RESEARCH PAPER
Impact of Neonatal Resuscitation Capacity Building of Birth Attendants
on Stillbirth Rate at Public Health Facilities in Uttar Pradesh, India
MANOJA KUMAR DAS1, CHETNA CHAUDHARY1, SANTOSH KUMAR KAUSHAL2, RAJESH KHANNA3 AND SUROJIT
CHATTERJI2
From 1The INCLEN Trust International, New Delhi; 2Save the Children, Lucknow, Uttar Pradesh; and 3Save the Children,
Gurgaon, Haryana; India.
Correspondence to: Dr Manoja Kumar Das, Director Projects, The INCLEN Trust International, F1/5, Okhla Industrial Area,
Phase 1, New Delhi 110 020, India. manoj@inclentrust.org.
Received: July 15, 2018; Initial review: December 03, 2018: Accepted: March 19, 2019.
Objective: To document the impact of neonatal resuscitation
capacity building of birth attendants at district and sub-district
level on fresh stillbirth within the public health system in India.
Design: An implementation research using pre-post study
design.
Setting: 3 high-infant and neonatal mortality districts (Gonda,
Aligarh and Raebareli) of Uttar Pradesh, India.
Participants: Pregnant women who delivered at the health
facilities and their newborns.
Interventions: An intervention package with (i) training on
essential newborn care resuscitation; (ii) skill laboratories
establishment for peer-interactive learning; (iii) better
documentation; and (iv) supportive supervision was implemented
at all health facilities in the districts.
G
lobally about 2.6 million stillbirths occur
annually apart from 2.7 million neonatal
deaths [1]. The decline in the stillbirth rates
has been slower than anticipated over last 2-
3 decades [2]. About 10-15% of the newborns require
resuscitation assistance at birth [3]. Emergency obstetric
and newborn care (EmONC) coupled with neonatal
resuscitation has been effective measure for reducing the
fresh stillbirths, early neonatal deaths and birth
asphyxias. In India, about 18-20% of newborn deaths are
due to birth asphyxia [4]. Helping Babies Breathe (HBB)
protocol has been used globally for training birth
attendants [5]. The impact of HBB or similar program
implementation in resource-poor settings has been
limited. There have been variation in reports in terms of
the settings used (facility- or community-level) and
parameters used for documenting impact (perinatal
death, early neonatal deaths, neonatal deaths, fresh
stillbirths and infant deaths). An implementation project
documented the impact of skill building of birth
INDIAN PEDIATRICS 369
Main outcome measures: Impact on fresh stillbirth rates and
resuscitation practices were documented at 42 health facilities
(Gonda-17, Aligarh-8 and Raebareli-17) over 12-18 months.
Results: Out of the 3.3% (4431/133627) newborns requiring
resuscitation, 58.5% (n=2599) were completely revived, 19%
(n=842) had some features of hypoxic insult after birth and 1.4%
(n=62) were stillbirths. There was 15.6% reduction in still birth rate
in the three districts with the intervention package.
Conclusion: The reduction in still birth rate and improvement in
newborn resuscitation efforts in the three districts indicated
feasibility of implementation and scalability of the intervention
package. However sustenance of the impact over longer period
needs documentation.
Keywords: Neonatal mortality, Perinatal mortality, Training.
attendants at district and sub-district public health
facilities in essential newborn care (ENC) and newborn
resuscitation practices (NRP) practices and outcome
including fresh stillbirths (FSBs). This article reports the
impact of the intervention on fresh stillbirth rate (FSBR)
at the health facilities in these districts.
Accompanying Editorial: Pages 365-67
METHODS
This implementation research was conducted in three
districts of Uttar Pradesh: Gonda, Aligarh and Raebareli.
The districts were chosen in consultation with state
government considering the levels of infant and neonatal
mortality rates. In these districts, 42 facilities (Gonda 17,
Raebareli 17 and Aligarh 8) with at least 100 deliveries
per month were selected for documentation of impact.
The protocol was reviewed and approved by Institute
Ethics Committee at The INCLEN Trust International.
As the data involved record review and no direct
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DAS, et al. IMPACT OF NEONATAL RESUSCITATION TRAINING OF BIRTH ATTENDANTS
information collection from the subjects, no informed
consent was required. Appropriate approval from the
competent state and district health authorities was
obtained.
The intervention package included: (i) training on
ENC and NRP using three-day module; (ii) development
of skill laboratories at four health facilities per district to
enable peer-interactive learning; (iii) better monitoring
and documentation of deliveries and peripartum events;
and (iv) supportive supervision. The training package
had more emphasis on skill building and hands-on
practice, compared to the existing two-day package
under government program. A cadre of master trainers at
state level was created through intense, hands-on
training workshop. These master trainers (four trainers
for 24 participants per batch) trained the birth attendants
from the facilities in batches at district level. The training
sessions were monitored by external monitors to ensure
quality and uniformity. The implementation was initiated
in Gonda and Aligarh in July 2014 and in Raebareli in
February 2015. The training of all staffs was completed
during July-September 2014 in Gonda and Aligarh and
during February-March 2015 in Raebareli. Additional
trainings were conducted to address staff turnovers, as
per need. The impact documentation was limited to the
selected 42 facilities, although all the birth attendants in
these districts were trained. Availability of resuscitation
kits (bag and masks) were ensured at all the delivery
points in the districts. Four skill laboratories were
established in each district including the district hospital
and three 24×7 first referral units. These skill
laboratories situated near labour room or maternity ward
were equipped with one radiant warmer, self-inflating
resuscitation bags with three size masks, one mannequin
(Laerdel Neonatalie), and other teaching and job-aids.
These skill laboratories were managed by one maternity
nurse with support from one doctor. The details about
deliveries were documented by the nurses in the registers
and case sheets indicating the mode of delivery, outcome
(livebirth or stillbirth–fresh/macerated), resuscitation
requirement for the newborn, outcome of resuscitation
(complete recovery, features of hypoxia) and
requirement of referral. The nurses and doctors from the
facilities were trained for appropriate documentation.
Non-breathing infants with gestation age weighing
>1000 grams without any signs of life (fetal heart rate
[FHR] or movement) or maceration and who were not
successfully revived were classified as a FSB. The
stillbirths were confirmed by the doctor on duty and
categorized as antepartum or intrapartum based on the
FHR documentation. The documentations in the
registers were verified with case sheets (including
INDIAN PEDIATRICS
partograph) weekly by the dedicated monitors (separate
for each district) not involved in service delivery. All
early neonatal deaths in these facilities were checked to
verify any misclassification. A monthly report was
compiled for the key parameters during October 2014 to
March 2016 for Gonda and Aligarh and during April
2015 to March 2016 for Raebareli using the delivery
registers (post-intervention periods). Additionally,
monitoring activities included direct supervision at
periodic intervals focusing on the clinical practice
adherence (through observation and record review),
documentation, availability of equipment and
maintenance, death audits, skill laboratory usage
(through record review), team building activities,
monthly review and feedback (during review meetings).
The data for 18 months (April 2013 to September 2014)
in Gonda and Aligarh and data for 12 months (April
2014- March 2015) in Raebareli prior to training were
collected from the registers and case records, which
represented the pre-intervention periods.
Statistical analysis: Double data entry was done using
excel sheet followed by quality check to ensure
correctness. The data entered was stored in a server with
restricted access. Descriptive statistics were used to
summarize the proportions and means. FSBR was
estimated per 1000 deliveries. The pre-and-post sample
means were compared using 2-sample t test. Data was
analyzed using Stata version 15.0 (StataCorp LLC,
Texas, USA).
We hypothesized that the intervention package
would reduce the FSBs by at least 15%. The FSBR in
pre-intervention period was 3.2% (2%-4%) of the total
deliveries. To document a 15% reduction in FSBR from
pre-intervention period with 80% power and 95%
confidence level, the required sample size was 17397.
RESULTS
In three districts, a total of 779 birth attendants including
69 doctors, 281 nurses and 429 auxiliary nurse midwives
(ANMs) at all level of facilities were trained. At the end
of the observation period, 98% of the birth attendants
and 93% of the doctors from trained pool were available
at the 42 observation facilities. No other training or new
activity on perinatal or newborn care was observed
during the same period in these districts. The deliveries
at the 42 health facilities between April 2013 and April
2016, were comparable.
The need for resuscitation varied from 2.8% to 3.6%
of the 4431 newborns who required resuscitation at birth,
2599 (58.5%) newborns were completely successfully
resuscitated. The resuscitation efforts were successful in
370 VOLUME 56__MAY 15, 2019
DAS, et al. IMPACT OF NEONATAL RESUSCITATION TRAINING OF BIRTH ATTENDANTS

WHAT IS ALREADY KNOWN?

• Training of birth attendants in essential newborn care and neonatal resuscitation may contribute to reducing
stillbirths and early neonatal deaths.
WHAT THIS STUDY ADDS?
• A three-day essential newborn care and neonatal resuscitation skill-based training coupled with skill laboratories
at facility level improved the resuscitation efforts at birth and reduced fresh stillbirths in India

82%, 35.1% and 39.7% of newborns requiring
resuscitation in the districts Gonda, Aligarh and
Raebareli, respectively. Post resuscitation, referrals were
needed in 17.2%, 61.8% and 59.1% of newborns in
Gonda, Aligarh and Raebareli districts respectively. The
impact on successful revival with resuscitation could not
be assessed due to absence of reliable data for pre-
intervention period.
Compared to the pre-intervention period, fresh still
births reduced by 15.6% in post-intervention period
(P<.001) (Table I). The overall risk reduction for FSB was
0.1 (RR 0.90; 95% CI 0.88-0.92) compared to pre-
intervention period. The risk reduction for the individual
districts ranged from 0.2 (RR 0.80; 95% CI 0.75-0.85) for
Aligarh; 0.09 for Gonda (RR 0.91; 95% CI 0.88-0.94) and
0.06 (RR 0.94; 95% CI 0.90-0.98) for Raebareli.
DISCUSSION
This implementation project documented the impact of
the intervention package (training on ENC and NRP;
skill laboratories; better documentation and supervision)
on FSB and outcome of resuscitation in three districts of
Uttar Pradesh, which is considered to have a weak public
health system. The overall improvement in the
identification of cases requiring resuscitation, successful
resuscitation and the degree of reduction in FSB was
promising. The variation across the districts probably
indicated the level of implementation by the birth
attendants and maturity. With time the resuscitation need
rate in the districts improved, indicating evolving
maturity. Lowest change in Raebareli may be explained
by shorter period of implementation. The higher change
in Aligarh district may be due to lower number of
facilities under study and higher proportion of deliveries
occurred at the district and sub-district hospitals,
compared to other two districts. The impact in Gonda
was comparable to the report from India [9]. Higher
referral of newborns post-resuscitation in Aligarh and
Raebareli districts could be due to the proximity to a
tertiary care facility.

TABLE I IMPACT ON THE STILLBIRTH PRE-AND POST-INTERVENTION ACROSS THE DISTRICTS

District Parameters Pre-intervention period* Post-intervention period@ Difference % (95% CI) P value
Gonda Deliveries, n 60192 58196 0.6% (0.38%, 0.81%) <0.001
Livebirths, n (%) 57839 (96.1) 56250 (96.7)
FSBs, n (%) 2353 (3.9) 1946 (3.3)
Aligarh Deliveries, n 37372 37627 0.7% (0.51%, 0.89%) <0.001
Livebirths, n (%) 36584 (97.9) 37091 (98.6)
FSBs, n (%) 788 (2.1) 536 (1.4)
Raebareli Deliveries, n 38346 37804 0.3% (0.05%, 0.54%) 0.016
Livebirths, n (%) 37101 (96.8) 36707 (97.1)
FSBs, n (%) 1245 (3.2) 1097 (2.9)
Pooled Deliveries, n 135910 133627 0.5% (0.37, 0.62%) <0.001
Livebirths, n (%) 131524 (96.8) 130048 (97.3)
FSBs, n (%) 4386 (3.2) 3579 (2.7)
Note: % of fresh stillbirths (FSBs) estimated using deliveries as denominator (including stillbirths).
*Pre-intervention period included 18 months (April 2013 to September 2014) for Gonda and Aligarh) and 12 months (April 2014- March 2015) for
Raebareli; @Post-intervention period included 18 months (October 2014 to March 2016) for Gonda and Aligarh) and 12 months (April 2015- March
2016) for Raebareli.

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DAS, et al. IMPACT OF NEONATAL RESUSCITATION TRAINING OF BIRTH ATTENDANTS
A study in India (two sites in Karnataka and
Maharashtra) and Kenya using the HBB newborn
resuscitation intervention documented 16% reduction in
stillbirth (pre-intervention and post-intervention 9 and 7.6
per 1000 births respectively) [6]. The changes were not
consistent across the sites and birth weight categories.
There were also reduction in perinatal deaths, first-day
deaths and early neonatal deaths in these area [6].
Implementation of HBB in eight hospitals in Tanzania
over 24 months resulted in decline of FSBs by 24%, from
19 to 14.5 per 1000 births (RR 0.76; 95% CI 0.64, 0.90; P
= 0.001) [10]. There was also decline in neonatal deaths
by 47% (RR 0.53; 95% CI 0.43, 0.65; P = 0.0001) over
the same period [7]. An evaluation of Neonatal
Resuscitation Program in Malaysia indicated minimal
reduction in stillbirth rates, 4.3 to 4.1 per 1000 deliveries
between 1996 and 2004 [8]. A NRP programme at 14
teaching tertiary care hospitals in India documented
improvement in resuscitation practices and significant
decline in asphyxia related deaths [9]. A metaanalysis
concluded that neonatal resuscitation training in facility
setting reduced intrapartum-related deaths by 30%
(RR=0.70; 95% CI 0.59, 0.84) [10]. HBB training in a
tertiary care hospital in Nepal resulted in reduction of
stillbirth rate from 9 to 3.2 per 1000 deliveries [11].
Our project has several strengths including a district-
wide implementation in three districts at different level
of newborn and infant mortality rates. The district-wide
implementation over one year with minimal external
input simulated real-field scenario. The training system
also simulated the real-field situation in these districts.
Thus the results can be generalized to many Indian states
and districts with reasonably high neonatal mortality.
Non-availability of information on changes according to
birthweights and early neonatal deaths are the
limitations. Also the potential effect of change in birth
attendant’s number and competency on resuscitation
effort, outcome and stillbirths could not be documented.
The determinants of variability of impact on fresh
stillbirths across the districts were not documented,
which may be a limitation. The quality and rigor of
documentation during pre-intervention period may be
different compared to the observation period. This
project documented the feasibility of implementation of
the newborn resuscitation program at scale with good
impact on the fresh stillbirths and possible newborn
outcome.
In conclusion, rapid scaling up of the essential
newborn care and neonatal resuscitation is critical for
newborns survival and reducing fresh stillbirths. This
successful experience of implementation within the
existing public health system in three districts, rise in
INDIAN PEDIATRICS
resuscitation attempts and positive impact on fresh
stillbirths demonstrated feasible and successful scaling
up of the intervention package. However additional
documentation may be undertaken to document the
degree of cost-benefit for the investments and long-term
impact.
Acknowledgements: We acknowledge the support of National
Health Mission, Government of Uttar Pradesh and District
Health Administration of the districts for their continued
facilitation and support. We highly appreciate the participation
of the doctors, nurses and ANMs in these districts.
Contributors: MKD: conceptualization, planning, tool
development, data analysis, manuscript writing; CC: data
collection and analysis; SKK: coordination of implementation,
supervision; RK: conceptualization, planning, supervision of
implementation; SC: coordination of implementation,
supervision. All the authors read and approved the final
manuscript before submission.
Funding: United States Agency for International Development
(USAID) under the FY12 Child Survival and Health Grant
Program: Saving Newborn Lives in Uttar Pradesh through
Improved Management of Birth Asphyxia (CS-28).
Competing Interest: None stated.
REFERENCES
1. Wang H, Liddell CA, Coates MM, Mooney MD, Levitz
CE, Schumacher AE, et al. Global, regional, and national
levels of neonatal, infant, and under-5 mortality during
1990-2013: A systematic analysis for the global Burden of
Disease Study 2013. Lancet. 2014; 384:957-79.
2. Blencowe H, Cousens S, Jassir FB, Say L, Chou D,
Mathers C, Hogan D, et al. National, regional, and
worldwideestimates of stillbirth rates in 2015, with trends
from 2000: A systematic analysis. Lancet Global Health.
2016;4:e98-108.
3. WHO, UNFPA, UNICEF, AMDD. Monitoring emergency
obstetric care: A handbook. Geneva: WHO; 2009. http://
whqlibdoc.who.int/publications/2009/ 9789241547734
_eng.pdf? ua=1. Accessed on August 25, 2017.
4. Bassani DG, Kumar R, Morris SK, Jha P, et al, for the
Million Death Study Collaborators. Causes of neonatal and
child mortality in India: A nationally representative
mortality survey. Lancet. 2010:376:1853-6.
5. Singhal N, Lockyer J, Fidler H, Keenan W, Little G,
Bucher S, et al. Helping Babies Breathe: Global neonatal
resuscitation program development and formative
educational evaluation. Resuscitation. 2012;83:90-6.
6. Bellad RM, Bang A, Carlo WA, McClure EM, Meleth S,
Goco N, et al. A pre-post study of a multi-country scaleup
of resuscitation training of facility birth attendants: does
Helping Babies Breathe training save lives? BMC
Pregnancy and Childbirth. 2016; 16:222-31.
7. Msemo G, Massawe A, Mmbando D, Rusibamayila N,
Manji K, Kidanto HL, et al. Newborn mortality and fresh
stillbirth rates in Tanzania after helping babies breathe
training. Pediatrics. 2013;131:e353-60.
8. Boo NY. Neonatal resuscitation programme in Malaysia:
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an eight-year experience. Singapore Med J. 2009;50:
152-9.
9. Deorari AK, Paul VK, Singh M, Vidyasagar D. Impact of
education and training on neonatal resuscitation practices
in 14 teaching hospitals in India. Ann Trop Paediatr.
2001;21:29-33.
10. Lee AC, Cousens S, Wall SN, Niermeyer S, Darmstadt GL,
Carlo WA, et al. Neonatal resuscitation and immediate
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newborn assessment and stimulation for the prevention of
neonatal deaths: A systematic review, meta-analysis and
Delphi estimation of mortality effect. BMC Public Health.
2011;11:S12.
11. KC A, Wrammert J, Clark RB, Ewald U, Vitrakoti R,
Chaudhary P, et al. Reducing perinatal mortality in Nepal
using Helping Babies Breathe. Pediatrics. 2016;137;
e20150117.
373 VOLUME 56__MAY 15, 2019
 RESEARCH PAPER
Normative Data of Optimally Placed Endotracheal Tube by Point-of-care
Ultrasound in Neonates
POONAM SINGH1, ANUP THAKUR1, PANKAJ GARG1, NEERAJ AGGARWAL2 AND NEELAM KLER1
From Departments of 1Neonatology and 2Pediatric Cardiac Sciences, Institute of Child Health, Sir Ganga Ram Hospital,
New Delhi, India.
Correspondence to: Dr Neelam Kler, Department of Neonatology, Institute of Child Health, Sir Ganga Ram Hospital, New Delhi,
India 110 060, India. drneelamkler@gmail.com.
Received: June 29, 2018; Initial review; August 13, 2018: Accepted: February 21, 2019.
Objective: To derive normative data of the distance between
optimally placed endotracheal tube tip and arch of aorta by
ultrasound in neonates across different weight and gestation.
Design: Cross-sectional study.
Setting: Tertiary care neonatal intensive care unit from April 2015
to May 2016.
Participants: All neonates requiring endotracheal intubation were
eligible for the study.
Methods: During intubation, insertional length was determined
using weight-based formula. The distance between endotracheal
tube tip and arch of aorta was measured by ultrasound.
Endotracheal tube position was confirmed by chest radiograph.
Results: Out of 133 enrolled infants, 101 (75.9%) had optimally
placed endotracheal tubes. The mean (SD) distance between
E
ndotracheal intubation is a commonly
performed procedure in neonates. Optimum
placement of endotracheal tube (ET) in the
trachea requires high degree of precision. Mal-
placement of ET results in complications including
pneumothorax, lung collapse, tracheal damage and
unplanned extubation [1]. Placement of ET up to
optimum depth (insertional length, IL) has been
predicted based on various anthropometric parameters
such as weight, gestation, sternal length (SL), nasal
tragus length (NTL), occipital frontal circumference
(OFC), crown heel length (CHL) and foot length [2-4].
However, despite using clinical predictors of IL, mal-
position of ET has been reported to be as high as 58%
[5]. The gold standard to confirm ET position is chest
radiograph. However, radiograph carries disadvantage
of radiation exposure, excessive handling of sick infants
and time delay [6]. In addition, it may not be feasible to
use X-ray when duration of intubation is brief e.g.,
during surfactant administration.
Point of care ultrasound (USG) has been found to be a
INDIAN PEDIATRICS
endotracheal tube tip and arch of aorta by ultrasound was 0.78
(0.21) cm in infants <1500 g and 1.04 (0.32) cm in infants ≥1500 g.
The regression equation to estimate insertional length from
weight, crown heel length (CHL), occipito-frontal circumference
(OFC), nasal tragus length (NTL) and sternal length (SL) were
Wt(kg)+4.95, 0.15×CHL(cm)+0.57, 0.22×OFC(cm)+0.49, 0.82×
NTL(cm)+1.24 and 0.75×SL(cm)+2.26, respectively.
Conclusion: Our study reports normative data of the distance
between optimally placed endotracheal tube tip and arch of aorta
by ultrasound in neonates. The distance between endotracheal
tube tip and arch of aorta increases with increase in weight and
gestation. Insertional length correlates strongly with all the
anthropometric parameters.
Keywords: Arch of aorta, Intubation, Insertional length.
Clinical Trial Registration: CTRI/2017/05/008718.
safe and feasible modality to determine ET tip position in
neonates [7-11]. An ET tip placed 0.5-1 cm above the arch
of aorta suggests its correct placement [7,11]; though, it
has been defined irrespective of weight and gestation
[16,20]. On the contrary, the length of the trachea has been
reported to be variable with weight, length and gestation
[12-14]. Therefore, the present study was planned to
derive normative data of the distance between optimally
placed ET tip and arch of aorta across different
birthweights and gestations by USG.
METHODS
The study was conducted in the neonatal intensive care
unit (NICU) of a tertiary-care centre in northern India
from April 2015 to May 2016. All neonates admitted in
NICU were screened for eligibility. Infants with known
tracheal, esophageal, cardiac and cranio-facial
anomalies and those with generalized edema were
excluded. Informed consent was obtained from either
parent of the infants, who were presumed to be at risk of
intubation by the treating neonatologist. Following
374 VOLUME 56__MAY 15, 2019
SINGH, et al.
intubation, infants were re-assessed by the principal or
co-investigator/s based on detailed clinical examination,
chest X-ray or echocardiographic findings. Subjects
found to be unsuitable for any of the anthropometric
examination or ultrasound measurement including even
minor abnormalities such as low set ear or depressed
nasal bridge were further excluded. Each eligible infant
was enrolled only once during the study period. The
study was approved by the Institutional ethics committee
and registered with Clinical trial registry of India
(CTRI). The primary objective of the study was to
measure the distance between optimally-placed ET tip
and arch of aorta by USG across different weights and
gestations. Secondary objective was to find out
correlation between IL of optimally placed ET and
various anthropometric parameters: weight, OFC, CHL,
NTL and SL.
All intubations were done through oral route as per
decision of the treating neonatologist. IL was decided by
Tochen’s formula (weight in (kg) + 6cm) [15].
Birthweight or current weight, whichever was higher,
was used to estimate IL. After intubation, ET was
readjusted by auscultation and fixed at a position where
air entry was bilaterally equal. Neck position was
maintained in slight extension with the help of a shoulder
roll during USG and radiograph. After intubation and
fixation of the ET, exact IL was calculated. The part of
ET present exterior to the lips (A) was measured from a
visible centimeter mark on the adapter end of ET to the
corner of the lip with a paper tape following curvature of
the ET. Exact IL was calculated by subtracting this length
(A) from length of ET (B) up to that mark (Web. Fig 1).
X-ray was ordered and USG was done to determine ET
tip position following intubation. No change in ET
position was done based on USG findings until X-ray
film was available.
ET tip position was determined by USG following
intubation using Sonosite M-Turbo portable ultrasound
machine with phase array probe of 8-4 MHz frequency.
To minimize the variability of USG measurements, only
two of the investigators conducted all USG after
appropriate training.
Mid-sagittal views were obtained by placing the
probe on the infant’s lower neck and upper sternum in
order to visualize the ET. Warm gel was applied to the
probe during USG. Care was taken to ensure adequate
oxygenation and temperature regulation throughout the
procedure. The bedside nurse was available to assist in
calming the infant. Arch of aorta was visualized by gray
scale and color Doppler. ET was identified as a linear
echo dense structure. The ET tip was reliably delineated
INDIAN PEDIATRICS
OPTIMUM ET PLACEMENT BY USG
by producing a minimal gentle movement with the help
of an assistant. Each image was zoomed and the distance
of the ET tip from superior border of arch of aorta was
measured in the line of ET (Fig. 1). A total of three
observations were made for each subject and average of
these measurements was taken. Both static images and
video clips were recorded and stored in the flashcard of
USG machine to be later transferred to the computer for
storage. Twenty percent of the videos were analyzed by a
pediatric cardiologist for validation. Time elapsed
between end of intubation and completion of last
measurement by USG was recorded.
In 25 intubated infants, USG was done in succession
by both the investigators blinded to each other’s
findings. A total of three observations were made by each
investigator for these infants. Intraclass correlation
coefficient (ICC) and Bland-Altman analysis were used
for measuring and testing the consistency, reliability and
agreement of USG measurements between the two
investigators. A strong intraclass correlation (ICC>0.9)
was also observed between average USG readings of
both the investigators (ICC 0.98; 95% CI 0.96 to 0.99). A
strong intraclass correlation was also observed for all the
three measurements of the investigators (ICC 0.93; 95%
CI 0.91, 0.95; and 0.97; 95% CI 0.95, 0.99). Bland-
Altman analysis (Web Fig. 2) showed mean difference of
-0.02 mm (95% CI -0.05 to -0.01) in the measurements
of the two co-investigators.
Corrective measure to place the ET in optimum
position was taken by the treating neonatologist after
availability of X-ray film. Time elapsed between
intubation and availability of X-ray film was noted. All
X-ray films were later reviewed by a radiologist and ET
was classified as optimum (ET tip located between upper
FIG. 1 Demonstrates linear echo bright structure confirmed to
be endotracheal tube (ET) by gently moving the tube; AA
distance of ET tip from arch of aorta measured in the line of ET.
375 VOLUME 56__MAY 15, 2019
SINGH, et al. OPTIMUM ET PLACEMENT BY USG

border of T1 and lower border of T2 vertebral body), low reproducibility (inter and intra observer variability
(ET tip lying below lower border of T2 vertebral body) between two observers) of USG measurements across
or high (ET tip situated above upper border of T1 the two examiners. The corresponding limits of
vertebral body) [16]. agreements were calculated by means of Bland-Altman
analysis after assuring the normality of the differences
All anthropometric measurements were made by a between two sets of results (i.e., the paired observations
single investigator on the day of enrolment. OFC was of principal investigator and co-investigator), which was
recorded with a paper tape placed posteriorly on external examined using Kolmogorov–Smirnov test.
occipital protuberance and anteriorly above supraorbital
ridges. CHL was measured with the help of a length RESULTS
board (Seca 210) with knee extended and foot A total of 1157 infants were admitted during the study
perpendicular to the ground. SL was measured from the period. Consent was obtained for 496 infants at risk of
suprasternal notch to the tip of the xiphoid process. NTL intubation, out of which 258 were intubated. During 68
was noted from the base of the nasal septum to the tip of intubations, investigators were not available and an
the tragus. A total of three readings were made for each additional 57 infants were excluded due to various other
parameter and mean of these readings was calculated. reasons (Fig. 2). A total of 133 infants were included, of
The primary outcome was to calculate the distance which 101 had optimally placed ET on X-ray.
between optimally placed ET tip and arch of aorta across The baseline characteristic of enrolled infants is
different weight and gestation by USG. Secondary described in Table I. Mean (SD) IL and USG distance
outcome included correlation between IL of optimally between optimally placed ET tip and arch of aorta in
placed ET and anthropometric parameters such as different weight and gestation groups is depicted in
weight, OFC, CHL, NTL and SL.
A pilot study was conducted in 15 infants to calculate Total admissions (n=1157)
the mean and standard deviation (SD) of the distance ↓
between optimally placed ET tip from arch of aorta by Consent obtained from infants at
USG. Among very low birth weight infants (birth weight risk of intubation (n=496)
<1500 g), mean (SD) was found to be 0.30 cm (0.11). ↓
Considering precision of 10% across the mean, sample Total number of infants intubated
size for very low birth infants was found to be 52. (n=258)
Similarly, for infants weighing >1500 g, mean (SD) was
Principal investigator not
0.60 cm (0.20) and considering a precision of 10%, → available (n=68)
sample size was found to be 42. Therefore, a total of 94 ↓
infants with optimally placed ET were required to derive Assessed for eligibility (n=190)
normative data of the distance between optimally placed
ET tip and arch of aorta by USG. Excluded (n=57)
Statistical analysis: Analysis of data was done using • Congenital anomalies - 18
• High frequency oscillatory
SPSS software version 20.0. Chi square or Fisher’s exact
ventilation - 17
test was used to compare categorical variables. Student’s
→ • Generalized edema - 3
t test and Mann Whitney test were applied to compare • Non visualization of ET on USG - 3
independent parametric and non-parametric variables, • Irritable baby during USG - 5
respectively. Non-parametric related samples were • Poor quality chest radiograph - 4
tested by Wilcoxon signed rank test. Two sided P value • Poor quality USG images - 7
<0.05 was considered significant. Pearson’s correlation ↓
and linear regression were used to analyze the Enrolled (n=133)
relationship between anthropometric data (weight, OFC,
CHL, NTL and SL) and IL. IL of correctly placed ET was → ET not optimally placed (n=32)
the dependent variable and anthropometric parameters
were independent variables for the correlation and ↓
Optimally placed ET (n=101)
regression analysis.
ET: Endotracheal tube; USG:Ultrasonography.
The intraclass correlation coefficient (ICC) was used
to determine the consistency, reliability and FIG. 2 Study flow chart.

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SINGH, et al. OPTIMUM ET PLACEMENT BY USG

TABLE I BASELINE CHARACTERISTICS OF OVERALL STUDY TABLE II INSERTIONAL LENGTH AND NORMATIVE DATA OF THE
POPULATION (N=133) DISTANCE BETWEEN OPTIMALLY PLACED ET TIP AND
ARCH OF AORTA BY USG ACROSS DIFFERENT WEIGHT
Parameter n (%)
AND GESTATION CATEGORIES (N=101)
Gestation in (wk)* 30.8 (4.6)
Categories Insertional USG distance
Birth weight in (g)* length (cm) Mean (SD) 95% CI
<1500 992.7 (272.0) Mean (SD)
≥1500 2480.1 (597.3) Weight (g)
PMA (wk)* 32.0 (5.3) <1000 (n=30) 5.80 (0.42) 0.73 (0.21) 0.65-0.80
Weight at enrolment (g)* 1000-1499 (n=27) 6.46 (0.46) 0.86 (0.18) 0.79-0.94
<1500 1028.9 (274.0) 1500-1999 (n=14) 6.97 (0.54) 0.94 (0.29) 0.77-1.12
≥1500 2456.1 (597.3) 2000-2499 (n=6) 7.26 (0.44) 0.98 (0.13) 0.84-1.13
Weight for gestation ≥2500 (n=24) 8.30 (0.54) 1.10 (0.35) 0.95-1.26
AGA 100 (75.2) PMA Gestation (wk)
SGA 26 (19.5) <28 (n=20) 5.83 (0.41) 0.65(0.19) 0.58-0.76
LGA 7 (5.3) 28-31 (n=25) 6.20 (0.56) 0.83 (0.15) 0.77-0.90
Gender 32-35 (n=20) 6.84 (0.58) 0.94 (0.22) 0.84-1.04
Male 99 (74.4) ≥36 (n=36) 7.78 (0.91) 1.04 (0.34) 0.93-1.16
Weight enrolment groups (g) ET: Endotracheal tube; PMA: Post menstrual age.
<1000 45 (33.8)
population was significantly less than the mean distance
1000-1499 38 (28.6) for infants with weight >1500g (0.78 (0.21) vs 1.04
1500-1999 16 (12.0) (0.32); P<0.001). Similarly, mean (SD) distance in
2000-2499 8 (6.0) infants with post menstrual age <32 wk was significantly
≥2500 26 (19.5) less as compared to the distance for the population ≥32
weeks (0.77 (0.18) vs 1.01 (0.30); P<0.001). Table III
PMA enrollment groups (wk)
illustrates centiles of the ultrasound distance between ET
<28 31 (23.3) tip and arch of aorta across different weight and
28-31 37 (27.8) gestation groups. The degree of correlation between IL
32-35 25 (18.8) and anthropometric parameters and the regression
≥36 40 (30.1) equation to predict insertional length from weight, OFC,
CHL, NTL and SL have been described in Table IV.
Data expressed as n (%) or *mean (SD); PMA: Post menstrual age;
AGA: Appropriate for gestational age; SGA: Small for gestational age;
Linear relationship between IL and various anthro-
LGA: Large for gestational age. pometric parameters has been displayed in the figure
(Web Fig. 3a-3e).
Table II. A total of 32 infants had malpositioned ET. DISCUSSION
Deep intubation was twice as common as high intubation
(15.10% (21/133) vs 8.30% (11/133); P=0.02). Endotracheal tube position can be confirmed by bedside
Proportion of malpositioned ET in infants <1500 g was USG without exposing the infant to radiation and
higher compared to infants ≥1500g (31.40% (26/83) vs handling [7,8,11,17]. Ultrasound studies have revealed
12% (6/50); P=0.01). Similarly, malposition was more that a distance of 0.5 to 1 cm between ET tip and arch of
common in infants of gestation <32 weeks compared to aorta suggests its correct placement [7,11]. However,
≥32 weeks (33.80% (23/68) vs 13.90% (9/65); P<0.01). this distance is likely to differ across different weight and
The median (IQR) time from intubation to completion of gestation due to variation in tracheal length [12-14]. We
three readings of USG was less than the time required for conducted an observational study with the primary
obtaining X-ray film (12.00 (8.00-15.00) min vs 98.00 objective to derive normative data of the distance
(64.00-132.00) min; P<0.001). between optimally placed ET tip from arch of aorta by
USG across different weight and gestation.
USG distance between ET tip and arch of aorta was
also compared in infants <1500g vs ≥1500g and <32 In our study, we determined the ET position by USG
weeks vs ≥32 weeks. Mean (SD) USG distance in VLBW in mid-sagittal view and measured the distance between

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SINGH, et al. OPTIMUM ET PLACEMENT BY USG

TABLE III DISTANCE BETWEEN ENDOTRACHEAL TUBE TIP AND ARCH OF AORTA BY ULTRASONOGRAPHY ACROSS DIFFERENT GESTATION
AND WEIGHT (N=101)

Percentiles
Parameter N 5th 10th 25th 50th 75th 90th 95th
Weight (g)
<1000 30 0.30 0.42 0.60 0.72 0.88 1.04 1.08
1000-1499 27 0.49 0.62 0.75 0.92 0.96 1.08 1.22
1500-1999 14 0.47 0.55 0.75 0.92 1.09 1.49 -
2000-2499 6 0.76 0.76 0.89 0.98 1.12 - -
≥ 2500 24 0.50 0.58 0.76 1.12 1.39 1.61 1.75
Post-menstrual age (wk)
<28 20 0.27 0.34 0.55 0.69 0.81 0.93 1.04
28-316/7 25 0.58 0.63 0.71 0.82 0.93 1.08 1.08
32-356/7 20 0.47 0.72 0.78 0.97 1.04 1.30 1.39
≥ 36 36 0.49 0.55 0.76 1.06 1.23 1.58 1.67

TABLE IV PEARSON’S CORRELATION (r) AND LINEAR radiograph [10,18]. Chowdhry, et al. [17] measured the
REGRESSION EQUATIONS FOR INSERTIONAL LENGTH distance from the point of maximal curvature of the arch
AND VARIOUS ANTHROPOMETRIC MEASUREMENTS
of aorta to the ET tip by USG and a minimum distance of
(N=101)
1 cm was used to define “not deeply placed ET”. This
Parameter r P value Regression equation (R2) distance was derived from preliminary analysis of
Weight (Kg) 0.906 <0.001 Wt (Kg) + 4.95
computed tomography scans of infants between zero to
three months of age. The study reported a concordance
OFC (cm) 0.903 <0.001 0.223×OFC (cm)+0.49
of 94.6% between USG and radiograph [17]. However,
NTL (cm) 0.898 <0.001 0.822×NTL (cm)+1.24 in none of these studies, authors reported variation in
CHL (cm) 0.896 <0.001 0.154×CHL (cm)+0.57 measurements across different weight and gestation.
STL (cm) 0.860 <0.001 0.752×STL (cm)+2.26
In our study, we found that the distance between
CHL: Crown heel length, OFC: Occipito frontal circumference, optimally placed ET tip from arch of aorta increases with
NTL: Nasal tragus length, STL: Sternal length, IL: Insertional length.
increase in weight and gestation. Anatomical studies can
explain our results. In a prospective study of routine
ET tip to arch of aorta in the line of ET. Our method was autopsies which included 274 fetuses (15-41 weeks) and
similar to that described by Slovis, et al. [7]. They 26 infants (0 to 3 months), anatomical measurements of
observed that the distance of ET tip to carina on X ray larynx and trachea showed linear relationship between
and ET tip to arch of aorta by USG had good degree of tracheal length and gestational age, body weight and
correlation. Sethi, et al. [11], using a similar method, length [13]. In term infants, trachea measures 5-6 cm,
found that the distance between ET tip to arch of aorta whereas in premature infants it can be as short as 3 cm
was 0.5–1 cm in 48 out of 53 correctly placed ET. [19, 20]. Therefore, a distance of 0.5-1 cm between ET
However, both the authors did not account for intra or tip and arch of aorta as suggested by previous authors
inter-observer variability during USG measurements. may not result in optimum placement of ET in all the
infants. Our study is in agreement with the biological
Lingle [8] described a modified technique to
plausibility of variation in tracheal length and reports
visualize the ET by using an USG ‘stand-off pad’ in 6
variation in the USG distance of ET tip from arch of
neonates, which obviated the need to extend the neck and
aorta in infants of various weight and gestation.
therefore reduce the risk of tube displacement. This
method was used only in six infants and lacks validation. We also compared the time required for obtaining X-
In two other studies, Dennington, et al. [10] and Najib, et ray film and USG. The mean (SD) time taken to conduct
al. [18] measured the distance between ET tip to an USG from the time of intubation was less than the time
anatomic equivalent of carina (superior portion of the required for availability of X ray film. Lesser mean (SD)
right pulmonary artery) and found good correlation with time taken to conduct USG as compared to radiograph

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SINGH, et al.
WHAT IS ALREADY KNOWN?
• Ultrasound is a feasible tool to determine endotracheal tube position and has good agreement with chest
radiograph.
WHAT THIS STUDY ADDS?
• This study provides normative data using ultrasound for the distance between endotracheal tube tip and arch
of aorta across various weight and gestation groups.
has previously been also reported [11]. The time required
for radiograph may vary depending on the setup,
availability of bedside machine and technician, and time
required to develop and deliver the X-ray film to the
clinician. On the contrary, point of care USG and
availability of personnel at the bedside avoids
unnecessary delay in confirmation of ET tip position.
In clinical practice, IL is predicted based on various
anthropometric parameters [2-4]. In our study, IL
correlated strongly with anthropometric parameters
(weight, CHL, OFC, NTL and SL). NRP guidelines till
2010 recommended weight-based formula given by
Tochen (Wt in kg + 6 cm) for deciding IL [21-23].
However, we found that the regression equation that best
predicted IL for optimum placement of ET is wt in kg +
4.95 cm. Our findings suggest that in our population
Tochen’s formula overestimates IL by approximately 1
cm. In another study from India, Tatwavedi, et al. [16]
also showed similar relationship between weight and IL
[IL=weight in kg +5 (r=0.81, P<0.001)]. Similarity in
Tatwavedi and our findings may be due to similar
population enrolled and the variation from other studies
may be attributed to racial difference in tracheal size [24-
26].
Weight may not be available immediately after birth
or during emergencies and may be fallacious in infants
who are edematous or dehydrated. One of the easily
measurable anthropometric parameter for prediction of
IL is NTL. It can be measured quickly as the two
landmarks, base of the nasal septum and tragus are well
defined and fixed. In addition, NTL measurement can be
done without disturbing the sick infant. As per the
regression equation in our study, IL can be predicted as
0.82 NTL (cm) +1.24. NRP 2015 guidelines also
endorse use of NTL to decide IL [27]. In our study, we
found good correlation between IL and other
anthropometric parameters (SL, OFC and CHL).
However, their regression equations seem complicated,
difficult to memorize and use in clinical practice. We
also observed that it was difficult to measure SL in
infants with marked chest retractions.
The mean and SD of the distance between ET tip and
INDIAN PEDIATRICS
OPTIMUM ET PLACEMENT BY USG
arch of aorta calculated in the pilot study are different
from the final results. Considering the final results,
sample size would have been smaller; however due to
paucity of literature, we were dependent on the pilot
study to calculate the required sample size. The
limitation of our study is that it only reports the
normative data but it does not validate what proportion
of ET would be optimally placed by using this data.
Our study reports the normative data of the distance
between optimally placed ET tip and arch of aorta by
USG in neonates. However, we emphasize that USG is a
skill-based technique and competency training is
required before this normative data can be used in
clinical practice. In addition, we conclude that IL can be
predicted based on various anthropometric parameters
such as weight, CHL, OFC, NTL and SL.
Contributors: AT: conceptualized and designed the study,
provided training to perform ultrasound, supervised data
collection, conducted statistical analysis and helped in
manuscript writing; PS, AT: performed ultrasounds, collected
data and drafted initial manuscript; NK, PG: study design,
supervised the conduct of the study and helped in manuscript
writing; NA: was involved in planning the study and analyzed
and validated the videos of ultrasound. All authors approved
the final manuscript.
Funding: None; Competing interest: None stated.
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15. Tochen ML. Orotracheal intubation in the newborn infant:
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16. Tatwavedi D, Nesargi SV, Shankar N, Rao S, Bhat SR.
Evaluation of body parameters for estimation of
endotracheal tube length in Indian infants. Eur J Pediatr.
2015;174:245-9.
17. Chowdhry R, Dangman B, Pinheiro JM. The concordance
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endotracheal tube position in infants. J Perinatol.
2015;35:481-4.
18. Najib K, Pishvs N, Amoozegar H, Pishdad P, Fallahzadeh
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E. Ultrasonographic confirmation of endotracheal tube
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Emergency Cardiovascular Care. Part 11: Neonatal
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Gallagher J, et al. Part 15: Neonatal Resuscitation:
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Tracheal length in adult human: The results of 100
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27. Wyckoff MH, Aziz K, Escobedo MB, Kapadia VS,
Kattwinkel J, et al. Part 13: Neonatal Resuscitation: 2015
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WEB FIG. 1 Measurement and calculation of insertional length (IL).

Average of ultrasound distances measured by observer 1 and observer 2

WEB FIG. 2 Bland-Altman plot of average distance measured by ultrasound by observer 1 versus observer 2.

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SINGH, et al. OPTIMUM ET PLACEMENT BY USG

(a) (b)

(c) (d)

(e)

WEB FIG. 3 (a) Linear relationship between insertional length and weight at enrolment, (b) Linear relationship between insertional
length and occipital frontal circumference, (c) Linear relationship between insertional length and nasal tragus length, (d) Linear
relationship between insertional length and crown heel length, and (e) Linear relationship between insertional length and sternal
length.

INDIAN PEDIATRICS VOLUME 56__MAY 15, 2019

 RESEARCH PAPER

Absolute Lymphocyte Count at the End of Induction as a Surrogate

Marker for Minimal Residual Disease in T-cell Acute Lymphoblastic
Leukemia
DEEPAM PUSHPAM1, ANITA CHOPRA2, VISHNUBHATLA SREENIVAS3, RAJIVE KUMAR2 AND SAMEER BAKHSHI1
From Departments of 1Medical Oncology, 2Laboratory Oncology and 3Biostatistics, Dr BRA Institute Rotary Cancer Hospital, All
India Institute of Medical Sciences, New Delhi, India.

Correspondence to: Dr Sameer
Bakhshi, Department of Medical
Oncology, Dr BR Ambedkar Institute
Rotary Cancer Hospital, All India
Institute of Medical Sciences, New
Delhi 110 029, India.
sambakh@hotmail.com
Received: June 06, 2018;
Initial review: June 28, 2018;
Accepted: January 24, 2019.
Objective: The relation of absolute lymphocyte count (ALC) with minimal residual disease
(MRD) in T cell – acute lymphoblastic leukemia (T-ALL) is not known. The objective of the
study was to correlate ALC with MRD, steroid-response and complete remission (CR).
Methods: De-novo T- ALL patients (age 1-18 y) recruited prospectively; 52 enrolled, 9
excluded, and 43 analyzed. 39 achieved CR and MRD was available for 28 patients; 23 were
MRD negative. Results: ALC did not correlate with steroid response and CR. Median (range)
ALC at the end of induction was significantly higher in patients who were MRD negative
compared to MRD positive [1.24 (0.12, 6.69) vs 0.62 (0.15, 0.87); P=0.03], respectively.
Patients having ALC ≥700 ×109 /L were significantly more likely to be MRD negative than
those with lower values (P= 0.028) Conclusion: Our study suggests that ALC is a favorable
factor, and may act as surrogate marker for MRD.
Keywords: Complete remission, Outcome, Prognosis, Steroid response.

Published online: March 17, 2019. PII:S097475591600127

A
bsolute lymphocyte count (ALC) has been
correlated with overall survival and event
free survival in acute lymphoblasti leukemia
(ALL). In T-cell Acute lymphoblastic
leukemia (T-ALL), studies have reported the data in
combination with B-ALL [1-7]; separate analysis of T-
ALL is lacking. Minimal residual disease (MRD) has
emerged as the most important predictor of outcome [8].
Current protocols use MRD for risk stratification and
treatment [9]. The drawback of MRD is that it is not
available at all centers. ALC is the most readily available
prognostic factor. Hence, we studied if ALC at baseline,
day 8, day 15 and at the end of induction (EOI) correlates
with MRD, steroid response (SR), complete response
(CR), and if it can be used as a surrogate marker for MRD
in T-ALL.
METHODS
We conducted a prospective study from July 2015 to June
2017. The study was approved by the institutional ethics
committee of our institute, and informed consent was
obtained from parents. Assent was obtained from those
patients who were above 7 years of age. Newly diagnosed
T-ALL cases with age ≥1 to 18 years were included.
Patients partially treated outside the institution and those
with relapsed ALL, were excluded.
Patients were treated on Indian Childhood
Collaborative Leukemia Group protocol (Unpublished-
CTRI/2015/12/006434). All patients were treated with
steroid prophase for seven days. Good SR was defined as
day 8 blasts in peripheral smear less than 1×109 /l. All
patients irrespective of SR and baseline features received
four doses of vincristine and eight doses of L-
asparaginase. MRD and bone marrow morphology was
assessed on day 35 at the EOI.
Total leukocyte count was done in MS95s [Melet
Schloesing Laboratories, Osny, France] at baseline, day
8, day 15 and day 35. Simultaneously, peripheral smear
was made and manual differential count was done to
determine the percentage of lymphocytes. ALC was
calculated as percentage of lymphocytes multiplied by
the total leukocyte count.
MRD assessment was done by 10-color flow
cytometry using stain-lyse-wash method [10]. Briefly,
bone marrow aspirate, was incubated for 15-20 minutes
in the dark at room temperature with antibody panel: anti-
CD20-FITC, CD123-PE, CD34-ECD, CD10-PC5, CD
19-PC7, CD58-APC, CD38-AF700, CD33-APC-AF
750, (Beckmann Coulter/Immunotech, Miami, FL,
USA), CD13-BV421, CD45-BV510 (Biolegend, San
Diego, USA). Samples were lysed using ammonium

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PUSHPAM, et al. MINIMAL RESIDUAL DISEASE IN T-ALL

chloridelysis buffer, then washed twice in phosphate- TABLE I BASELINE CHARACTERISTICS AND INDUCTION
buffered saline, re-suspended in 1 ml of phosphate- OUTCOMES IN CHILDREN WITH T-ALL (N=43)
buffered saline and analyzed by flow cytometry. All Characteristics Values
samples were processed within 24 hours after collection.
For flow cytometry analysis, at least 10,00,000 cells were *Age (y) 5 (1-18)
acquired in all cases, and data were stored in list mode Male sex (%) 37 (86)
files. Data analysis was done using Kaluza software *Hemoglobin (g/L) 90 (40-147)
(version 1.3). The cutoff levels used for MRD positivity *Total leucocyte count (×109 cells/L) 69 (1.3-500)
were ≥0.01%. MRD was said to be not evaluable when
*Platelet (×109 cells /L) 41 (5-505)
two consecutive bone marrow samples were not
analyzable for MRD. *ALC- day 0 (n=41) 5.01 (0-80.64)
*ALC- day 8 (n=43) 1.22 (0.117-15.142)
The association between continuous and categorical
*ALC- day 15 (n=37) 1.1 (0.248-12.384)
data was examined by Mann-Whitney U-test. For
analyzing cut-off of ALC with MRD Fisher’s Exact test *ALC- day 35 (n=39) 1.09 (0.117-6.691)
was used. Statistical analyses were carried out using Stata Steroid response
ver. 14 software. The criterion for significance in all Good 16 (37.2)
analyses was P <0.05.
Poor 27 (62.8)
RESULTS Complete remission 39 (91)

A total of 52 patients presented during the study period. Minimal residual disease (n=28)
Nine patients were excluded (5 deaths during induction Negative 23 (82)
and 4 opted for no treatment); and remaining 43 were ALC: Absolute lymphocyte count in X109 cells/L; Values in n (%) except
analyzed. Baseline characteristics and induction outcome *median (range).
are shown in Table I. Out of 43 patients, 39 (90.7%)
achieved CR. MRD data were available for 28 patients. were analyzed. Patients having ALC ≥0.7×109/l (n=16,
Correlation of ALC with a day 8 SR, CR and MRD is MRD negative 15) were significantly more likely to be
shown in Table II. At the EOI ALC was significantly MRD negative than patients with ALC<0.7×109/l (n=8,
higher in patients who were MRD negative, so cutoffs MRD positive 4), P= 0.028.

TABLE II CORRELATION OF ABSOLUTE LYMPHOCYTE COUNT WITH VARIOUS OUTCOME MEASURES IN CHILDREN WITH T-ALL (N=43)

ALC day 0 ALC day 8 ALC day 15 ALC day 35

*Steroid response
Poor 25 27 21 24
Median (range) 4.92 ( 0-60) 1.15 (0.12-4.90) 0.94 (0.24-12.38) 1.24 (0.12-6.50)
Good 16 16 16 15
Median (range) 6.35 (0.69-806.4) 1.72 (0.13-15.14) 1.19 (0.30-2.5) 0.87 (0.15-6.69)
*Complete remission
Yes 37 39 33 35
Median (range) 5.01 (0-80.64) 1.22 (0.12-15.14) 1.06 (0.25-12.38) 1.09 (0.12-6.69)
No 4 4 4 4
Median (range) 6.20 (0.69-25.65) 1.69 (0.78-2.99) 1.29 (0.93-1.80) 1.06 (0.53-1.94)
#MRD

Negative 23 23 19 19
Median (range) 5.01 (0-80.64) 1.26 (0.13-15.14) 0.94 (0.27-12.38) 1.24 (0.12-6.69)
Positive 5 5 3 5
Median (range) 2.88 (0.78-6.99) 0.8 (0.12-6.99) 0.45 (0.36-1.73) 0.62 (0.15-0.87)
ALC: Absolute lymphocyte count; MRD: Minimal residual disease. The numbers may not add up to 43 due to missing data; *P>0.5; #P=0.03 for
Minimal residual disease with ALC at day 35.

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PUSHPAM, et al.
DISCUSSION
We found that ALC at any point during induction does not
correlate with SR and CR. We propose following
explanations for our negative results. Mutations in
interleukin 7 receptor signalling pathways form the basis
of steroid resistance in T ALL [11]. So, lymphocytes may
have a limited role in the attainment of SR. In the
achievement of CR, instead of ALC, specific lymphocyte
subsets could be responsible in eradicating leukemic
cells. Since lymphocyte subsets were not analyzed and
correlated with CR, it might explain the lack of
relationship between CR and ALC. Another possibility is
that the number of patients, who failed to achieve CR,
was too small to yield statistically significant results.
MRD at the EOI is a favorable factor in T-ALL [12].
We interpret that ALC ≥0.7×109/l is a favorable factor
and ALC may act as a surrogate marker of MRD at the
EOI. Rolf, et al. [13] have shown that ALC at the EOI in
B-ALL has a very high correlation with CD3+T cells and
dendritic cells, which are known to mediate potent
antileukemic activity. Probably, lymphocytes have a role
in eradicating residual leukemic cells in T-ALL at the
EOI, as higher ALC correlated with MRD negative status.
The limitations of this study are that sample size is
small and follow-up duration is short for event free
survival, relapse and overall survival. Current study
provides evidence that in T-ALL, lymphocytes may have
an important role to play in attaining MRD negative
status at the EOI. As patients who are MRD negative at
the end of consolidation have more favorable outcomes
even if they had been MRD positive at the EOI, we
suggest further studies of on correlation of ALC with
MRD at the end of consolidation.
Contributors: DP: performed the research, designed the
research study, analysed the data and wrote the paper; AC:
performed the research, contributed essential reagents or tools;
VS: analyzed the data; RK: performed the research, contributed
essential reagents or tools; SB: performed the research, designed
the research study, analyzed the data, wrote the paper.
Funding: None; Competing Interest: None stated.
REFERENCES
1. Gupta A, Kapoor G, Jain S, Bajpai R. Absolute lymphocyte
count recovery independently predicts outcome in
childhood acute lymphoblastic leukemia: Experience from
a tertiary care cancer center of a developing country. J
Pediatr Hematol Oncol. 2015;37:e143-9.
2. De Angulo G, Yuen C, Palla SL, Anderson PM, Zweidler-
McKay PA. Absolute lymphocyte count is a novel
prognostic indicator in ALL and AML: Implications for
INDIAN PEDIATRICS
MINIMAL RESIDUAL DISEASE IN T-ALL
risk stratification and future studies. Cancer.
2008;112:407-15.
3. Anoceto Martínez A, González Otero A, Guerchicoff de
Svarch E, Arencibia Nuñez A, Jaime JC, Dorticos E, et al.
Contaje absoluto de linfocitos como factor pronóstico en la
leucemia aguda linfoblástica del niño. Ann Pediatría.
2012;76:10.e1-10.e6. [English abstract]
4. Hirase S, Hasegawa D, Takahashi H, Moriwaki K, Saito A,
Kozaki A, et al. Absolute lymphocyte count at the end of
induction therapy is a prognostic factor in childhood acute
lymphoblastic leukemia. Int J Hematol. 2015;102:594-601.
5. Rubnitz JE, Campbell P, Zhou Y, Sandlund JT, Jeha S,
Ribeiro RC, et al. Prognostic impact of absolute
lymphocyte counts at the end of remission induction in
childhood acute lymphoblastic leukemia: Absolute
lymphocyte counts in ALL. Cancer. 2013;119:2061-6.
6. Farkas T, Müller J, Erdelyi DJ, Csoka M, Kovacs GT.
Absolute lymphocyte count (ALC) after induction
treatment predicts survival of pediatric patients with acute
lymphoblastic leukemia. Pathol Oncol Res. 2017;23:889-
97.
7. Zeidler L, Zimmermann M, Moricke A, Meissner B,
Bartels D, Tschan C, et al. Low platelet counts after
induction therapy for childhood acute lymphoblastic
leukemia are strongly associated with poor early response
to treatment as measured by minimal residual disease and
are prognostic for treatment outcome. Haematologica.
2012;97:402-9.
8. Berry DA, Zhou S, Higley H, Mukundan L, Fu S, Reaman
GH, et al. Association of minimal residual disease with
clinical outcome in pediatric and adult acute lymphoblastic
leukemia: A meta-analysis. JAMA Oncol. 2017;3:
e170580.
9. Alexander S. Clinically defining and managing high-risk
pediatric patients with acute lymphoblastic leukemia. ASH
Educ Program Book. 2014;2014:181-9.
10. Li Y, Buijs-Gladdines JGCAM, Canté-Barrett K, Stubbs
AP, Vroegindeweij EM, Smits WK, et al. IL-7 Receptor
mutations and steroid resistance in pediatric T cell acute
lymphoblastic leukemia: A genome sequencing study.
PLOS Med. 2016;13:e1002200.
11. Chopra A, Pati H, Mahapatra M, Mishara P, Seth T, Kumar
S. Flow cytometry in myelodysplastic syndrome: analysis
of diagnostic utility using maturation pattern-based and
quantitative approaches. Ann Hematol. 2012;91:1351-62.
12. Schrappe M, Valsecchi MG, Bartram CR, Schrauder A,
Panzer-Grumayer R, Moricke A, et al. Late MRD response
determines relapse risk overall and in subsets of childhood
T-cell ALL: results of the AIEOP-BFM-ALL 2000 study.
Blood. 2011;118:2077-84.
13. Rolf N, Smolen KK, Kariminia A, Velenosi A, Fidanza M,
Strahlendorf C, et al. Absolute lymphocyte counts at end of
induction correlate with distinct immune cell
compartments in pediatric B cell precursor acute
lymphoblastic leukemia. Cancer Immunol Immunother.
2018;67:225-36.
383 VOLUME 56__MAY 15, 2019
 RESEARCH PAPER

Gallbladder Disease in Children: A 20-year Single-center Experience

ZENON POGORELIC1,2, MAJA ARALICA2, MIRO JUKIC1, VANDA ZITKO3, RANKA DESPOT3 AND IVO JURIC1,2
From Departments of 1Pediatric Surgery and 3Pediatrics, University Hospital of Split, Spinciceva 1; and 2University of Split,
School of Medicine, Soltanska 2; Split, Croatia..

Correspondence to: Zenon Pogorelic,
Department of Pediatric Surgery,
Head, University Hospital of Split,
Spinciceva 1, 21 000 Split, Croatia.
zpogorelic@gmail.com
Received: July 12, 2018;
Initial review: December 17, 2018;
Accepted: February 21, 2019.
Objective: Aim of this study was to examine the changes in incidence of pediatric
cholecystectomies. Methods: Based on a review of hospital-records, children were divided
into two groups regarding year of surgery (Group I: 1998-2007; Group II: 2008-2017) and their
characteristics were compared. Results: Number of cholecystecomies increased from 11 to
34. Median age increased from 11 to 15.5 years and mean BMI increased from 19.2 kg/m2 to
23.0 kg/m2. Hereditary spherocytosis decreased from 63.6% to 11.8% (P=0.001) of
indications for cholecystectomy, while proportion of cholesterol stones increased from 27.3%
to 70.6% (P=0.006). Frequency of laparoscopic cholecystectomy increased from 36.4% to
85.3% (P=0.001). Duration of hospital stay shortened from 8 to 4 days (P=0.008).
Conclusions: Number of pediatric cholecystectomies has significantly increased in the last
20 years, as well as average BMI of the observed population This probably signifies a
correlation between rising obesity rates and increase in frequency of symptomatic
cholelithiasis in children.
Keywords: Association; Cholecystectomy; Hereditary spherocytosis; Obesity.

Published online: March 17, 2019. PII:S097475591600128

I
n recent years, gallstone disease has been
increasingly diagnosed in the pediatric population
and the spectrum of pediatric biliary tract disease
has been changing [1]. Until recently, the majority
of pediatric gallstones were pigmented stones, related to
hemolytic diseases such as hereditary spherocytosis
[2,3]. In recent decades, the incidence of gallstone
disease in children has risen, principally related to the
epidemic of pediatric obesity [2,3]. Other than obesity,
improved survival of critically ill neonates who have
received long-term total parenteral nutrition or have
underlying abnormalities resulting in short-gut syndrome
is the new factor that may have led to an increased
incidence of cholelithiasis in the pediatric population [3].
The objective of our study was to document the
apparent increase in frequency of cholecystectomies in
children, and to establish the epidemiological,
demographic and clinical characteristics of children who
underwent cholecystectomy.
METHODS
The case records of 45 children (12 males) who underwent
cholecystectomy due to cholelithiasis from January 1998
to December 2017 at the Department of Pediatric Surgery,
University Hospital of Split, were retrospectively
reviewed. All patients with symptomatic cholelithiasis
who underwent cholecystectomy younger than 18 years of
age were enrolled in the study. Preoperative MR
cholangiography was performed in all of the patients.
Intraoperative cholangiogram was performed only in
patients with choledocholithiasis. The patients were
divided into two groups (children who were operated on
between 1998 and 2007 and children who underwent
surgery between 2008 and 2017) and compared by
demographic and anthropometric data, clinical findings,
risk factors for the disease, indications for surgery,
procedure type, treatment outcomes and complications.
The patient data are summarized in Table I.
Statistical analysis: The data were analyzed using the
Microsoft Excel for Windows Version 11.0 (Microsoft
Corporation) and SPSS 19.0 (IBM Corp, Armonk, NY)
software programs. Differences in median values of
quantitative variables between the groups of patients
were tested with Mann-Whitney U test. The Chi-square
test was used for the statistical analysis of categorical
data. All values of P<0.05 were considered to indicate
statistical significance.
RESULTS
Out of 45 cholecystectomies, 11 (24.4%) were conducted
in the first half of the study period (1998-2007) and 34
(75.6%) were conducted in the following 10 years (2008-
2017), resulting in a 3.1-fold increase in the incidence of
cholecystectomies (P=0.002). In both groups patients

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POGORELIC, et al. GALL BLADDER DISEASE IN CHILDREN IN CROATIA

TABLE I DEMOGRAPHIC AND CLINICAL CHARACTERISTICS OF DISCUSSION

CHILDREN UNDERGOING CHOLECYSTECTOMY, CROATIA.
Cholelithiasis in children used to be a rarity, but we have
Variable 1998-2007 2008-2017 found that that is no longer the case: in our study there
Baseline data was a three-fold increase in the number of children who
*Age, y 11 (10, 15) 15.5 (13, 16) underwent cholecystectomy in the last 10 years compared
Male sex 2 (18.2) 10 (29.4) to the previous 10-year period, thus affirming our
#height, cm hypothesis. The age median of children in our study has
144.7 (10.1) 169.5 (15.3)
#Weight
increased, while female sex has consistently prevailed in
42.9 (16.8) 66.9 (18.7)
both observed periods. The median BMI of the observed
#BMI, kg/m2 19.2 (3) 23.0 (5.1) children has increased in the last two decades, providing a
<18.5 1 (9.1) 8 (23.5) significant between-group difference, with an even more
18.5-25 8 (72.7) 11 (32.4) significant proportion of children falling into the obese
25-30 2 (18.2) 10 (29.4) category. Spectrophotometric analysis of gallstones
>30 0 5 (14.7) found in observed children’s gallbladders revealed that
Main indication for surgery cholesterol stones, which have been a rarity in the first
Biliary colic 4 (36.4) 17 (50.0) observed period, have increased to a vast majority in the
more recent decade.
Hereditary spherocytosis 7 (63.6) 4 (11.8)
Acute pancreatitis 0 7 (20.6) Retrospective character is the main limitation of this
Acute cholecystitis 0 4 (11.8) study, although we have implemented multiple
Obstructive jaundice 0 2 (5.9) plausibility checks and cross validations in our data
collection tool. Also, there is a relatively small number of
All data in no. (%) except *median (IQR) and #mean (SD). All P<0.05
for baseline data except sex difference. Indications for surgery all
patients included in the study, so further studies are
P>0.05 except hereditary spherocytosis (P<0.001). needed to analyze the same parameters on a larger
sample.
were predominantly female (81.8% vs 70.6%). The The increase in the incidence of cholecystectomies in
median age was 11 (range 6-17) years in the period 1998- the pediatric population was already recorded in multiple
2007, with an increase to 15.5 (range 7-17) years in the
period 2008-2017 (P=0.001). TABLE II MANAGEMENT AND OUTCOME IN CHILDREN
UNDERGOING CHOLECYSTECTOMY IN CROATIA.
The median body weight of children in the first group
was 42.9 (IQR 27.25, 54.75) kg, while it was 66.9 (IQR Variable 1998-2007 2008-2017
55, 79) kg in the second group (P=0.004). In the first *Laparoscopic surgery, n( %) 4 (36.4) 29 (85.3)
group the body weight of only 3 (27.3%) children was
Procedures, n( %)
above the 90th percentile, while there were 14 (41.2%)
such children in the second group, with 6 (17.6%) Intraoperative cholangiogram 2 (18.2) 3 (8.8)
children above the 97th percentile (P=0.02). The median Complications, n( %)
BMI of children in the first group was 19.2 kg/m2, with no Bile duct injury 0 (0) 1 (2.9)
obese children. In the second group, the average BMI was Hospitalization (median, IQR)
23.0 kg/m2, with 5 (14.7%) obese children (P=0.012). #Hospitalization, d 8 (2, 11) 4 (2, 5)
The most common indication for cholecystectomy in Laparoscopic procedure 4 (2, 5.5) 3 (2, 5)
the first 10-year period was hereditary spherocytosis Open procedure 12 (2, 13) 10 (6, 12)
(63.6%), while biliary colic (50.0%) was main indication #Type of gallstone, n( %)
in the second 10-year period, followed by acute Pigment 7 (63.6) 5 (14.7)
pancreatitis (20.6%), thus moving hereditary
Cholesterol 3 (27.3) 24 (70.6)
spherocytosis to the third place (11.8%).
Mixed 1 (9.1) 5 (14.7)
The results of spectrophotometric analysis of Histopathology report, n( %)
gallstones removed during the first observed period
Normal 4 (36.4) 3 (8.8)
showed a significantly greater proportion of pigment
Acute cholecystitis 0 (0) 4 (11.8)
stones (63.6%), whereas in the second observed period
the majority of gallstones were cholesterol stones Chronic cholecystitis 7 (63.6) 27 (79.4)
(70.6%) (P=0.006) (Table II). *P<0.001; #P<0.05.

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POGORELIC, et al.
studies carried out in the last three decades [2,6,7]. A
suggestion was found in literature that the reason for such
a rise could lie in the increasing use of abdominal
ultrasound, with a consequent increase in the ability to
identify gallstones and establish a diagnosis of
cholelithiasis [6,8]. Some authors speculated that the
most likely cause for this condition lied in the increase of
average BMI of children with gallstones based on its
increase in the general pediatric population [2,5,9-10]. In
an attempt to additionally correlate excessive body mass
with the increased incidence of cholelithiasis, the
composition of gallstones found in observed children’s
gallbladders was compared to other studies, in which
pigment stones were found to represent a vast majority
[11,12]. However, these studies were conducted in the
years accordant to the first period of our study, thus
yielding results consistent with our findings in the said
period. The prevalence of hereditary spherocytosis, and
consequently, its proportion among the indications for
cholecystectomy, strongly depends on geography, which
makes it inappropriate for comparison [2,4-5,7,10].
However, the trend of decrease in the share of hereditary
spherocytosis among the indications for cholecystectomy
is consistent with the findings in many other published
studies [6,13-15]. Children who do not suffer from
haemolysis, and therefore, most likely do not have
pigment but cholesterol gallstones, have a significantly
higher BMI [10]. This relates high BMI to cholesterol
stones, showing that the average BMI of children who
underwent cholecystectomy probably increased on
account of children who do not have a haemolytic
anaemia, which fits the changes of the share of hereditary
spherocytosis among the indications for the procedure.
In conclusion, this study suggests an association
between rising obesity rates in the pediatric population
and the increase in frequency of symptomatic
cholelithiasis in children. If this hypothesis is proven by
larger epidermiological studies, it would provide an
impetus to efforts to prevent this risk factor with lifestyle
changes.
Contributors: ZP: concepted and designed the study, analyzed
data, supervised and revised manuscript critically for important
intellectual content; MA: collected the data, helped in data
analysis, performed literature review and drafted the manuscript;
MJ: drafted the manuscript; VZ: collected the data and revised
manuscript critically; RD: collected the data and revised
manuscript critically.
INDIAN PEDIATRICS
GALL BLADDER DISEASE IN CHILDREN IN CROATIA
Funding: None; Competing interest: None stated.
REFERENCES
1. Peric B, Perko Z, Pogorelic Z, Kraljevic J. Laparoscopic
cholecystectomy in Cantonal Hospital Livno, Bosnia and
Herzegovina and University Hospital Center Split, Croatia.
Coll Antropol. 2010;34:125-8.
2. Khoo AK, Cartwright R, Berry S, Davenport M.
Cholecystectomy in English children: Evidence of an
epidemic (1997-2012). J Pediatr Surg. 2014;49:284-8.
3. Rothstein DH, Harmon CM. Gallbladder disease in
children. Semin Pediatr Surg. 2016;25:225-31.
4. Tuna Kirsaclioglu C, Cuhaci Cakir B, Bayram G, Akbiiik
F, Isik P, Tunc B. Risk factors, complications and outcome
of cholelithiasis in children: A retrospective, single-center
review. J Paediatr Child Health. 2016;52:944-9.
5. Campbell S, Richardson B, Mishra P, Wong M,
Samarkkody U, Beasley S, et al. Childhood cholecystec-
tomy in New Zealand: A multicenter national 10 year
perspective. J Pediatr Surg. 2016;51:264-7.
6. Waldhausen JHT, Benjamin DR. Cholecystectomy is
becoming an increasingly common operation in children.
Am J Surg. 1999;177:364-7.
7. Murphy PB, Vogt KN, Winick-Ng J, McClure JA, Welk B,
Jones SA. The increasing incidence of gallbladder disease
in children: A 20-year perspective. J Pediatr Surg.
2016;51:748-52.
8. Bogue CO, Murphy AJ, Gerstle JT. Risk factors,
complications, and outcomes of gallstones in children: A
single-center review. J Pediatr Gastroenterol Nutr.
2010;50:303-8.
9. Mehta S, Lopez M, Chumpitazi B. Clinical characteristics
and risk factors for symptomatic pediatric gallbladder
disease. Pediatrics. 2012;129:82-8.
10. Walker SK, Maki AC, Cannon RM, Foley DS, Wilson KM,
Galganski LA, et al. Etiology and incidence of pediatric
gallbladder disease. Surgery. 2013;154:927-31.
11. Kleiner O, Ramesh J, Huleihel M, Cohen B, Kantarovich
K, Levi C, et al. A comparative study of gallstones from
children and adults using FTIR spectroscopy and
fluorescence microscopy. BMC Gastroenterol. 2002;2:3.
12. Stringer MD, Taylor DR, Soloway RD. Gallstone
composition: Are children different? J Pediatr.
2003;142:435-40.
13. Rescorla FJ, Grosfeld JL. Cholecystitis and cholelithiasis
in children. Sem Pediatr Surg. 1992;1:98-106.
14. Bailey PV, Connors RH, Tracy TJ Jr, Sotelo-Avila C,
Lewis JE, Weber TR. Changing spectrum of cholelithiasis
and cholecystitis in infants and children. Am J Surg.
1989;158:585-8.
15. Holcomb GW Jr, Holcomb GW III. Cholelithiasis in
infants, children, and adolescents. Pediatr Rev.
1990;11:268-74.
386 VOLUME 56__MAY 15, 2019
 RESEARCH PAPER

Association between Helicobacter pylori Infection and Iron Deficiency

Anemia in School-aged Iranian Children
MOZHGAN ZAHMATKESHAN1, MEHRAN KARIMI2, BITA GERAMIZADEH3, SOMAYEH ESLAMINASAB4, ATEFEH
ESMAILNEJAD5 AND ALI REZA SAFARPOUR1
From 1Gastroenterohepatology Research Center, 2Hematology Research Center, 3Transplant Research Center, and 4Department of
Pediatrics, Shiraz University of Medical Science, Shiraz, Iran; and 5Department of Pathobiology, School of Veterinary Medicine,
Shiraz University, Shiraz, Iran.
Correspondence to: Objective: To find the relationship between Helicobacter pylori infection and iron deficiency
Dr Ali Reza Safarpour, Department of anemia in school-aged children. Methods: 71 children with dyspepsia, epigastric and vague
Internal Medicine, abdominal pain attending a tertiary medical center in Iran underwent upper gastrointestinal
endoscopy and were investigated for H. Pylori infection. Hemoglobin, mean corpuscular
Gastroenterohepatology Research
volume (MCV), serum ferritin, total iron binding capacity (TIBC) and serum iron levels were
Center, Shiraz University of Medical compared between children with or without H. pylori infection. Results: H. pylori infection was
Sciences, Shiraz, Iran. detected in 42 (59.1%) patients. Proportion of children with iron deficiency anemia was not
Asafarpour@sums.ac.ir statistically different between two groups (26.2% vs. 14.3%; P=0.48). While hemoglobin was
Received: July 16, 2018; significantly lower in children with H. pylori infection (P=0.01), there were no significant
Initial review: January 03, 2019: differences in serum level of ferritin, iron, mean corpuscular volume and total iron binding
Accepted: March 19, 2019. capacity. Conclusion: Presence of H. pylori does not seem to play an important role in the
pathophysiology and development of iron deficiency anemia in school-aged Iranian
population.
Keywords: Complications, Endoscopy, School children.

I
ron deficiency anemia is among the most common
nutritional deficiencies in the world, with an
estimated prevalence of more than 50% in children
living in developing countries [1]. Although poor
nutritional status, lack of access to iron supplements and
parasitic infestations have been proposed as major
etiologies of iron deficiency anemia in children [1,2],
infections agents have also been reported as contributory
factors [3]. Epidemiological studies have demonstrated
that the prevalence of Helicobacter pylori infection is
extremely high in the areas with high prevalence of iron
deficiency anemia [4-6]. Patients infected with H. pylori
are considered at a higher risk of iron deficiency and
reduced iron reserves [7,8]. On the other hand, some
studies have shown that resolution of H. pylori infection
would not significantly improve the iron status or reduce
the iron deficiency anemia in young children [9,10].
Thus, the exact relationship between H. pylori infection
and iron deficiency anemia is still a matter of debate. This
study aimed to evaluate the association between H. pylori
infection and iron deficiency anemia in school-aged
Iranian children.
METHODS
This cross-sectional study was conducted in the Pediatric
Gastroenterology Ward at Namazi Hospital, Shiraz
University of Medical Sciences, Iran, between November
2016 and May 2017. All children (age <18 y) who were
referred due to dyspepsia, epigastric and vague
abdominal pain and had not responded to medical therapy
(high dose H2 blockers or proton pump inhibitors (PPI)
were included. Patients having celiac disease, chronic or
hemorrhagic diseases, weight loss or inappropriate
weight gain, chronic diarrhea, intractable vomiting,
recent PPI or acid suppression therapy, and
gastrointestinal bleeding were excluded. All
experimental procedures were approved by the Ethical
Committee of Shiraz University of Medical Sciences.
Written informed consent was obtained from the parents
or guardians.
Detailed dietary history and history of intake of iron
supplements in the first two years of life was recorded. All
partcipants underwent an upper gastrointestinal
endoscopy, and two biopsy specimens were obtained
from the gastric antrum. The diagnosis of H. pylori
infection was based on presence of H. pylori in the
histopathological specimen, and concurrent positive
rapid urease examination. According to the above
mentioned criteria, patients were divided into two study
groups: H. pylori positive and H. pylori negative.
Hematological studies included hemoglobin (Hb),

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ZAHMATKESHAN, et al.
WHAT THIS STUDY ADDS?
• The presence of Helicobacter pylori infection may not play an important role in the pathophysiology and
development of iron deficiency anemia in children.
hematocrit, mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), and mean corpuscular
hemoglobin concentration (MCHC) were recorded for all
patients. Serum ferritin level was measured using electro
chemiluminescence (Elec Sys 2010 analyzer; Roche
Diagnostics, Mannheim, Germany), and Serum iron level
and total iron binding capacity (TIBC) were determined
by CobasIntegra700 analyzer (Roche Diagnostics, Basel,
Switzerland). Iron deficiency anemia (IDA) was defined
as serum ferritin level of <10 µg/L along with hemoglobin
level <-2SD for age [11].
Statistical analyses were performed using SPSS
software, version 19 (SPSS Inc., Chicago, IL, USA).
Independent student t test was used for comparison of
quantitative variables between groups. Chi-square or
Fisher’s exact test was used to compare the proportions.
P<0.05 was considered statistically significant.
RESULTS
Seventy-one children (27 boys) with a mean (SD) age of 8
(4.2) years were evaluated. Demographic characteristics
of all patients are presented in Table I. H. pylori infection
was detected in 42 (59.1%) participants. Proportion of
children with IDA was comparable between H. pylori
positive and negative patients (26.2% vs. 14.3%;
P=0.48). Serum level of hemoglobin was significantly
different between two study groups (P=0.01). No
significant differences were observed regarding MCV,
TIBC, serum ferritin and serum iron levels between H.
pylori positive and negative patients (Table II).
DISCUSSION
In the present study, no significant association was found
between H. pylori infection and iron deficiency anemia in
TABLE I DEMOGRAPHIC CHARACTERISTIC OF H. PYLORI POSITIVE
AND NEGATIVE IRANIAN SCHOOL CHILDREN
H. pylori positive H. pylori negative P value
(n=42) (n=29)
Age (y)* 8.5 (4.2) 8.9 (4.3) 0.88
Male sex* 16 (38.1) 11 (37.9) 0.33
Weight 26.6 (12.9) 31.2 (14.1) 0.40
Body mass index 16.7 (3.2) 17.1 (3.2) 0.62
Data in mean (SD) or *n (%).
INDIAN PEDIATRICS
H. PYLORI INFECTION AND ANEMIA
a group of Iranian school-aged children. Though the
mean hemoglobin was lower in H.pylori positive cases,
the difference was not significant for other hematological
parameters, including serum ferritin, MCV and TIBC.
These results should be interpreted with caution in
view of small sample size and observational data. Larger
studies and interventional trials may further clarify
association between iron deficiency and H. pylori
infection in children. These results are in contrast with
some recent studies indicating some associations between
H. pylori infection and iron deficiency anemia [7,8].
However, in agreement with our findings, H. pylori
seropositivity was not associated with iron deficiency in
Estonian children aged 7–18 years [9]. H. pylori infection
was neither a cause of iron deficiency anemia nor a reason
for treatment failure of iron supplementation in
Bangladeshi children [10]. Zamani, et al. [12] also
reported no significant association between serum ferritin
level and antibody titer against H. pylori bacteria in
school-aged children in Tehran province, Iran. Variations
among different studies might be as a result of
confounding variables such as different species of H.
pylori bacteria. Cag-PAL positive isolates are mostly
associated with peptic ulcer and gastrointestinal
symptoms, while some H. pylori species are related to the
gastric ulcer and iron deficiency anemia [13].
Furthermore, serum ferritin which is used for identifying
the iron deficiency anemia is an acute phase protein and its
level is influenced by other factors besides iron deficiency.
In conclusion, presence of Helicobacter pylori may
not play an important role in the pathophysiology and
TABLE II HEMATOLOGICAL INDICES IN H. PYLORI POSITIVE AND
NEGATIVE IRANIAN SCHOOL CHILDREN
H. pylori positive H. pylori negative P value
(n=42) (n=29)
Low (<10 µg/L) 0 5 (14.3) 0.06
ferritin*
Low serum iron* 14 (34.1) 9 (31.1) 1.00
MCV 87.9 (5.99) 77.9 (6.71) 0.85
Hemoglobin (g/dL) 12.1 (1.3) 12.9 (1.2) 0.01
Low TIBC* 1 (2.4) 0 1.00
Data presented as mean (SD) on *n (%); MCV: mean corpuscular
volume; TIBC: total iron binding capacity.
388 VOLUME 56__MAY 15, 2019
ZAHMATKESHAN, et al.
development of iron deficiency anemia in school-aged
Iranian population. At present, severity for H. pylori
infection in children with iron deficiency anemia is not
justified.
Contributors: MZ and BC: conception and design of study,
interpretation of data; MK: acquisition, analysis, and
interpretation of data; BG: design of study, interpretation of data;
SE: acquisition of data, drafting the manuscript; AE:
experimental procedures, statistical analysis of the manuscript;
ARS: conception and design of study, critical revision of the
manuscript for important intellectual content. He could also be
approached for access to the raw data. All authors contributed to,
and approved the final version of the manuscript.
Funding: None; Competing interest: None stated.
REFERENCES
1. Stoltzfus RJ. Iron deficiency: Global prevalence and
consequences. Food Nutr Bull. 2003;24:S99-103.
2. Stoltzfus RJ, Chwaya HM, Tielsch JM, Schulze KJ,
Albonico M, Savioli L. Epidemiology of iron deficiency
anemia in Zanzibari schoolchildren: The importance of
hookworms. Am J Clin Nutr. 1997;65:153-9.
3. Baggett HC, Parkinson AJ, Muth PT, Gold BD, Gessner
BD. Endemic iron deficiency associated with Helicobacter
pylori infection among school-aged children in Alaska.
Pediatrics. 2006;117:396-404.
4. Muhsen K, Barak M, Shifnaidel L, Nir A, Bassal R, Cohen
D. Helicobacter pylori infection is associated with low
serum ferritin levels in Israeli Arab children: A
seroepidemiologic study. J Pediatr Gastroenterol Nutr.
2009;49:262-4.
5. Muhsen K, Cohen D. Helicobacter pylori infection and
INDIAN PEDIATRICS
H. PYLORI INFECTION AND ANEMIA
iron stores: A systematic review and meta-analysis.
Helicobacter. 2008;13:323-40.
6. Qu XH, Huang XL, Xiong P, Zhu CY, Huang YL, Lu LG,
et al. Does Helicobacter pylori infection play a role in iron
deficiency anemia? A meta-analysis. World J
Gastroenterol. 2010;16:886-96.
7. Seo JK, Ko JS, Choi KD. Serum ferritin and Helicobacter
pylori infection in children: a sero-epidemiologic study in
Korea. J Gastroenterol Hepatol. 2002;17:754-7.
8. Mourad-Baars P, Hussey S, Jonel NL. Helicobacter pylori
Infection and childhood. Helicobacter. 2010;15:53-9.
9. Vendt N, Kool P, Teesalu K, Lillemäe K, Maaroos HI,
Oona M. Iron deficiency and Helicobacter pylori infection
in children. Acta Paediatrica. 2011;100:1239-43.
10. Sarker SA, Mahmud H, Davidsson L, Alam NH, Ahmed T,
Alam N, et al. Causal relationship of Helicobacter pylori
with iron-deficiency anemia or failure of iron
supplementation in children. Gastroenterology. 2008;
35:1534-42.
11. Suoglu OD, Gokce S, Saglam AT, Sokucu S, Saner G.
Association of Helicobacter pylori infection with
gastroduodenal disease, epidemiologic factors and iron-
deficiency anemia in Turkish children undergoing endo-
scopy, and impact on growth. Pediatr Int. 2007;49:858-63.
12. Zamani A, Shariat M, Oloomi Yazdi Z, Bahremand S,
Akbari Asbagh P, Dejakam A. Relationship between
Helicobacter pylori infection and serum ferritin level in
primary school children in Tehran-Iran. Acta Med Iran.
2011;49:314-8.
13. Baysoy G, Ertem D, Ademoglu E, Kotiloglu E, Keskin S,
Pehlivanoglu E. Gastric histopathology, iron status and
iron deficiency anemia in children with Helicobacter pylori
infection. J Pediatr Gastroenterol Nutr. 2004;38:146-51.
389 VOLUME 56__MAY 15, 2019
 RESEARCH PAPER

Association between Helicobacter pylori Infection and Iron Deficiency

Anemia in School-aged Iranian Children
MOZHGAN ZAHMATKESHAN1, MEHRAN KARIMI2, BITA GERAMIZADEH3, SOMAYEH ESLAMINASAB4, ATEFEH
ESMAILNEJAD5 AND ALI REZA SAFARPOUR1
From 1Gastroenterohepatology Research Center, 2Hematology Research Center, 3Transplant Research Center, and 4Department of
Pediatrics, Shiraz University of Medical Science, Shiraz, Iran; and 5Department of Pathobiology, School of Veterinary Medicine,
Shiraz University, Shiraz, Iran.
Correspondence to: Objective: To find the relationship between Helicobacter pylori infection and iron deficiency
Dr Ali Reza Safarpour, Department of anemia in school-aged children. Methods: 71 children with dyspepsia, epigastric and vague
Internal Medicine, abdominal pain attending a tertiary medical center in Iran underwent upper gastrointestinal
endoscopy and were investigated for H. Pylori infection. Hemoglobin, mean corpuscular
Gastroenterohepatology Research
volume (MCV), serum ferritin, total iron binding capacity (TIBC) and serum iron levels were
Center, Shiraz University of Medical compared between children with or without H. pylori infection. Results: H. pylori infection was
Sciences, Shiraz, Iran. detected in 42 (59.1%) patients. Proportion of children with iron deficiency anemia was not
Asafarpour@sums.ac.ir statistically different between two groups (26.2% vs. 14.3%; P=0.48). While hemoglobin was
Received: July 16, 2018; significantly lower in children with H. pylori infection (P=0.01), there were no significant
Initial review: January 03, 2019: differences in serum level of ferritin, iron, mean corpuscular volume and total iron binding
Accepted: March 19, 2019. capacity. Conclusion: Presence of H. pylori does not seem to play an important role in the
pathophysiology and development of iron deficiency anemia in school-aged Iranian
population.
Keywords: Complications, Endoscopy, School children.

I
ron deficiency anemia is among the most common
nutritional deficiencies in the world, with an
estimated prevalence of more than 50% in children
living in developing countries [1]. Although poor
nutritional status, lack of access to iron supplements and
parasitic infestations have been proposed as major
etiologies of iron deficiency anemia in children [1,2],
infections agents have also been reported as contributory
factors [3]. Epidemiological studies have demonstrated
that the prevalence of Helicobacter pylori infection is
extremely high in the areas with high prevalence of iron
deficiency anemia [4-6]. Patients infected with H. pylori
are considered at a higher risk of iron deficiency and
reduced iron reserves [7,8]. On the other hand, some
studies have shown that resolution of H. pylori infection
would not significantly improve the iron status or reduce
the iron deficiency anemia in young children [9,10].
Thus, the exact relationship between H. pylori infection
and iron deficiency anemia is still a matter of debate. This
study aimed to evaluate the association between H. pylori
infection and iron deficiency anemia in school-aged
Iranian children.
METHODS
This cross-sectional study was conducted in the Pediatric
Gastroenterology Ward at Namazi Hospital, Shiraz
University of Medical Sciences, Iran, between November
2016 and May 2017. All children (age <18 y) who were
referred due to dyspepsia, epigastric and vague
abdominal pain and had not responded to medical therapy
(high dose H2 blockers or proton pump inhibitors (PPI)
were included. Patients having celiac disease, chronic or
hemorrhagic diseases, weight loss or inappropriate
weight gain, chronic diarrhea, intractable vomiting,
recent PPI or acid suppression therapy, and
gastrointestinal bleeding were excluded. All
experimental procedures were approved by the Ethical
Committee of Shiraz University of Medical Sciences.
Written informed consent was obtained from the parents
or guardians.
Detailed dietary history and history of intake of iron
supplements in the first two years of life was recorded. All
partcipants underwent an upper gastrointestinal
endoscopy, and two biopsy specimens were obtained
from the gastric antrum. The diagnosis of H. pylori
infection was based on presence of H. pylori in the
histopathological specimen, and concurrent positive
rapid urease examination. According to the above
mentioned criteria, patients were divided into two study
groups: H. pylori positive and H. pylori negative.
Hematological studies included hemoglobin (Hb),

INDIAN PEDIATRICS 387 VOLUME 56__MAY 15, 2019

ZAHMATKESHAN, et al.
WHAT THIS STUDY ADDS?
• The presence of Helicobacter pylori infection may not play an important role in the pathophysiology and
development of iron deficiency anemia in children.
hematocrit, mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), and mean corpuscular
hemoglobin concentration (MCHC) were recorded for all
patients. Serum ferritin level was measured using electro
chemiluminescence (Elec Sys 2010 analyzer; Roche
Diagnostics, Mannheim, Germany), and Serum iron level
and total iron binding capacity (TIBC) were determined
by CobasIntegra700 analyzer (Roche Diagnostics, Basel,
Switzerland). Iron deficiency anemia (IDA) was defined
as serum ferritin level of <10 µg/L along with hemoglobin
level <-2SD for age [11].
Statistical analyses were performed using SPSS
software, version 19 (SPSS Inc., Chicago, IL, USA).
Independent student t test was used for comparison of
quantitative variables between groups. Chi-square or
Fisher’s exact test was used to compare the proportions.
P<0.05 was considered statistically significant.
RESULTS
Seventy-one children (27 boys) with a mean (SD) age of 8
(4.2) years were evaluated. Demographic characteristics
of all patients are presented in Table I. H. pylori infection
was detected in 42 (59.1%) participants. Proportion of
children with IDA was comparable between H. pylori
positive and negative patients (26.2% vs. 14.3%;
P=0.48). Serum level of hemoglobin was significantly
different between two study groups (P=0.01). No
significant differences were observed regarding MCV,
TIBC, serum ferritin and serum iron levels between H.
pylori positive and negative patients (Table II).
DISCUSSION
In the present study, no significant association was found
between H. pylori infection and iron deficiency anemia in
TABLE I DEMOGRAPHIC CHARACTERISTIC OF H. PYLORI POSITIVE
AND NEGATIVE IRANIAN SCHOOL CHILDREN
H. pylori positive H. pylori negative P value
(n=42) (n=29)
Age (y)* 8.5 (4.2) 8.9 (4.3) 0.88
Male sex* 16 (38.1) 11 (37.9) 0.33
Weight 26.6 (12.9) 31.2 (14.1) 0.40
Body mass index 16.7 (3.2) 17.1 (3.2) 0.62
Data in mean (SD) or *n (%).
INDIAN PEDIATRICS
H. PYLORI INFECTION AND ANEMIA
a group of Iranian school-aged children. Though the
mean hemoglobin was lower in H.pylori positive cases,
the difference was not significant for other hematological
parameters, including serum ferritin, MCV and TIBC.
These results should be interpreted with caution in
view of small sample size and observational data. Larger
studies and interventional trials may further clarify
association between iron deficiency and H. pylori
infection in children. These results are in contrast with
some recent studies indicating some associations between
H. pylori infection and iron deficiency anemia [7,8].
However, in agreement with our findings, H. pylori
seropositivity was not associated with iron deficiency in
Estonian children aged 7–18 years [9]. H. pylori infection
was neither a cause of iron deficiency anemia nor a reason
for treatment failure of iron supplementation in
Bangladeshi children [10]. Zamani, et al. [12] also
reported no significant association between serum ferritin
level and antibody titer against H. pylori bacteria in
school-aged children in Tehran province, Iran. Variations
among different studies might be as a result of
confounding variables such as different species of H.
pylori bacteria. Cag-PAL positive isolates are mostly
associated with peptic ulcer and gastrointestinal
symptoms, while some H. pylori species are related to the
gastric ulcer and iron deficiency anemia [13].
Furthermore, serum ferritin which is used for identifying
the iron deficiency anemia is an acute phase protein and its
level is influenced by other factors besides iron deficiency.
In conclusion, presence of Helicobacter pylori may
not play an important role in the pathophysiology and
TABLE II HEMATOLOGICAL INDICES IN H. PYLORI POSITIVE AND
NEGATIVE IRANIAN SCHOOL CHILDREN
H. pylori positive H. pylori negative P value
(n=42) (n=29)
Low (<10 µg/L) 0 5 (14.3) 0.06
ferritin*
Low serum iron* 14 (34.1) 9 (31.1) 1.00
MCV 87.9 (5.99) 77.9 (6.71) 0.85
Hemoglobin (g/dL) 12.1 (1.3) 12.9 (1.2) 0.01
Low TIBC* 1 (2.4) 0 1.00
Data presented as mean (SD) on *n (%); MCV: mean corpuscular
volume; TIBC: total iron binding capacity.
388 VOLUME 56__MAY 15, 2019
ZAHMATKESHAN, et al.
development of iron deficiency anemia in school-aged
Iranian population. At present, severity for H. pylori
infection in children with iron deficiency anemia is not
justified.
Contributors: MZ and BC: conception and design of study,
interpretation of data; MK: acquisition, analysis, and
interpretation of data; BG: design of study, interpretation of data;
SE: acquisition of data, drafting the manuscript; AE:
experimental procedures, statistical analysis of the manuscript;
ARS: conception and design of study, critical revision of the
manuscript for important intellectual content. He could also be
approached for access to the raw data. All authors contributed to,
and approved the final version of the manuscript.
Funding: None; Competing interest: None stated.
REFERENCES
1. Stoltzfus RJ. Iron deficiency: Global prevalence and
consequences. Food Nutr Bull. 2003;24:S99-103.
2. Stoltzfus RJ, Chwaya HM, Tielsch JM, Schulze KJ,
Albonico M, Savioli L. Epidemiology of iron deficiency
anemia in Zanzibari schoolchildren: The importance of
hookworms. Am J Clin Nutr. 1997;65:153-9.
3. Baggett HC, Parkinson AJ, Muth PT, Gold BD, Gessner
BD. Endemic iron deficiency associated with Helicobacter
pylori infection among school-aged children in Alaska.
Pediatrics. 2006;117:396-404.
4. Muhsen K, Barak M, Shifnaidel L, Nir A, Bassal R, Cohen
D. Helicobacter pylori infection is associated with low
serum ferritin levels in Israeli Arab children: A
seroepidemiologic study. J Pediatr Gastroenterol Nutr.
2009;49:262-4.
5. Muhsen K, Cohen D. Helicobacter pylori infection and
INDIAN PEDIATRICS
H. PYLORI INFECTION AND ANEMIA
iron stores: A systematic review and meta-analysis.
Helicobacter. 2008;13:323-40.
6. Qu XH, Huang XL, Xiong P, Zhu CY, Huang YL, Lu LG,
et al. Does Helicobacter pylori infection play a role in iron
deficiency anemia? A meta-analysis. World J
Gastroenterol. 2010;16:886-96.
7. Seo JK, Ko JS, Choi KD. Serum ferritin and Helicobacter
pylori infection in children: a sero-epidemiologic study in
Korea. J Gastroenterol Hepatol. 2002;17:754-7.
8. Mourad-Baars P, Hussey S, Jonel NL. Helicobacter pylori
Infection and childhood. Helicobacter. 2010;15:53-9.
9. Vendt N, Kool P, Teesalu K, Lillemäe K, Maaroos HI,
Oona M. Iron deficiency and Helicobacter pylori infection
in children. Acta Paediatrica. 2011;100:1239-43.
10. Sarker SA, Mahmud H, Davidsson L, Alam NH, Ahmed T,
Alam N, et al. Causal relationship of Helicobacter pylori
with iron-deficiency anemia or failure of iron
supplementation in children. Gastroenterology. 2008;
35:1534-42.
11. Suoglu OD, Gokce S, Saglam AT, Sokucu S, Saner G.
Association of Helicobacter pylori infection with
gastroduodenal disease, epidemiologic factors and iron-
deficiency anemia in Turkish children undergoing endo-
scopy, and impact on growth. Pediatr Int. 2007;49:858-63.
12. Zamani A, Shariat M, Oloomi Yazdi Z, Bahremand S,
Akbari Asbagh P, Dejakam A. Relationship between
Helicobacter pylori infection and serum ferritin level in
primary school children in Tehran-Iran. Acta Med Iran.
2011;49:314-8.
13. Baysoy G, Ertem D, Ademoglu E, Kotiloglu E, Keskin S,
Pehlivanoglu E. Gastric histopathology, iron status and
iron deficiency anemia in children with Helicobacter pylori
infection. J Pediatr Gastroenterol Nutr. 2004;38:146-51.
389 VOLUME 56__MAY 15, 2019