vitamin B
12
(pg/mL)
Geometric mean (95%
CI)
Vitamin B
12
deficiency % (95%
CI)
Boys
310.3
a
(301.4-319.3)
14.3
a
(11.4-17.7)
297.3
a
(290.0-304.7)
16.7
a
(14.7-18.9)
241.6
a
(235.2-248.2)
35.0
a
(31.8-38.3)
Girls
313.3
a
(304.1-322.8)
13.3
a
(10.9-16.2)
291.7
a
(283.5-300.2)
17.9
a
(15.3-20.8)
257.1
b
(250.6-263.8)
27.0
b
(24.4-29.7)
Total
311.7
*
(305.0-318.5)
13.8
†
(11.7-16.2)
294.6
#
(288.2-301.0)
17.3
†
(15.4-19.3)
249.2
$
(243.9-254.6)
31.0
‡
(28.7-33.5)
Superscripts ab in the same column indicates estimates with non-overlapping CIs.
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Same with superscripts *#$&†‡ for serum vitamin B12 levels and vitamin B12 deficiency respectively in the same row.
Table 2B. Erythrocyte folate levels and prevalence of folate deficiency among children and adolescents stratified based on sex and age groups.
Sex
1-4 years (n=11,004)
5-9 years (n=14,125)
10-19 years (n=13,621)
Folate (ng/mL)
Geometric mean (95%
CI)
Folate
deficiency %
(95% CI)
Folate (ng/mL)
Geometric mean (95%
CI)
Folate
deficiency %
(95% CI)
Folate (ng/mL)
Geometric mean (95%
CI)
Folate deficiency
% (95% CI)
Boys
245.8
a
(233.4-258.9)
22.6
a
(19.7-25.8)
217.7
a
(208.0-228.0)
27.7
a
(25.1-30.4)
173.3
a
(164.4-182.6)
38.3
a
(35.3-41.4)
Girls
237.8
a
(225.1-251.1)
22.9
a
(20.2-25.9)
212.2
a
(201.8-223.2)
27.6
a
(25.1-30.3)
196.7
b
(186.6-207.3)
32.9
a
(30.2-35.7)
Total
241.9
*
(231.4-252.9)
22.8
†
(20.5-25.2)
215.0
#
(206.4-224.1)
27.6
‡
(25.5-29.9)
184.5
$
(176.3-193.0)
35.6
§
(33.1-38.2)
Superscripts ab in the same column indicates estimates with non-overlapping CIs.
Same with superscripts *#$&†‡§for Erythrocyte folate levels and folate deficiency respectively in the same row.
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9
Figure 2. Serum vitamin B12 and erythrocyte folate concentrations (left panel) and prevalence of vitamin B12 and folate deficiency (right panel) as a function of age and
gender. The line indicates geometric mean and the shaded area 95% confidence bands.
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10
3.2. State-based, rural-urban and regional differences in prevalence of B12 and FA deficiencies
The point prevalence of B12 deficiency varied across the states: while highest in Gujarat (pre-
schoolers-29.2%, CI: 20.3-40.0; adolescents-47.6%, CI: 37.3-58.2) and Punjab (school-age children-32.4%,
CI: 25.2-40.5) and lowest in West Bengal (pre-schoolers-1.9%, CI: 0.4-8.5) and Kerala (school-age
children-0.9%, CI: 0.2-3.6; adolescents-2.3%, CI: 1.0-5.5) (Figure 3). Similarly, the point prevalence of FA
deficiency was highest in Nagaland (pre-schoolers-71.1%, CI: 55.5-83.0; adolescents-85.9%, CI: 63.3-
94.9), and Andhra Pradesh (school-age children-67.8%, CI: 60.4-74.3) and lowest in Sikkim for all age
groups (0.1-0.8%) (Figure 3).
A significant positive relationship of B12 and FA deficiency prevalence between the age groups by
state was noted (1-4 vs 5-9 years: r=0.888, p<0.001 & r=0.961, p<0.001; 1-4 vs 10-19 years: r=0.747, p<0.001
& r=0.936, p<0.001; 5-9 vs 10-9 years: r=0.938, p<0.001 & r=0.967, p<0.001, respectively) (Figure S1A &
S1B).
Further, there was wide regional variation in the prevalence of B12 and FA deficiency. While, the
prevalence of B12 deficiency was high in central region across all the age groups (pre-schoolers-21.0%,
CI: 14.9-28.7; school-age children-29.1%, CI: 24.5-34.2; adolescents-42.7%, CI: 37.4-48.2), the prevalence
of FA deficiency was high in north-east for pre-schoolers (47.4%, CI: 39.1-55.8), west for school-age
children (54.9%, CI: 50.2-59.5) and south region for adolescents (71.6%, CI: 66.8-76.0) (Figure 4).
However, the B12 and FA prevalence were similar between rural and urban locations across all age
groups (Table S2).
3.3. B12 and FA deficiency by socio-demography, WASH characteristics, undernutrition and morbidity
The prevalence of B12 deficiency was higher in school-age children of mothers who had lower
education (Table S2). While no association was observed between B12 deficiency and WASH variables,
the prevalence of FA deficiency was higher with unimproved drinking water among all the age groups
(pre-schoolers: 35.8%, CI: 23.0-51.0, school-age children: 39.2%, CI: 30.4-48.8 and adolescents: 54.7%, CI:
45.7-63.5). In all age groups, no association was observed between B12 deficiency and the wealth index.
However, children and adolescents (5-19 years) from richer households (school-age children: 29.6%, CI:
26.6-32.8 and adolescents: 42.0%, CI: 37.2-47.0) had higher FA deficiency than those from poorer
households (school-age children: 20.7%, CI: 15.4-27.1 and adolescents: 27.0%, CI: 21.6-33.2). (Table S2).
There was no association between anthropometric undernutrition and B12 and FA deficiency
(Table 3). Children with diarrhoea (pre-schoolers: 14.9%, CI: 11.5-19.1 and school-age children: 20.2%,
CI: 15.8-25.5), and fever (school-age children: 22.8%, CI: 19.6-26.4), in the two weeks preceding the
survey, had significantly lower FA deficiency than those without morbidity, while no association was
found between B12 deficiency and morbidity (Table 3). In preschool age children, FA deficiency was
higher in those who did not receive the IFA supplement compared to those who had received this in
the previous week (Table 4).
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11
Figure 3. Prevalence of vitamin B12 and folate deficiency in children and adolescents across geographical states of India. The dot indicates mean and bars 95% CI. NCT:
National Capital Territory.
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13
Table 3. Prevalence of vitamin B12 and folate deficiency by under-nutrition and morbidity variable.
Characteristics 1-4 years 5-9 years 10-19 years
Vitamin B12
deficiency % (95%
CI)
Folate deficiency
% (95% CI)
Vitamin B12
deficiency
% (95% CI)
Folate deficiency
% (95% CI)
Vitamin B12
deficiency %
(95% CI)
Folate
deficiency
% (95% CI)
Stunting
No Stunting
(HAZ < -2SD)
12.7
a
(10.2-15.8)
22.9
a
(20.5-25.5)
17.8
a
(15.8-20.0)
28.1
a
(25.9-30.4)
31.2
a
(28.8-33.7)
37.2
a
(34.4-40.1)
Moderate
(HAZ: -3 to -2SD)
15.1
a
(12.2-18.5)
23.5
a
(19.9-27.5)
18.9
a
(15.2-23.2)
27.2
a
(23.4-31.4)
31.5
a
(26.3-37.3)
31.8
a
(27.9-35.9)
Severe
(HAZ < -3SD)
18.9
a
(12.7-27.3)
21.6
a
(16.8-27.4)
8.2
b
(5.2-12.8)
22.2
a
(16.7-28.8)
27.6
a
(20.3-36.3)
29.1
a
(23.5-35.5)
Wasting/Thinness
Not present
(WAZ < -2SD)
14.8a
(12.3-17.6)
22.8a
(20.3-25.5)
18.0a
(15.9-20.3)
27.4a
(25.1-29.9)
32.9a
(30.0-35.9)
35.8a
(32.9-38.7)
Moderate
(WAZ: -3 to -2SD)
11.1
a
(8.2-14.8)
21.5
a
(17.5-26.2)
17.0
a
(13.7-20.8)
29.1
a
(25.6-32.9)
26.8
ac
(23.0-30.8)
35.0
a
(31.1-39.1)
Severe
(WAZ < -3SD)
7.8
a
(4.7-12.7)
24.0
a
(17.1-32.7)
12.1
a
(8.0-18.0)
26.4
a
(20.5-33.4)
21.6
bc
(16.6-27.5)
34.0
a
(28.4-40.1)
Underweight
Not present
(WHZ < -2SD)
13.6
a
(11.1-16.5)
23.1
a
(20.5-25.9)
-
-
-
-
Moderate
(WHZ: -3 to -2SD)
15.8
a
(12.2-20.2)
22.6
a
(18.9-26.8)
-
-
-
-
Severe
(WHZ < -3SD)
11.8
a
(8.1-16.9)
20.4
a
(16.5-24.9)
-
-
-
-
History of diarrhoea in
the two weeks prior to
survey
Yes
19.1
a
(12.1-28.8)
14.9
a
(11.5-19.1)
23.8
a
(18.1-30.5)
20.2
a
(15.8-25.5)
-
-
No
12.9
a
(11.1-15.0)
24.2
b
(21.8-26.7)
16.6
a
(14.8-18.6)
28.4
b
(26.1-30.7)
-
-
History of fever in the
two weeks prior to
survey
Yes
16.9
a
(11.6-23.9)
19.6
a
(16.4-23.3)
15.8
a
(12.5-19.8)
22.8
a
(19.6-26.4)
-
-
No
12.5
a
(10.7-14.4)
24.2
a
(21.5-27.1)
17.7
a
(15.9-19.7)
29.0
b
(26.7-31.4)
-
-
Superscripts abc in the same column indicates estimates with non-overlapping CIs. HAZ; height-for-age, WHZ; weight-for-height, WAZ; weight-for-age.
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Table 4. Prevalence of vitamin B12 and folate deficiency with IFA supplementation in all age
groups.
Superscripts ab in the same column indicates estimates with non-overlapping CIs.
4. Discussion
This is the first study from India providing the serum B12 and erythrocyte FA levels and their
prevalence estimates, in a representative sample of children and adolescents, at the national, state
and regional level. The prevalence of B12 deficiency was high among adolescents (31%), with ~50%
lower prevalence in preschool (13.8%) and school age (17.3%) children. Similarly, the prevalence of
FA deficiency was also higher in adolescents (35.6%) compared to preschool (22.8%) and school-age
(27.6%) children.
In the present study, the prevalence of B12 deficiency in children and adolescents was lower,
while the prevalence of FA deficiency was almost similar to estimates from previous Indian and other
studies [13,14,25-27]. However, excepting the very recent study by Awasthi et al [28], these previous
studies had small sample sizes and were not nationally representative. Interestingly, the recent
nationally representative study conducted soon after this CNNS study (2019-21) also reported almost
similar trends of B12 deficiency but FA deficiency was slightly lower. Further, similar to the findings
reported in this study, Awasthi et al [28] also found higher prevalence of B12 and FA deficiency
increased with age and more so in boys. However, this study is not state representative and did not
capture the regional differences and associated factors. Nationally representative surveys in other
countries have also demonstrated a low prevalence of B12 deficiency among 1-6 y children in Mexico
(7.7%) [29], and school-aged children in Venezuela (11.4%) [30,31], although a Guatemalan study
reported the prevalence deficiency of B12 and FA to be 22.5 and 33.5% respectively, among children
aged 6-59 months [6]. In countries where animal foods constitute ∼5–10% of the energy intake, the
prevalence of B12 deficiency was high. For example, the prevalence of B12 deficiency was >70% in
school children in Kenya [32], and 27% in pre-schoolers of New Delhi [13]. A study in Nepal revealed
41% of B12 deficiency (serum B12 <150 pmol/L) plus 16% depletion (150–200 pmol/L) in 6–35-month-
old children with acute diarrhoea [25]. Our recent studies among apparently healthy adults showed
a high prevalence of B12 deficiency (~40%) along with suboptimal dietary intakes [10,15].
The differences in the magnitude of the prevalence of deficiency among the earlier studies might
be multifactorial including methodological issues. For example, in case of FA, the data availability is
complicated due to the large differences in the assay approaches (e.g., microbiological, immunoassay,
and chromatography-based), analytes (e.g., total compared with major circulating types of folate),
antibodies used for immunoassay approaches and because of the measurement of FA in serum or
RBC. For B12, it has been argued that its measurement lacks sensitivity or specificity and that
biomarkers such as serum methylmalonic acid (MMA) and homocysteine (Hcy) could be more
IFA 1-4 years 5-9 years 10-19 years
Vitamin B12
deficiency %
(95% CI)
Folate
deficiency %
(95% CI)
Vitamin B12
deficiency %
(95% CI)
Folate
deficiency %
(95% CI)
Vitamin B12
deficiency %
(95% CI)
Folate
deficiency %
(95% CI)
Yes 11.2a
(7.2-16.9)
14.6a
(9.9-21.0)
18.1a
(13.8-23.2)
28.9a
(23.4-35.1)
28.5a
(23.6-34.0)
38.9a
(33.2-45.0)
No 14.0a
(11.7-16.7)
23.3b
(21.0-25.7)
17.2a
(15.4-19.2)
27.5a
(25.3-29.9)
31.2a
(28.7-33.7)
35.5a
(32.9-38.1)
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sensitive measures of B12 deficiency [33]. Serum holotranscobalamin concentration is an early marker
with a better representation of the actual B12 status [34,35]. In a recent study in apparently healthy
adults, we found higher prevalence of B12 deficiency (46%) with holotranscobalamin compared to
total B12 measurement (37%) [11]. These differences make it difficult to choose an appropriate
threshold to define deficiency.
Although deficiency of these vitamins can occur primarily as a result of insufficient dietary
intake or malabsorption, various other factors such as gender, age, genetic, ethnic and sociocultural
backgrounds are likely to influence their status. Further, predominantly cereal-based and low
vegetables and fruits or animal food diets could contribute to the deficiency of these vitamins. The
prevalence of B12 deficiency in this study was higher in school-age children of mothers who had lower
education. Similarly, the FA prevalence was higher among the participants with unimproved
drinking water. However, in the present study, intriguing trends were observed with higher
prevalence of FA deficiency in participants from higher socio-economic status (SES) households
(indicated by richer wealth quintiles than those from the lower SES. Nevertheless, a similar pattern
of lower B12 deficiency in low SES group was reported by a study on rural school children in Raigad,
India [36].
Rural or urban residence represents an aggregate of multiple factors with rural residents more
likely to be from low SES households with poorer WASH facilities. Although overall prevalence of
B12 and FA was similar between urban and rural participants, significant regional differences were
observed. Central region showed higher B12 prevalence across all age groups. Likewise, state-wise
prevalence of B12 and FA showed perplexing trends with relatively richer states (in terms of per capita
income as well consumption of milk and dairy products) like Punjab and Gujarat showing higher
prevalence. The inverse socio-economic gradient of B12 and FA prevalence are difficult to explain and
need further exploration.
While moderately stunted school-children had higher prevalence of B12 deficiency, no such
difference was observed for FA in this study. Further, in the frame of anemia, B12 and FA deficiency
were earlier shown to be associated with 19-25% of anaemia prevalence in children and adolescents
in this survey [37]. In a more recent study on the same data sets it was found that folate deficiency
was negatively associated with anaemia while vitamin B12 deficiency was not associated with
anaemia [38]. In addition to increased growth requirements in children and adolescents, chronic low
intakes through predominantly vegetarian diets and poor absorption could induce the risk of these
vitamin deficiencies.
Adolescent boys had higher prevalence of B12 deficiency compared to girls, which is in line with
previous study in Venezuela, where the B12 deficiency prevalence was higher in adolescents
compared to infants and children [30]. Similarly, other studies in adult population showed higher
prevalence of vitamin B12 in males compared to females commensurate with higher Hcy in males
[10,11,15]. A higher prevalence of vitamin B12 deficiency among boys may be explained by a higher
requirement of micronutrients among them to sustain rapid muscular growth during adolescence, as
compared to girls. Furthermore, in a separate regional study, dietary vitamin B12 intake was lower
among boys compared to girls [16]. The inverse relation between male sex or age with vitamin B12
status could be interpreted in light of greater requirements for more rapid growth in boys than in
girls and in older than in younger children that are not being met with adequate dietary intake. At
least in Colombia, older children were shown to be less likely to adhere to an animal protein intake
pattern supports this possibility [39].
The strengths of our study include a large sample, representative at regional, state and national
level covering the wide range of age (1-19 years) and information on the B12 and FA prevalence of
deficiency estimates. The important limitations include smaller sample sizes in some of the states and
lower proportion of 1-2 y old children in the study sample, which may have resulted in
underestimation of B12 and FA prevalence in 1-4 y age group. Another limitation pertains to the lack
of data on other biomarkers such as MMA, Hcy and holotranscobalamin.
5. Conclusions
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 15 May 2023 doi:10.20944/preprints202305.1011.v1
Our study bridges a critical information gap on the prevalence of B12 and FA in Indian children
and adolescents and demonstrates that about a third of the adolescent boys are likely to be deficient
in B12 and FA. The prevalence of these deficiencies, however, is lower in younger age groups. These
findings are important to inform nutrition policy in India. More consistent use of thresholds to define
deficiency is needed in order to assess the realistic public health significance of FA and B12
deficiencies.
Abbreviations: CNNS, Comprehensive National Nutrition Survey; CRP, C-reactive protein; SES,
socioeconomic status; WASH, water, sanitation and hygiene; IFA, iron-folic acid; NFHS, National Family
Health Survey; BMI, body mass index; HAZ, height-for-age; WHZ, weight-for-height; WAZ, weight-for-age.
Supplementary Materials: The following supporting information can be downloaded at:
https://www.mdpi.com/ , Table S1: Comparison of characteristics of the study (vitamin B12 and folate) sample
with the total survey sample; Table S2: Prevalence of vitamin B12 and folate deficiency in children and
adolescents by socio-demographic and WASH variables; Figure S1A: Association between the vitamin B12
prevalence by state across all the age groups (A) 1-4 years vs 5-9 years (B) 1-4 years vs 10-19 years and (C) 5-9
years vs 10-19 years; Figure S1B: Association between the folate prevalence by state across all the age groups
(A) 1-4 years vs 5-9 years (B) 1-4 years vs 10-19 years and (C) 5-9 years vs 10-19 years.
Author contributions: TS, RP, SG, BK, RH, HSS, AVK and GBR performed initial statistical analyses on the
CNNS data; further comments and iterations involved all authors. While TS, RP, HSS, AVK and GBR wrote
and edited the manuscript, all authors were involved at every iteration of all analyses, and approved the final
manuscript.
Funding: The Comprehensive National Nutrition Survey (CNNS-2016-18) was conducted by the Ministry of
Health and Family Welfare, Government of India, and the UNICEF, with support from the Mittal Foundation.
Institutional Review Board Statement: The CNNS was conducted after obtaining due International Ethical
approval from the Population Council’s International Review Board, New York, USA and National Ethical
approval from Post Graduate Institute of Medical Education and Research, Chandigarh, India (IEC #
PGI/IEC/2015/1508) [17].
Informed Consent Statement: Informed consent from the parent/caregiver of children under 10 years,
informed consent of parent/caregiver of adolescents (11-17y) as well as the latter’s assent, and informed
consent of adolescents above 17y were obtained.
Data Availability Statement: The data that support the findings of this study are available from the Ministry
of Health and Family Welfare (MoHFW), Government of India, but restrictions apply to the availability of
these data, which were used under license for the current study, and so are not publicly available. Data are
however available from the authors upon reasonable request and with permission of [MoHFW].
Conflict of Interest: HSS designed the draft protocol of the CNNS with consultancy support from the
UNICEF, India. HSS and AVK were members of the Technical Advisory Committee of the CNNS, constituted
by the Ministry of Health and Family Welfare of the Government of India, to oversee its conduct and analysis.
SG has consultancy support for statistical analyses from UNICEF, India. There were no other conflicts to
declare. The views expressed here by the authors are in their individual capacity but not of the Institutions the
authors belong to.
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