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ASSOCIATION BETWEEN SERUM ASCORBIC ACID
LEVELS AND SEVERITY OF DENGUE IN CHILDREN – A
CROSS SECTIONAL OBSERVATIONAL STUDY
Dissertation Submitted to
THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY, CHENNAI
In the fulfilment of the regulations for the award of the degree
Doctor of Medicine in Paediatrics
By
Dr. S. VIKRAM
(Reg. No: 201717502)
Under the guidance of
Dr. A. JAYAVARDHANA,
Professor of Paediatrics.
PSG INSTITUTE OF MEDICAL SCIENCE AND RESEARCH
THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY CHENNAI,
TAMILNADU
MAY 2020
ASSOCIATION BETWEEN SERUM ASCORBIC ACID
LEVELS AND SEVERITY OF DENGUE IN CHILDREN – A
CROSS SECTIONAL OBSERVATIONAL STUDY
Dissertation Submitted to
THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY, CHENNAI
In the fulfilment of the regulations for the award of the degree
Doctor of Medicine in Paediatrics
By
Dr. S. VIKRAM
(Reg. No: 201717502)
Under the guidance of
Dr. A. JAYAVARDHANA,
Professor of Paediatrics.
PSG INSTITUTE OF MEDICAL SCIENCE AND RESEARCH
THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY CHENNAI,
TAMILNADU
MAY 2020
CERTIFICATE
This is to certify that the thesis entitled “ASSOCIATION BETWEEN
SERUM ASCORBIC ACID LEVELS AND SEVERITY OF DENGUE IN
CHILDREN – A CROSS SECTIONAL OBSERVATIONAL STUDY” is a
bonafide work of Dr. S. VIKRAM done under the direct guidance and
supervision of Dr. A. JAYAVARDHANA, Professor, Department of
Paediatrics, PSG Institute of Medical Sciences and Research, Coimbatore in
fulfilment of the regulations of The Tamil Nadu Dr.MGR Medical University
for the award of M.D. degree in Paediatrics.
Prof. Dr. NEELAKANDAN Dr. RAMALINGAM
Head of the Department DEAN
Dept. of Paediatrics PSGIMS&R
PSGIMS&R
CERTIFICATE BY THE GUIDE
This is to certify that the thesis entitled “ASSOCIATION BETWEEN
SERUM ASCORBIC ACID LEVELS AND SEVERITY OF DENGUE IN
CHILDREN – A CROSS SECTIONAL OBSERVATIONAL STUDY” is a
bonafide work of Dr. S. VIKRAM done under my direct guidance and
supervision in the department of Paediatrics, PSG Institute of Medical Sciences
and Research, Coimbatore in the fulfilment of the regulations of The Tamil
Nadu Dr.MGR Medical University for the award of MD degree in Paediatrics.
Dr. A. JAYAVARDHANA,
Professor
Dept. of Paediatrics,
PSGIMS&R.
DECLARATION
I, Dr. S. Vikram hereby declare that this dissertation entitled
“ASSOCIATION BETWEEN SERUM ASCORBIC ACID LEVELS AND
SEVERITY OF DENGUE IN CHILDREN – A CROSS SECTIONAL
OBSERVATIONAL STUDY” was prepared by me under the direct guidance
and supervision of Dr. A. JAYAVARDHANA, Professor, Department of
Paediatrics, PSG Institute of Medical Sciences and Research, Coimbatore.
The dissertation is submitted to The Tamil Nadu Dr.MGR Medical
University, Chennai, in fulfilment of the University regulations for the award of
M.D. degree in Paediatrics. This dissertation has not been submitted for the
award of any other Degree or Diploma.
DR. S. VIKRAM
ETHICAL COMMITTEE APPROVAL
PLAGIARISM SOFTWARE REPORT
CERTIFICATE – II
This is to certify that this dissertation work titled
“ASSOCIATION BETWEEN SERUM ASCORBIC ACID LEVELS AND
SEVERITY OF DENGUE IN CHILDREN – A CROSS SECTIONAL
OBSERVATIONAL STUDY” of the candidate Dr. S. VIKRAM with
Registration Number 201717502 for the award of MD degree in the
branch of Paediatrics. I personally verified the urkund.com website for
the purpose of plagiarism check. I found that the uploaded thesis file
contains from introduction to conclusion pages and results shows 2
percentage of plagiarism in the dissertation.
Guide & Supervisor Sign with Seal,
ACKNOWLEDGEMENT
I am extremely grateful to my guide, Dr. A. Jayavardhana, for his
timely advice and guidance at various stages of my dissertation right from
choosing the topic till analysis of results.
I would like to extend my whole-hearted gratitude to all faculties in the
department; First and foremost, to my HOD, Dr. Neelakandan for his
supervision and constant encouragement. I also thank my teachers Professor
Dr. K. Jothilakshmi, Dr. S. Ramesh, Dr. P. Venkateswaran and Dr. N.T
Rajesh for their care and constant support in completion of this thesis.
I thank Dr. Bharathi Elangovan for her unflinching support and timely
help while writing the dissertation. I also thank her father Dr. Elangovan for
his valuable data analysis.
I cannot forget all the help made by my Assistant Professors Dr.
Nirmala, Dr. Indumathi, Dr. Muruganantham, Dr. Vadivel Vinoth, Dr.
Gayathri, Dr. Sudhakar, Dr. Saranyaa, Dr. Suchitra, Dr. Lavanya, Dr.
Deepthi Shetty and Dr. Sumathi and would like to use this opportunity to
profusely thank all of them.
I would like to make a special mention of Dr. Veda Senthil Velan, Dr.
Shruthi Ravikumar, Dr. Jayamkondan and all seniors who were very
supportive and friendly. I thank them all for the help they did during the initial
stages of my thesis.
I would also like to thank my colleagues Dr. Christina, Dr. Induja, Dr.
Lavanya, Dr. Arun Prasath, Dr. Naveenkumar, Dr. Lingeshwaran and Dr.
Srinidhi, Dr. Aruna Rani and all other juniors for helping me in sample
collection and covering up for me during my ward duties.
This study would not be possible without funding and logistics from the
institution for which I express my sincere gratitude to the dean Dr.
Ramalingam and Dr. Sudha Ramalingam. A special mention to all the staff at
CMMT lab who were always happy to help.
I express my sincere gratitude to Dr. Arun Padmanandan, Assistant
Professor, Department of SPM for his valuable time for the data analysis.
I thank Dr. Gayathri, Associate Professor, Department of Biochemistry
for helping me procure reagents and for teaching me about vitamin C
estimation.
I cannot forget the help of Mr. Sivakumar of Stanes Phytolab and his
team who were very co-operative and I take this opportunity to thank them.
I would like to exceptionally thank all the staffs in the Paediatric ward,
PICU and special wards for helping me obtain samples for my study. I thank the
staffs in the OPD for all their help and support.
I thank my wife, Dr. Janaki. V for always being there for me. Her part in
the completion of this thesis was very crucial and valuable.
I would be failing in my duty if I do not immensely thank my beloved
parents, Mr.R. Sriram and Mrs. Jaya Sriram and my brother, Mr.
Vaikunth.S and his wife Mrs. V. Suryalakshmi for making me what I am
today, and offering constant support all along without which this thesis would
not have been completed.
And I thank the almighty for His grace. I also thank my spiritual guru
Mr. Anandan for moral support. I thank all my friends and well-wishers.
Last but not the least I thank all my patients without whose consent, I
would not have been able to complete this study.
TABLE OF CONTENTS
SL.NO. CONTENT PAGE NO.
1 INTRODUCTION 1 - 14
2 RESEARCH QUESTION 15
3 REVIEW OF LITERATURE 16 - 37
4 MATERIALS AND METHODS 38 - 47
5 RESULTS 48 - 69
6 DISCUSSION 70 - 74
7 CONCLUSION 75
8 BIBLIOGRAPHY
9
ANNEXURES
A) Consent Forms
B) Proforma
C) Vitamin C Estimation by DCPIP
D) Master chart
E) List of Abbreviations
LIST OF TABLES
Table 1: Agewise distribution of cases and controls.........................................50
Table 2: Genderwise distribution of cases and controls....................................53
Table 3: Comparison of laboratory parameters between cases and controls.....54
Table 4: Comparison of mean Vitamin C levels in Dengue and in
controls................................................................................................................57
Table 5: Distribution of cases according to severity of dengue.........................58
Table 6: Distribution of primary and secondary dengue based on severity.......60
Table 7: Comparison of baseline characteristics among mild, moderate and
severe dengue......................................................................................................61
Table 8: Comparison of baseline laboratory parameters among mild, moderate
and severe dengue...............................................................................................62
Table 9: Comparison of Vitamin C levels in mild, moderate and severe
dengue.................................................................................................................64
Table 10: Association between severity of dengue and serum ascorbic acid
levels...................................................................................................................65
Table 11: Association between mild dengue and ascorbic acid levels..............67
Table 12: Association between moderate dengue and ascorbic acid levels.......68
Table 13: Association between severe dengue and ascorbic acid levels...........69
LIST OF FIGURES
Figure 1Dengue around the world (Source: CDC) ............................................. 3
Figure 2 Incidence of dengue in India - From 1998 to 2014 (Source: Dengue
burden in India by Mutheneni et al) (4) .............................................................. 5
Figure 3 Seasonal Trend of occurrence of cases. (Source: National Guidelines
for clinical management of dengue fever 2014 - NVBDCP) .............................. 6
Figure 4 Course of the illness (Source: CDC) ................................................... 8
Figure 5 Patho-physiology of dengue fever (Source: NVBDCP National
guidelines for clinical management of dengue fever) ....................................... 10
Figure 6 Classification of dengue fever (Source: NVBDCP guidelines for
clinical management of dengue fever)................................................................43
Figure 7 Flowchart (Proposed methodology)....................................................47
Figure 8 Flowchart of the actual study..............................................................49
Figure 9 Agewise distribution of Dengue cases................................................51
Figure 10 Agewise distribution of Control........................................................52
Figure 11 Genderwise distribution of Cases and Controls................................53
Figure 12 Distribution of Vitamin C levels among Cases.................................55
Figure 13 Distribution of Vitamin C levels among Control..............................56
Figure 14 Classification of Dengue cases clinically..........................................59
Figure 15 Primary and Secondary Dengue........................................................60
Figure 16 Outcome of all dengue cases.............................................................63
Figure 17 Vitamin C levels in mild, moderate and severe dengue – comparison
using cut-off of 0.6mg/dl....................................................................................64
Figure 18 Vitamin C levels in mild dengue – number of cases in normal and
above normal range............................................................................................67
Figure 19 Vitamin C levels in moderate dengue – number of cases in normal
and above normal range......................................................................................68
Figure 20 Vitamin C levels in severe dengue – number of cases in normal and
above normal range............................................................................................67
INTRODUCTION
1
Introduction:
Dengue fever is a viral infection caused by arthropod-borne virus of the
flaviviridae family, occurring in countries with temperate climate (1).
There are four identified serotypes of dengue virus (DENV1, DENV2, DENV3,
and DENV4). Each serotype has several genotypes – 3 in DENV1, 2 in
DENV2, 4 in DENV3 and 4 in DENV4. The virus has three structural protein
genes (coding for core, membrane associated and an envelope protein of the
nucleocapsid) and seven non-structural proteins-NS1, NS2A, NS2B, NS3,
NS4A, NS4B and NS5, of which only NS1 antigen is found to interact with host
immune system and the function of others is not well-characterised (2).
The disease is spread by the bite of Aedes mosquitoes (1). Aedes aegypti is the
predominant vector species in dengue fever although other species like Aedes
albinopictus are significant vectors. These mosquitoes are usually day-time
biters and breed in clean water. Trans-ovarian transmission of the virus (i.e., the
ability of mosquitoes born out of infected mosquitoes to act as vectors) also
contribute to the exponential infection occurring during outbreaks (3). The
mosquitoes usually fly only over a short distance and this is the reason why
there is clustering of cases noted over a small well-defined geographical area
during outbreaks (2).
2
The clinical presentation of dengue shows a wide spectrum of disease
manifestation, varying from asymptomatic infection (80%) to severe dengue
(less than 5% of total infections) (4). The occurrence of fatality is less than 1%
of all infections and occurs in a fraction of the severe cases who do not receive
appropriate and timely treatment(4).
The immune reaction to the virus is implicated in the severity of the disease.
The central hallmark of severe dengue is “capillary leaks” wherein there is
increased capillary permeability causing leakage of plasma from the
intravascular compartment to the interstitial and extravascular compartment
leading to the manifestations of “Dengue-shock syndrome”.
Background of the problem
The WHO has considered dengue as a global threat in tropic and subtropical
nations (1). It is a major public health concern in south-east Asian countries,
especially in India over the last decade (1).
Figure 1Dengue around the world (Source: CDC)
The incidence of dengue has increased 30
expansion to new countries and from urban to rural setting. An estimated 50
million infections occur annually and 2.5 million people are at risk in endemic
countries (1). The increase in incidence
dengue over the last decade
overcrowding, population growth rate, inefficient control of mosquitoes and
lack of access to proper health
The disease was seen in people of all age groups and had no racial or ethnical
predilections. The severity of the disease had b
proportionately more mortality in infants and the elderly. In children the risk of
severe dengue is more than that in adults probably due to their robust immune
response and co-morbidities in elderly complement the disease severit
Dengue around the world (Source: CDC)
The incidence of dengue has increased 30-fold in last 50 years with geographic
expansion to new countries and from urban to rural setting. An estimated 50
million infections occur annually and 2.5 million people are at risk in endemic
The increase in incidence of infection and deaths related to
has been attributed to unplanned urbanization,
overcrowding, population growth rate, inefficient control of mosquitoes and
lack of access to proper health-care facilities (1).
The disease was seen in people of all age groups and had no racial or ethnical
predilections. The severity of the disease had bimodal distribution, with
proportionately more mortality in infants and the elderly. In children the risk of
severe dengue is more than that in adults probably due to their robust immune
morbidities in elderly complement the disease severit
3
fold in last 50 years with geographic
expansion to new countries and from urban to rural setting. An estimated 50
million infections occur annually and 2.5 million people are at risk in endemic
and deaths related to
has been attributed to unplanned urbanization,
overcrowding, population growth rate, inefficient control of mosquitoes and
The disease was seen in people of all age groups and had no racial or ethnical
imodal distribution, with
proportionately more mortality in infants and the elderly. In children the risk of
severe dengue is more than that in adults probably due to their robust immune
morbidities in elderly complement the disease severity (1).
4
There was no significant difference noted in the number of cases and proportion
of severity between urban and rural areas (5).
In developing countries like India, it causes significant economic burden to the
country. In the context of dengue vaccine still under trial, it is important to
identify a modifiable risk factor for development of severe dengue, so that we
can prevent occurrence of severe manifestations (6).
The Problem Statement
South-East Asia including India is declared to be endemic to Dengue by the
WHO(1). Dengue is endemic in all states of India except Lakshadweep (figure
2) (2). The economic burden amounts to 548 million USD every year on
medical expenses for dengue and indirect costs taking into account DALYs lost
for the disease leads to a whopping 1.11 billion USD every year (7).
5
Figure 2 Incidence of dengue in India - From 1998 to 2014 (Source: Dengue
burden in India by Mutheneni et al) (4)
6
Cyclical outbreaks or epidemics are reported in India, with an increase in
infection occurring during monsoon seasons (Figure3). During the rainy season,
water is collected in outdoor reservoirs like plastic cups, coconut kernels,
mortars, buckets etc., which acts as a source for mosquito breeding(8). The
sources of mosquito breeding in urban areas were refrigerator trays, air-
conditioners, flower vases, pots for storing drinking water etc. In India, seasonal
outbreaks has been occurring with increased numbers of symptomatic cases
especially with increasing morbidity and mortality in various parts of India
including Delhi, Uttar Pradesh, Maharashtra, Karnataka and Tamil Nadu. The
latest outbreak in South India was in 2017, mainly in Chennai and surrounding
areas (5).
Figure 3 Seasonal Trend of occurrence of cases. (Source: National
Guidelines for clinical management of dengue fever 2014 - NVBDCP)
7
Overview of clinical course of dengue
The severity of the disease varies from asymptomatic sero-conversion to
symptomatic infection. It is a dynamic infection and systemic disease.
Symptomatic infection again has a wide clinical spectrum which may be
undifferentiated fever to severe clinical manifestations.
The diagnosis of the disease in resource-limited countries and management is
mainly clinical.
Capillary leakage is the main clinical feature and thrombocytopenia is the usual
laboratory finding in dengue fever.
The illness begins abruptly after the incubation period which usually lasts for 4
to 10 days.
There are three phases during the illness viz., febrile phase, critical phase and
recovery.
The following diagram explains the course of the disease through each phase
along with the timeline and changes in platelet, hematocrit and serological
status of patients with dengue.
8
Figure 4 Course of the illness (Source: CDC)
The febrile phase lasts for 2-7 days and is usually associated with non-specific
symptoms resembling a flu-like illness. Fever is usually biphasic and is
associated with rash occurring on first day of illness as a generalised
erythematous maculopapular rash which blanches under pressure and disappears
in 24-48 hours.
9
Many children have severe retro-orbital pain, backache, headache, myalgia and
tiredness. Nausea and vomiting occurs in many children. There is loss of
appetite and poor oral intake. Dehydration maybe manifest in children with high
grade fever and poor oral intake. In infants high fever may cause
encephalopathy and febrile seizures.
Following febrile phase, critical phase occurs around the time of defervescence
usually between the 3rd
and 7th
days of illness. This is characterised by capillary
leaks (leaky phase) and severe forms like dengue hemorrhagic fever and dengue
shock syndrome manifests.
It is essential to monitor children for warning signs and severe manifestations of
dengue. Organ damage may occur during this phase. The cornerstone of
management at this stage is optimal fluid resuscitation in order to maintain
adequate intravascular volume and to maintain perfusion to vital organs.
Recovery phase occurs after 24 to 48 hours after critical phase and is
characterised by the reabsorption of leaked fluid back into the intravascular
compartment.
Hemodynamic stability and diuresis ensues with an improvement in appetite
and well-being. If during critical phase, fluids were administered in excess or
intravenous fluids administered during recovery phase, fluid overload features
like pulmonary edema or congestive cardiac failure maybe seen in recovery
10
phase, contributing to the mortality if not identified promptly and managed
appropriately. A recovery rash described as “isles of white in sea of red” (9)
maybe seen in this phase. The child usually recovers from the infection after
this phase.
Primary and secondary dengue
Infection with any of the dengue serotypes for the first time is termed as
primary dengue whereas re-infection with another serotype is called as
secondary dengue. The clinical importance is that secondary dengue infection is
usually seen with increasing severity of the disease. This is because of the
immunopathogenesis that has been explained in the diagram below (2):
Figure 5 Patho-physiology of dengue fever (Source: NVBDCP National
guidelines for clinical management of dengue fever)
11
The increasing severity of the illness with secondary dengue infection is
attributed to the infection-enhancing antibodies with absence of cross-reactive
neutralising antibodies. There is rapid activation of complementary system in
secondary dengue leading on to cytokine storm with increased release of
Tumour necrosis factor, Interferon-γ and interleukin-2 which contributes to the
increase in capillary permeability in addition to the viremia, thus resulting in
severe forms of dengue. The internal redistribution of fluids together with fluid
deficit caused by fasting, vomiting and anorexia leads to hemoconcentration and
is the basis of dengue shock syndrome and dengue hemorrhagic fever occurring
in secondary dengue.
Dengue case classification
According to 2014 national guidelines of National Vector Borne Disease
Control Programme (NVBCDP)of the Ministry of Health and Family Welfare
(MoHFW), Government of India (GOI), the classification of clinical dengue is
of three types – mild, moderate and severe dengue (2).
Mild dengue is undifferentiated dengue fever without evidence of capillary
leakage.
12
Moderate dengue is dengue fever in certain high risk groups like in infants,
pregnancy, immune-compromised, chronic illnesses etc., and with capillary
leaks as seen by certain warning signs and raising hematocrit.
Severe dengue is dengue hemorrhagic fever with significant bleeds, dengue
shock syndrome and expanded dengue syndrome (with severe organ
involvement). The classification is discussed in further details in methodology
section.
Risk Factor for severe dengue
There is no proven risk factor for severe dengue except the presence of
enhancing antibodies due to previous infection with a different serotype of
dengue virus.
There have been studies implicating certain high-risk groups who could develop
severe dengue like infants, pregnant women, elderly people, immune-
compromised and people with chronic diseases.
The severity in infants has been attributed to maternal antibodies that enhance
the infection. In all other high-risk groups, it is primarily because of the
immune-dysregulation.
13
Capillary leaks in burns and septic shock
In burns patients, there is destruction of tissues causing capillary leaks resulting
in hypovolemia and shock.
Similarly in sepsis, there is cytokine storm similar to dengue fever causing
capillary leaks into extravascular compartment manifesting as shock.
In the above conditions, it is essential to administer iv fluids and vasopressors to
prevent organ damage due to hypotensive shock.
Death due to fluid overload contributes to a significant proportion of burns and
septic shock patients. Recent studies (10,10–13) have shown that by treatment
with high doses of intravenous ascorbic acid, the fluid requirement is
significantly reduced in these patients.
The mechanism of action of vitamin c is not clearly understood but assumed to
be probably the function of vitamin c in maintaining capillary permeability.
Vitamin C and capillary integrity
Vitamin C is a water soluble vitamin found in abundance in citric fruits like
oranges, lemon, Indian gooseberries, tomatoes etc.(14) Unlike animals, humans
cannot synthesize vitamin C in the body due to the absence of the enzyme l-
gulonolactone oxidase and must be obtained from diet(14). Vitamin C has
14
antioxidant roles and more importantly, it is required for synthesis of collagen,
l-carnitine and certain neurotransmitters. It also helps in protein metabolism.
Non-synthetic functions include role in immune function and absorption of non-
heme iron. (15) The antioxidant function prevents free radical injury to
endothelium and in addition to it collagen biosynthesis helps in wound-healing
and thus helps in maintaining endothelial integrity by many mechanisms.
Insufficient vitamin C intake causes a deadly disease called scurvy which
manifests with tiredness, lassitude, widespread connective tissue weakness and
capillary fragility leading on to mucosal bleeds like petechiae, purpurae, gum
bleeds etc.(14) In certain scorbutic patients, there was plasma leakage noted
(similar to patients with dengue fever) like pleural effusion, joint effusions,
pedal edema, clubbing, congestive cardiac failure and various other
manifestations. Vitamin C deficiency occurs in children with inadequate oral
supplementation of vitamin C, infants only on cow’s milk, critical illness, burns,
sepsis, ARDS, pancreatitis etc.(14)
In all the above conditions, vitamin C deficiency might have an exacerbating
effect on the disease severity and by supplementing with vitamin C could
improve outcomes as seen in burns patients and sepsis patients treated with
high-dose of parenteral vitamin c.
RESEARCH
QUESTION
15
Research question
“Is there any association between serum ascorbic acid levels in children infected
with dengue and the severity of the illness?”
The rationale behind the question is that we do not know the levels of serum
ascorbic acid in children who are infected with dengue virus. There is a
possibility that it could be low because of poor oral intake and vomiting in
children with the disease. Low ascorbic acid levels in scorbutic patients has
been found to contribute to plasma leaks (14). Hence the capillary leaks in
dengue maybe attributed to low levels of ascorbic acid in children with severe
disease. So we hypothesized that the ascorbic acid levels in children with severe
dengue could be low when compared with healthy children and children with
milder forms of the disease.
REVIEW OF
LITERATURE
16
Review of literature
The dengue infection irrespective of primary or secondary dengue starts usually
as an undifferentiated fever and later progresses to cause capillary leaks
manifesting as dengue hemorrhagic fever and dengue shock syndrome (severe
dengue).
Prediction of whether a child with dengue infection would proceed to have
severe dengue will be helpful in management of the case by early intervention
and rigorous monitoring. Many studies have been done previously to identify
risk factors that could predict progression to severe forms of dengue in children
as well as in adult patients.
Although a few studies have given certain clinical and lab parameters that could
predict the severity of the disease, a vast majority could not cite out specific
parameters that could identify risk factors for developing severe dengue.
Preventing transmission
Dengue management in developed countries starts with dengue prevention and
control of outbreak. This is aimed at curbing transmission of the disease by
implementing vector control measures. This is done by use of insecticides and
destroying potential sources for breeding of aedes mosquitoes.
17
Certain genetic modifications and sterilisation has been carried out in the
mosquitoes with much less beneficial effect than expected (16).
In developing countries like India, it is not possible to achieve vector control
mainly because of lack of resources. In a study in Rajasthan, it was noted that
water storage habits including cement tanks during arid and semi-arid seasons
in urban households were notable mosquito breeding sites (3).
Another study in Maharashtra noted that outdoor non-potable water storage
were more important breeding sites than indoor potable water storage (17).
The susceptibility pattern of mosquitoes to various insecticides were studied by
sampling mosquitoes from Jodhpur, Delhi, Mumbai, Chennai and Coimbatore
showed susceptibility to all insecticides, mainly temephos.(18).
But it requires a short course of fogging especially indoor fogging. This again is
not always possible due to lack of public awareness of dengue and acceptance
of control measures as noted in a study in Chennai by Ashokkumar et al. (19)
The lack of adequate vector control in India has caused the rise in geographical
distribution of the disease across the country and also the incidence of dengue
fever in the last decade (16). This has indirectly caused an increase in
occurrence of severe dengue compared to previous outbreaks for reasons
unknown and hence has led to increased morbidity and mortality.
18
Vaccine developed against dengue is currently in Phase 3 trials and approved
for use in children above 9 to 16 years with laboratory confirmed previous
infection with dengue virus and living in endemic areas (20). It is not available
in India currently.
Inadequate vector control and lack of an effective vaccine has hurdled the
prevention of disease transmission. Hence, rigorous monitoring to look for early
signs of worsening and aggressive management remains to be the mainstay of
prevention of complications in dengue patients (16).
Possible risk factors for severe dengue
Several studies have concurred that children who have secondary dengue
infections, infection with DENV2 serotype and increased viral load will have
severe dengue infection (21) (22).
In the study by Libraty et al, it was shown that circulating levels of NS-1
antigen had correlation with severity of the disease (21). But in another study
done in this institution (PSG Hospitals) by Lavanya et al, there was no
significant correlation between NS-1 titres and severity of the disease although,
secondary dengue was frequently associated with severe capillary leaks as
observed in other studies.
19
The challenge in prediction of severity in resource limited countries like India is
the cost and availability of test to identify the serotype of infection. Moreover,
detection of NS-1 antigen may indicate presence of infection but does not
correlate with the severity.
Serology to find out of it is a secondary or primary infection will not be useful
in the clinical setting to predict severity because the antibodies appear only after
the capillary leaks manifest. This makes serological tests practically useless to
predict if a child with the infection would progress to severe dengue. Thus
although the above studies could point out certain risk factors, it cannot be used
in a clinic setting to predict worsening of the disease (23).
Phakhounthong et al., had tried to formulate a decision tree in order to manage
children presenting with dengue like illness (24). In his study he has mentioned
that in resource limited endemic nations the differentiation of dengue fever from
other febrile illnesses require training and education to health care providers.
The revised classification of dengue fever by WHO in the year 2009 to identify
if there were warning signs or severe dengue had room for improvement (24).
Hence in this single-centered study among just 198 children with dengue, they
had identified 5 risk factors at very early stage of the disease or at admission
that could progress to severe dengue (24). The algorithm was not further
evaluated or validated.
20
In another study done in this institution (PSG Hospitals) by Bharathi et al, it
was shown that severe dengue usually presented with hypotension and cases of
severe dengue had low platelets, absolute neutrophil count and ESR at
admission when compared with children who were diagnosed to have scrub
typhus.
Kedia et al. in his retrospective in a PICU in a tertiary care centre in South India
had mentioned few risk factors for severe dengue (25). Female sex was
associated with severe dengue with an Odds Ratio of 1.6. Bleeding tendency
had significant association with severe dengue (OR = 5.7) and hepatomegaly
and severe thrombocytopenia (Platelet count < 50,000 cells/mm3) also had
significant association. Except for female sex, all the other factors mentioned
were already termed as warning signs or defined as moderate dengue as per
NVBCDP guidelines of 2014. Hence this study did not yield any extra
information to predict severe dengue.
Genetic and immunological risk factors in dengue
Study of genetic factors that could predispose to severe dengue infections have
shown mutations of genes coding for certain cytokines and their receptors,
mainly TNF-α, IL-6, IFN-γ, TGF-β1 and IL-10.
21
A specific mutation in TNF-α 308A allele has been observed in a study in
patients with dengue fever who had manifestations of severe dengue (20). The
mutation allows expression of higher levels of TNF-α contributing to increased
amounts of vascular permeability, hemorrhage and severe dengue.
Other genetic factors that have been shown to affect disease severity include
certain HLA alleles, variations in the vitamin D receptor and Fc gamma
receptor IIa, and also CD209 (27).
Other immunological parameters that may have a role in the pathogenesis of
severe dengue include mast cell activation and mast-cell-derived mediators,
mainly vascular endothelial growth factor, and antibody-immune complexes
(27).
Other risk factor
The other risk factors for severe dengue are host factors like age of the host with
children likely to develop plasma leakage and shock and adults could
experience significant bleeding and organ impairment (27).
Elderly and people with co-morbidities like Diabetes Mellitus and Hypertension
experience severe symptoms due to pre-existing endothelial dysfunction in this
group (27).
22
Obesity and pregnancy is also associated with increased risk for severe dengue
due to immeasurable extra-vascular volume and the tendency to get dehydrated
faster.
In immune-compromised people, the immune-dysregulation could predispose to
an unpredictable immune response to dengue and secondary infections that may
directly influence on the severity of the disease(27).
All the above studies have identified risk factors for developing severe dengue
but the risk factors are not modifiable. The presence of these risk factors would
mean stringent monitoring and fluid management in these cases to ameliorate
the severity of the illness (27).
The purpose of identifying such risk factors like high viral load or infection
with specific serotype etc., are virtually useless to the clinician because of the
non-availability of these tests in the general clinical setting and the cost of these
tests. Secondary dengue is usually identified by presence of both IgG and IgM
to dengue, which usually takes 5 to 7 days to be detectable. But by that time, the
disease would have progressed with manifestations of severe capillary leaks.
This has led to further research to identify therapeutic options to prevent dengue
infection from progressing to severe disease.
23
Treatment options for mitigating severity of dengue fever
Treatment of dengue is usually supportive with iv fluids, mainly crystalloids
and in severe cases, colloids and plasma replacement is indicated (1). The fluid
management must be optimized during the critical phase to maintain adequate
perfusion and restriction of fluid during recovery phase to prevent fluid
overload (1).
It has been established that minimal fluid support with appropriate inotropic
support would drastically reduce mortality and improve clinical outcomes (27).
Therapeutic trials at earlier stage of the disease with antiviral agents have not
been shown to be effective in termination of the disease progression (27).
There are two studies that have studied the use of antivirals like celgosivir and
balapiravir in dengue infection. Ngyuen et al. studied the use of balapiravir, a
protease inhibitor tried in treatment of hepatitis C in dengue patients in Vietnam
(28). The drug at dosage appropriate to bring about a reduction in viral load in
chronic hepatitis C patients, failed to show a decrease in viral load in dengue
patients as measured by NS-1 levels twice daily during the course of treatment
(28). The other study by Low et al. in Singapore, studying the effect of a α-
glucosidase inhibitor, Celgosivir in dengue patients, showed no effect in
virological log reduction when compared to placebo.
24
Adjunctive therapies also have been tried in dengue patients and again no
satisfactory results have been obtained.
The anti-malarial drug chloroquine has shown promising in vitro antiviral
effects. But in a randomised control trial by Tricou et al. in adult patients with
dengue, it has shown no significant reduction in viral load (29).
Corticosteroids which are helpful in immune thrombocytopenic purpura to
improve platelet counts have shown no effect on thrombocytopenia in dengue
patients. According to a randomized placebo-controlled trial by Tam et al. in
Vietnamese patients with dengue, short courses of oral steroids did not increase
viremia as expected but did not have any improvement in clinical outcomes also
(30). Another study done in pediatric patients with dengue shock syndrome with
single dose of intravenous methylprednisolone was not effective in reducing
mortality (31).
Statins inhibited dengue virion assembly in vitro and hence lovastatin was
studied in a randomised control trial which showed no significant reduction in
dengue severity (32).
Intravenous immunoglobulin also seemed to be ineffective in a study in adult
patients in Vietnam (33).
Platelet transfusions are obsolete for treatment of thrombocytopenia unless the
patient has severe bleeding manifestations (2).
25
Nutritional factors in dengue
The presence of malnutrition in the countries with dengue endemicity has posed
a great risk for dengue mortality.
Although there is no clear association between the host nutritional status and the
risk of dengue virus infection, malnutrition by itself is a cause of childhood
mortality under 5 years of age (34).
Malnourished children have less robust immune response when compared to
well-nourished children and since dengue severity depends on host immune
response, it was proposed that malnutrition could be a protective factor against
severe dengue (34).
Although in previous studies, it was shown that malnourished children had less
chances of severe dengue when compared to well-nourished child, recent
studies by Marón et al have refuted the theory (35).Based on anthropometric
measurements and comparing it with standard charts to classify children as
normal nutritional status, malnourished and overweight did not show any
significant association with severity of the disease. It means children in either
group had equal chances to have severe dengue. (35)
The complex interplay between nutrition and other infections is well-
established, and modulation of nutritional status by community intervention and
nutritional rehabilitation often presents a simple low-cost solution to interrupt
26
transmission, reduce susceptibility, and/or ameliorate disease severity to a
significant extent.
Ahmed et al. in his study wanted to examine the effects of micronutrients in
dengue fever and whether the levels of micronutrients and vitamins in the blood
had any significance in reducing transmission of the virus or reducing the
severity of the infection (34).
Alagarasu et al. in his study (36) had investigated for vitamin D levels and
dengue severity. It was shown that though there was no significant association
between vitamin D levels and primary and secondary dengue or dengue
hemorrhagic fever and undifferentiated dengue. But vitamin D levels in infected
people were higher than that of healthy controls. The higher levels in
symptomatic infection when compared to asymptomatic individuals were
explained by the role of vitamin D in immunomodulation.
In yet another study, the authors have proved the association of vitamin D
receptor gene polymorphisms with the occurrence of dengue and dengue shock
syndrome when compared with healthy controls. In another study by Fatima et
al. (37), it was shown that infection with dengue causes lower levels of vitamin
D, vitamin K, thrombopoietin and angiotensin. Though the study did not
correlate severity of the manifestation with lower levels, it found a significantly
low value of vitamin K and vitamin D in children infected with the virus when
compared to healthy controls.
27
Villamor et al. in his study (38), has mentioned that low levels of vitamin D
could be a possible risk factor for the development of severe dengue.
Zinc is yet another micronutrient that has an important role in immune
regulation. Its use has been proven in pneumonia and diarrhoea in decreasing
the severity of the illness and length of hospital stay.
In an Indonesian study (39), serum zinc levels in children infected with dengue
was compared in differing severity of the disease. The study had shown that
zinc levels were significantly lower in children with dengue shock syndrome
and dengue hemorrhagic fever compared to dengue fever. But zinc levels were
not proved as a risk factor for manifesting severe dengue.
Studies to find out if there could be a significant association between vitamin A
levels (40), iron (41) and chromium (42) between severity of dengue had shown
no significant results.
Vitamin A levels were found to be higher in severe dengue due to a turbulence
of the anti-oxidant system (40).
Iron levels could not be accurately assessed in dengue patients because serum
ferritin is elevated in most patients with all forms of dengue as it would be
expected since it is an acute phase reactant. High ferritin levels (>1200ng/ml)
had an increased association with dengue hemorrhagic fever (41).
28
Chromium promotes action of insulin and regulates blood sugar and also it
influences immune response by its effects on T and B-lymphocytes, antigen
presenting cells and cytokine release. Chromium and DENV infection had a
possible association in a study done by Shrivatsava et al. in mice population
(42). Exposure to chromium in dengue infected mice showed less severe
outcomes and a significantly faster and robust increase in platelet counts (42).
Alternative medicine in Dengue
The administration of alternative medicines in dengue fever has also been
researched.
“Nilavembu” extract is a siddha preparation made from 8 indigenous herbs, and
has been extensively propagated for use in Tamil Nadu in patients affected with
dengue. It has antipyretic, anti-inflammatory, analgesic and immune-stimulant
action which is used to manage initial symptoms of dengue fever in siddha
medicine (43).
It has been shown to have substances that possess anti-viral activity against
DENV-2 and chickungunya virus and shown to decrease severity of the disease
(44).
29
It has also shown protective effects in uninfected individuals and hence used in
prophylaxis. But in females, it has less protective effect probably due to
metabolic pathways not clearly known (45).
Carica Papaya leaf extract was demonstrated to have a significant improvement
in platelet counts in patients with dengue according to a systematic review done
by Charan et al (46). The platelet counts do not predict severity of the disease.
But its nadir correlates with severity of plasma leakage and its improvement
also correlates with clinical improvement.
It was postulated that papaya leaf extract could contain flavonoids, alkaloids,
enzymes and minerals that could have immuno-modulatory and anti-oxidant
effects. Since data of safety was not available, the study could not suggest use
of these herbal preparations without evidence from further trials (46).
Diet therapy in prevention and control of dengue (47) is a naturopathy
treatment. It involves careful selection of diet to meet micronutrient and
macronutrient demand during dengue fever and to decrease occurrence of
gastritis and vomiting. Although no clear scientific evidence was quoted, citrus
fruits especially lemon helped in lessening nausea and improving outcomes.
30
Effects of micronutrients supplementation on course of illness
Very few experimental studies have been done in dengue with micronutrient
supplementation. Although there is no replacement for an effective vaccine, it
must be noted that modulation of nutritional status and micronutrient
supplementation could be a cheaper and cost-effective alternative.
In a study by Sánchez-Valdéz et al in Mexico (48), it was shown that
supplementation with calcium and vitamin D can improve outcomes in dengue
infection. It was explained by the platelet aggregatory effect of calcium and the
ability of vitamin D to alter IL-12 expression and dendritic cell maturation.
Vitamin E supplementation has been beneficial in improving platelet count in
children with dengue as shown by the study done by Vaish et al (49), but did not
alter disease severity.
As mentioned in the article by Ahmed et al (34) in his concluding remarks, the
role of multivitamin supplementation (Vitamin B and C) needs to be studied in
dengue.
Vitamin C especially has proven benefits in treatment of respiratory tract
infections and pneumonia due to its anti-oxidant properties. Vitamin C has been
found to play important role in increasing gut iron absorption, folate
metabolism, and essential roles in amino acid and hormone metabolism (50).
31
The role of vitamin C in collagen formation and maintaining capillary integrity
in addition shows promise in treatment of dengue though there have been no
studies in this regard till date (34)
Therapeutic role of vitamin C in conditions with capillary leaks
Vitamin C has been tried in various scenarios that are associated with conditions
associated with loss of capillary integrity leading on to plasma leakage. High
dose of ascorbic acid in sepsis and burns patients is a recently emerged therapy
in adults.
The underlying mechanisms for the effect of ascorbate on these conditions have
been demonstrated in in vitro studies with cultured endothelial cells.
Ascorbate decreases oxidative stress in endothelial cells by reducing the
production reactive oxygen species. Reactive Oxygen Species increase
endothelial permeability causing edema and contributing to organ dysfunction.
Ascorbate can tighten the endothelial barrier through several pathways.
There are very few studies regarding the effect of high dose of ascorbic acid to
prevent the capillary leak.
32
The role of ascorbic acid in dengue capillary syndrome is never studied. The
following studies studied the effect of ascorbic acid in preventing endothelial
syndrome and capillary leakage in sepsis and burns patients and in animal
models.
Alpha A Fowler III et al has done a phase 1 randomized control safety trial of
intravenous ascorbic acid in adult medical intensive care unit patients with
severe sepsis and showed positive impact on the extent of multiple organ failure
and biomarkers of inflammation and endothelial injury (51). This phase I trial
shows that aggressive repletion of plasma ascorbic acid levels in patients with
severe sepsis is safe. This early work in septic patients suggests that
pharmacologic ascorbic acid repletion reduces the extent of multiple organ
failure and attenuates circulating injury biomarker levels.
Mohadeseh Hosseini Zabet et al have studied the effect of high-dose ascorbic
acid on vasopressor drug requirement in surgical critically ill patients with
septic shock (10). They suggested that high-dose of ascorbic acid (25 mg/kg
intravenously every 6 h for 72 h) with its probable anti-oxidant, anti-
inflammatory, cortisol sparing, nitric oxide synthase inhibitory and increasing
catecholamine synthesis in the brain, and adrenal medulla properties may be
considered as an effective and safe adjuvant therapy in critically ill surgical
patients with septic shock.
33
A randomized, prospective study by Tanaka et al. (13) evaluated the use of
continuous ascorbic acid infusion in burn patients using a group of 37 patients
with greater than 30% TBSA burns. Investigators compared resuscitation fluid
volume requirements and overall edema formation. A significant reduction in
fluid volume requirements, weight gain, and wound edema was noted, along
with an overall improvement in pulmonary function, demonstrated by a
significant reduction in mechanical ventilation days.
Matsuda T et al studied the hemodynamic effects of antioxidant therapy with
high-dose administration (170 mg/kg/24 h) in guinea-pigs with 70 per cent body
surface area deep dermal burns (11). They have demonstrated that there was
significant reduction of plasma leakage and decreased need of fluid requirement
with high dose of ascorbic acid.
Kremer et al (12) demonstrated the reduction capillary leakage in burns animal
models with high dose of ascorbic acid. They have also concluded that half dose
of ascorbic acid is inefficient to reduce the endothelial damage. They have
suggested that high-dose ascorbic acid should be considered for parenteral
treatment in every burn patient to reduce capillary permeability.
A randomized, double-blinded study by Horton JW (52) demonstrated a
significant reduction in net fluid balance and plasma lipid peroxidation among
sheep sustaining 40% TBSA burns who were resuscitated with either Lactated
34
Ringer’s solution or hypertonic saline in conjunction with a high-dose infusion
of ascorbic acid.
Gonzalez et al had reported the use of high-dose intravenous ascorbic acid in a
54 year lady with chickungunya infection and noted that her symptoms abated
early. He had ascribed the effects to the anti-oxidant property and the property
of vitamin C to maintain capillary integrity. He noted that treatment with high
dose vitamin C could be useful in infections similar to chikungunya and needs
to be studied.
Currently, there is an ongoing randomised control trial in Srilanka by Herath et
al. with oral liposomal vitamin C in dengue infected patients to find out if it
reduces the morbidity of dengue infected patients above 12 years of age.
Vitamin C in critically ill patients
According to a systematic review done by Zhang et al (53), Low plasma levels
of vitamin C are associated with adverse outcomes and especially increased
mortality in critically ill patients. Though the study was done in adults, the
study points out the importance of vitamin C in critically ill patients.
The patients included in the studies in the meta-analysis included septic shock,
severe neurological trauma, post cardiac-surgery patients, ARDS etc.
35
Vitamin C administration parenterally decreased the need for inotropes and
mechanical ventilation.
The mechanism proposed was that vitamin C is an important co-factor in
synthesis of endogenous vasopressors (54,55). The endogenous synthesis of
adrenaline, dopamine etc., requires ascorbic acid as a co-factor.
According to Carr et al (56), the parenteral administration of high dose ascorbic
acid can improve endogenous vasopressor production especially in states of
hypotension like septic shock and thus reduce exogenous vasopressor
requirement.
Since severe dengue usually presents with shock, which is similar to septic
shock, administration of vitamin C can reduce need of IV fluids and
vasopressors.
The need to identify a modifiable risk factor to prevent severe dengue
Dengue poses a great global problem wherein the progress of disease in an
infected individual cannot be prevented but it has been shown that the severity
can be mitigated to a certain extent by early identification of critical phase,
hospitalisation, judicious fluid administration and intensive monitoring (57).
36
In countries like India where there is high incidence of dengue and inadequate
trained health personnel to meet the health needs of the population, there is
occasional faltering in monitoring patients and resulting in inadvertent fluid
overload. It would be wise to devise a treatment protocol with judicious use of
intravenous fluids, optimum use of fluids to achieve the best outcome.
The early identification of cases that would have severe capillary leaks and
severe disease can be managed well in High Dependency Units (58). If it is
possible to identify a risk factor that could predict progression of disease before
clinical worsening happens, resources could be allocated with priority to
monitor/ manage these patients in intensive care units and prevent the disease
progression thereby preventing prolonged hospital stay and mortality (58).
Distinguishing patients with dengue infection who will develop more severe
forms of disease remains a clinical challenge and is an area yet to be explored
and researched intensively.
While studies have shown positive results with high dose ascorbic acid and
reduced need for IV fluids in burns and sepsis patients, a similar result may be
expected in dengue. If by increasing vitamin C levels, the outcome might
improve, then it is safe to assume that low vitamin C levels might predispose to
severe dengue and hence we would like to explore the possibility of low levels
of vitamin C to be a risk factor for severe dengue.
37
Vitamin C estimation:
Vitamin C (Ascorbic acid) is found in higher concentrations in citrus fruits and
is absorbed in the intestines by a sodium dependent carrier mediated mechanism
(SVCT-1) which is dose dependent. Dehydroascorbate present in the diet is
absorbed by sodium independent mechanisms that is competitively inhibited by
hexoses (59).
The absorbed ascorbic acid is transported in the blood as ascorbate anion. A
normal serum level of 0.6-2 mg/dl is maintained and excess ascorbate is
excreted renally. If there is deficiency of ascorbic acid in the plasma, renal
reabsorption is increased.
Vitamin C is accumulated in almost all human tissues (60). The plasma levels of
ascorbate in fasting state provides a surrogative measure of adequacy of vitamin
C stores in tissues. Vitamin C in plasma is also stored in WBCs and platelets.
Measure of Vitamin C levels in WBCs gives a more accurate measure of
deficiency but requires expertise.
High Pressure Liquid Chromatography is the gold standard procedure to
measure Vitamin C levels (61). Biosensors are latest devices to measure
ascorbic acid levels quickly and reliably. Cheaper alternatives like dye
reduction with DCPIP, Bromate etc., are traditional method employed in most
research labs.
MATERIALS AND
METHODS
38
Materials and methods:
Aims and objectives:
Primary objective:
To determine ascorbic acid level in children with dengue fever
Secondary objective:
To determine the correlation of serum ascorbic acid level with severity of
dengue fever in children.
39
Study method:
Cross-sectional observational study.
Sample size: 160 children
(By using 4pq/d2; with 5% prevalence and d-7%, 40 cases of severe dengue is to
be studied)
By quota sampling method, 40 children will be recruited from each category
namely mild dengue, moderate dengue and severe dengue. 40 children between
the age group of 1-15 years with normal nutritional status (based on age and sex
appropriate IAP growth charts and WHO criteria) without chronic illnesses will
be taken as a control group.
Study Area: Department of Pediatrics, PSGIMSR
Study Subject: Children and adolescents between 1-15 years of age
Study Period: January 2018 to November 2019
40
Inclusion criteria:
All children aged 6 months to 18 years admitted with the diagnosis of mild
dengue, moderate dengue or severe dengue (as per NVBCD guidelines for
management of dengue) in PSG Hospitals, Coimbatore, irrespective of any
treatment received outside before admission.
Exclusion criteria:
1. Children who are classified as severe acute malnutrition and moderate acute
malnutrition as per WHO criteria (weight for height between -2 SD and – 3 SD
and weight for height less than -3 SD respectively).
2. Children with pre-existing systemic illness/chronic underlying medical
illness.
41
Operational definitions:
� Probable Dengue Fever – Any child presenting during dengue outbreak
with clinical features of dengue fever, i.e., an acute febrile illness of 2-7
days with two or more of the following manifestations:
o Headache
o Retro-orbital pain
o Myalgia
o Arthralgia
o Rash
o Hemorrhagic manifestations.
(Or) non-ELISA based NS-1 or IgM positivity.
� Dengue Hemorrhagic fever: Clinical criteria of dengue fever with
Hemorrhagic tendencies (Petechiae, ecchymoses, purpura; bleeding from
mucosa, GIT, injection sites), thrombocytopenia (platelets <
1lakhs/cu.mm) and evidence of plasma leakage (pleural effusion, ascites,
pedal edema, hemoconcentration etc.)
42
� Dengue Shock Syndrome: Dengue Hemorrhagic Fever with evidence of
circulatory failure.
The definition of confirmed cases of dengue is made when:
� Isolation of dengue virus by viral culture from serum, plasma, leucocytes.
� Demonstration of IgM antibody by ELISA positive in single serum
sample in significant titre (>9IU/L).
� Demonstration of dengue virus antigen (NS1) in serum sample by
ELISA.
� IgG seroconversion in paired sera with four fold increase of IgG titre after
2 weeks.
� Detection of viral nucleic acid by PCR (polymerase chain reaction).
The classification of dengue is made based on the above clinical diagnosis as
follows:
43
Figure 6 Classification of dengue fever (Source: NVBDCP guidelines for
clinical management of dengue fever)
44
Methodology:
It is a cross-sectional observational study. After obtaining informed consent
from a parent/guardian and assent (as applicable) from children, 2ml of blood
sample was taken for measurement of ascorbic acid in all children admitted in
the department of paediatrics, PSGIMSR with the diagnosis of mild dengue/
moderate dengue/ severe dengue (as per NVBDCP guidelines on management
of dengue) on Day 4 of illness or day of admission, whichever is earlier. All
children were monitored for the evidence of onset, progression or regression of
plasma leakage in the form of pedal edema, facial puffiness, ascites, pleural
effusion and shock. Patients were managed as per discretion of treating
pediatrician. All the parameters were entered in a predesigned proforma.
Controls were selected from healthy well-nourished in-patients other than
probable dengue fever with normal nutritional status (as per IAP growth charts
and WHO criteria for malnutrition), and must be without chronic illnesses,
2ml blood sample, from all study participants, was obtained in a vaccutainer
with heparin, plasma separated and acidified with 2ml of freshly prepared 10%
metaphosphoric acid and stored at -70 °C. The samples were sent in batches
transported with dry ice for analysis in Stanes laboratory, Coimbatore.
Vitamin C levels were estimated in the serum samples using DCPIP
(dichlorophenolindophenol) method which is a dye reduction method (62).
45
The principle in this method is that ascorbic acid reduces DCPIP which is
usually a blue-colour solution to a colourless base. The sample with unknown
concentration of ascorbic acid is titrated against the dye in the presence of
oxalic acid and the end-point is appearance of pink colour (as the dye changes
into pink colour in acidic medium) and compared against the volume of dye
required to titrate a standard solution of ascorbic acid. Then the concentration of
ascorbic acid is given by the formula:
Amt of ascorbic acid, mg/100g of sample= 0.5/V1 X V2/5 X 100/ W X 100
(V1 – Volume of dye used for STD ascorbic acid solution
V2- Volume of dye used for sample.
W- Weight of sample taken for test.)
Funding:
Expenses involved in this study for storage, transportation and analysis of
Vitamin C levels in the serum samples were met by intramural funding from the
institution.
Conflicts of interest:
None
46
Statistical analysis:
All statistical analyses will be performed using SPSS 25.0. The mean values of
serum ascorbic acid between dengue children and control group was compared
by student T-test and between mild, moderate and severe groups by one-way
ANOVA.
Outcome of study:
If ascorbic acid level is found to be low in children with severe dengue or in
dengue with warning signs, further study will be on ascorbic acid
supplementation in children with dengue to prevent plasma leakage.
The dose and route of administration of vitamin C will be based on evidence of
improvement in patients with other diseases who had received treatment with
vitamin C.
47
Figure 7 Flowchart (Proposed Methodology)
Exclusion criteria:
1. Children with malnourishment and pre
existing systemic illness
2. Children on chronic medication
Mild dengue
40 cases
Moderate
dengue
40 cases
Severe
Dengue
40 cases
Assess for
eligibility
Collect 2ml blood
sample on Day 4 of
illness or on admission
whichever is earlier.
Control
40 children with
normal nutritional
status
Store acidified plasma at -
70oC.
Send Samples to Stanes
Lab, Coimbatore in
batches in dry ice.
Collect reports
Collect 2ml
blood sample
Analyze
RESULTS
48
Results:
A total of 73 dengue patients were admitted to the hospital during the study
period which was less than the required sample size. Of the 73 children, one
child had congenital heart disease and was excluded from the study. Of the
remaining 72 children, 3 parents did not give consent and hence were not
included. The rest 69 children were enrolled in the study. 2ml of heparinised
sample was obtained from each participant in a vacuutainer. The samples were
centrifuged immediately and serum was separated and acidified with freshly
prepared metaphosphoric acid which was then stored in -70oC.
Of the 69 dengue children included in the study, there were 10 (14.5%) severe
cases, 31(44.9%) moderate and 28 (40.6%) of mild dengue cases. There were
totally 26 (37.7%) primary dengue and 43 (62.3%) secondary dengue children.
By quota sampling method, 40 controls were chosen from in-patients with
minor illnesses. Most of the controls were children with acute respiratory tract
illnesses or viral fevers other than dengue admitted in view of parental concern.
These children were carefully selected that they did not have vomiting or
diarrhoea before admission. 2ml of heparinised sample was obtained from each
patient on day of admission and stored and analysed for Vitamin C levels as
mentioned before.
Data was recorded for all children as per protocol and analysed.
49
Figure 8 Flowchart (Proposed Methodology)
Statistical analysis of the results and completion of study.
73 Dengue children 40 Controls
1 child excluded
due to Congenital
Heart Disease
Parents of 3
children did not
give consent for
study
2ml Heparinised sample
obtained from all children
after obtaining consent (and
assent as needed).
Plasma stored and processed in Stanes Lab in
batches and reports obtained.
69 children included
in the study
Mild - 28 Moderate - 31 Severe - 10
50
The mean (±SD) age of the dengue was 7.29(±4.34) whereas that in the control
group was 6.1(±4.4) in years which was not statistically significant. The least
age in the cases group was 8 months and the highest age was 15 years. There
were three infants included in the study group. In the control group the lowest
and the highest age among the children included were 1 year and 17 years
respectively. No infant was included in the control group.
The following table gives distribution of the cases and controls based on their
age:
Table 1: Agewise distribution of cases and controls
Age in years
Cases Controls
Frequency Percentage
(%) Frequency
Percentage
(%)
0-5 24 34.8 22 55
5.1-10 24 34.8 08 20
11.1-15 21 30.4 10 25
Total 69 100 40 100
The table shows that the number of children with dengue in the age group of 0-5
years was less than that in the control group probably because infants and
toddlers with minor illness are usually hospitalised than older children due to
parental concern.
51
Figure 9 Agewise distribution of Dengue cases
52
Figure 10 Agewise distribution of controls
53
Table 2: Genderwise distribution of cases and controls
Sex
Cases Controls
Frequency Percentage
(%)
Frequency Percentage (%)
Male 43 72.5 29 62.3
Female 26 27.5 11 37.7
Total 69 100 40 100
Both the groups (cases and controls) had male predominance. But there was no
statistical difference in the distribution of boys and girls among cases and the
controls chosen.
Figure 11 Genderwise distribution of controls
43
29
0
5
10
15
20
25
30
35
40
45
50
Cases Control
Males
Females
54
Table 3: Comparison of laboratory parameters between cases and controls
Lab Parameters Cases (Mean ± SD) Controls (Mean ± SD)
Haemoglobin (in g/dl) 13.1 ± 1.8 12.1 ± 1.6
Hematocrit (in %) 39.8 ± 5.4 37.1 ± 4.5
Platelets (in cells/cu.mm) 91,900 ± 81,300 230,000 ± 1,46,200
SGOT (in U/L) 301.76 ± 850.4 NA
SGPT (in U/L) 118.07 ± 311.3 NA
Vitamin C Levels (in
mg/dl)
2.36 ± 1.47 1.73 ± 0.99
The baseline parameters at admission between dengue children and the control
group showed a significant difference in platelet value as expected (p<0.001).
The hematocrit was comparable.
The Vitamin C level distribution between cases and controls are given by the
figures below:
55
Figure 12 Distribution of Vitamin C levels among cases
56
Figure 13 Distribution of Vitamin C levels among controls
57
Table 4: Comparison of mean Vitamin C levels in Dengue and in controls
N Mean SD
Dengue 69 2.36 1.47
Control 40 1.73 0.99
P = 0.01 (Students t test)
Statistical significance: P < 0.05
The comparison of mean Vitamin C levels between control group and dengue
children showed a statistically significant lower value in the control group when
compared to the study group (P = 0.01).
58
Table 5: Distribution of cases according to severity of Dengue: (N=69)
Dengue severity Frequency Percentage (%)
Mild Dengue 28 40.6
Moderate Dengue 31 44.9
Severe Dengue 10 14.5
Total 69 100
Dengue children were classified clinically into mild, moderate and severe
dengue as mentioned in the methodology section at admission. The children
were closely monitored and reclassified if the disease severity progresses.
Finally at discharge, the child was diagnosed as having either mild or moderate
or severe dengue which was considered for analysis.
In that respect, out of the 69 children admitted in the study period, mild dengue
was diagnosed in 28 children, moderate in 31children and severe dengue in 10
children.
59
Figure 14 Classification of dengue cases clinically
Also, in all dengue children, serology was done before discharge and based on
IgG and IgM titres, they were classified as primary dengue (only IgM positive)
or secondary dengue (both IgG and IgM positive). In this study, there were
totally 26 primary dengue and 43 secondary dengue.
Dengue
Mild
Moderate
Severe
60
Figure 15 Primary and secondary Dengue
Table 6: Distribution of primary and secondary dengue based on severity
Primary Dengue (n=26) Secondary (n=43)
Mild Dengue (no / %) 12 (46) 16 (37.2)
Moderate Dengue (no /
%)
11 (42.5) 20 (46.5)
Severe Dengue (no / %) 3 (11.5) 7 (16.3)
46% of primary dengue was mild whereas 46.5% of secondary dengue was
moderate. The results are as expected with primary dengue being less severe.
70% of severe dengue is secondary dengue and 66% of moderate dengue is
secondary.
Dengue
Primary
Secondary
61
Table 7: Comparison of baseline characteristics among mild, moderate and
severe dengue
Characteristic Mild Dengue
(n=28)
Moderate Dengue
(n=31)
Severe Dengue
(n=10)
Age (in Years)
Mean ± SD
6.99 ± 4.37 7.35 ± 4.37 7.98 ± 4.55
Male (no./%) 20 (71.5) 18 (58) 5 (50)
Day of illness at
admission (Mean
± SD in days)
4.5 ± 1.37 4.81 ± 1.35 4.1 ± 1.1
Location:
Coimbatore
Tiruppur
Others
6 9 2
21 19 6
1 3 2
The mean age was lower in the mild dengue group and higher in severe dengue.
Percentage of males was significantly higher in mild dengue group.
Tiruppur is a neighbouring district with high dengue load and consequently,
more number of cases in all 3 groups were from Tiruppur district. We had cases
from Nilgiris, Erode, Salem, Dindugal and also from Nagaipattinam District.
The mean duration of illness at admission was slightly lower in severe dengue.
62
Table 8: Comparison of baseline laboratory investigations among mild,
moderate and severe dengue cases
Investigations Mild Dengue Moderate Dengue Severe Dengue
Haemoglobin (in
g/dl)
12.7 ± 1.5 13.1±1.7 14.3 ±2.1
Hematocrit (in %) 38.4 ± 4.8 39.7±5.3 43.7±6
Platelets (in
cells/cu.mm)
98200 ± 75100 84500±76300 97200 ±115000
SGOT (in U/L) 103 ±56.9 210 ±253 1133.5±2016.4
SGPT (in U/L) 40.4 ±25 91±108.1 416±750
Outcome All Improved and
discharged
All Improved and
discharged
One died
The clinical parameters show a significantly high PCV in the severe dengue
group and elevated liver enzymes when compared to the other two groups.
The outcome of treatment showed all cases improved and discharged except
two in severe dengue group. One died and another was discharged against
medical advice. In the severe dengue group, 4 children required colloids, 4
required inotrope support and 2 children needed mechanical ventilation.
63
Figure 16 Outcome of all dengue cases
64
Table 9: Comparison of Vitamin C levels in mild, moderate and severe dengue
N Mean SD
Mild 28 2.35 1.29
Moderate 31 2.17 1.55
Severe 10 2.99 1.62
P = 0.31 (One way ANOVA)
The comparison of vitamin C levels in mild, moderate and severe dengue
groups showed no statistical difference although the value is numerically greater
in the severe dengue group.
The normal range of vitamin C is 0.6 to 2 mg/dl according to previous studies.
Any value below 0.3 mg/dl is considered as scurvy and values more than
3mg/dl is considered high (63).
65
Table 10: Association between severity of dengue and ascorbic acid levels:
Outcome
Vit C levels P value
<0.6 >0.7
0.163
Mild Dengue 0 28
Moderate Dengue 3 29
Severe Dengue 0 9
Total 3 66
Significance level is P value <0.05
The above table shows number of patients with serum ascorbic acid levels less
than 0.6 mg/dl (considering the normal range of serum ascorbic acid) and above
0.7 mg/dl.
66
Figure 17 Vitamin C levels in mild, moderate and severe dengue –
comparison using cut-off of 0.6mg/dl
67
Table 11: Association between Mild dengue and ascorbic acid levels:
Outcome
Vitamin C levels P value
0.6-2 >2.1
0.456 Mild Dengue 18 09
Total 18 09
Significance level is (P value <0.05)
Figure 18 Vitamin C levels in mild dengue – number of cases in normal and
above normal range
0
2
4
6
8
10
12
14
16
18
20
0.6-2 >2.1
Mild Dengue - Vitamin C Levels
68
Table 12: Association between Moderate dengue and ascorbic acid levels:
Outcome
Vitamin C levels P value
0.6-2 >2.1
0.245 Moderate Dengue 22 09
Total 22 09
Significance level is (P value <0.05)
Figure 19 Vitamin C levels in moderate dengue – number of cases in
normal and above normal range
0
5
10
15
20
25
0.6-2 >2.1
Moderate Dengue - Vitamin C levels
69
Table 13: Association between Severe dengue and ascorbic acid levels:
Outcome
Vitamin C levels P value
0.6-2 >2.1
0.126 Severe Dengue 04 06
Total 04 06
Significance level is (P value <0.05)
Figure 20 Vitamin C levels in severe dengue – number of cases in normal
and above normal range
0
1
2
3
4
5
6
7
0.6-2 >2.1
Severe Dengue - Vitamin C levels
DISCUSSION
70
Discussion:
Dengue causes significant morbidity as well as mortality in children in India
(7). The disease has a dynamic course and the severity may vary during the
course of illness.
The only identified risk factor till date that could predict the progression of
severity is the presence of infection-enhancing antibodies (64).
Severe dengue usually presents with hypotension that would necessitate fluid
boluses, colloids, inotropes and mechanical ventilation (65).
ECMO (Extra-Corporeal Membrane Oxygenation) and CRRT (Continuous
Renal Replacement Therapy) have changed the outcomes of severe dengue that
end with ARDS (Acute Respiratory Distress Syndrome) or AKI (Acute Kidney
Injury). But it requires expertise and centres with facilities (66). Moreover, the
cost of treatment is too high and burdening on the caregivers.
An effective and a cheap agent that could prevent the capillary leaks associated
with dengue fever and thus alter the hemodynamic imbalance that occurs in the
critical phase is the need of the hour.
We considered vitamin C could be such an agent based on evidences shown in
burns and sepsis models (10-13). Hence our study aimed at finding a correlation
between vitamin C levels and severity of dengue.
71
The normal value of Serum Vitamin C was 0.6-2 mg/dl. In our study, out of 109
children enrolled (Cases and controls included), only 7 had values less than 0.6
(4 from control group and 3 from cases) of which only 2 children had <0.3 (1
from either group).
The mean value of vitamin C levels (in mg/dl) in the cases [2.36 ± 1.47] was
high when compared to the control group [2.36 ± 1.47] which was statistically
significant.
This meant that children with dengue fever had higher levels of vitamin C when
compared to controls, thus contradicting the proposed hypothesis of the study.
The probable reasons for the findings could be:
1. The consumption of extra fluids by dengue children in the form of fruit
juices when compared to children with other viral fevers (especially
respiratory infections where there is a misconception that consumption of
fruit juices could worsen the infection) (67)
2. Moreover, the use of Nilavembu extract and papaya leaf extract is
common in Tamil Nadu and has been advocated by the state government
during dengue epidemics. The presence of alkaloids and vitamins in these
herbal preparations could have caused the increased vitamin C levels in
dengue.
72
3. Multivitamins consumed during illness could have contributed to the
significantly higher ascorbic acid levels in dengue children.
4. In previous studies in critically ill patients, it has been shown lower levels
of vitamin C are associated with severity of illnesses (68,69). Vitamin C
is a powerful anti-oxidant and helps in maintaining capillary
integrity. Although due to increased oxidative stress, vitamin C
requirements are greater in this population, levels may be restored to
normal and sometimes supra-normal, with parenteral supplementation
(53).
The high levels of vitamin C in dengue children in our study can be attributed to
the compensatory mechanisms to counteract free oxygen radical injury and the
occurrence of capillary leaks. With good enteral supplementation of
multivitamins, there could have been an acute increase in levels due to the need
for increased anti-oxidant activity.
Though we had collected data regarding nutritional status of participants, we
had not collected data regarding consumption of commercially available
multivitamin supplements, fruit juices, diet and herbal preparations like papaya
leaf extract or nilavembu extract.
73
The secondary objective of our study was to analyse the levels of vitamin C in
mild, moderate and severe forms of dengue. The levels (mean ± SD in mg/dl)
were 2.35 ± 1.29, 2.17 ± 1.55, 2.99 ± 1.62 respectively.
On comparing the mean values with one-way ANOVA, there was no statistical
significance although the mean value in severe dengue was comparatively
higher when compared to the other two groups.
The difference in the mean between the groups can be easily made out by
comparing the charts 10-12. The chart shows an increased number of children in
the group of vitamin c > 2.1mg/dl in the severe dengue group when compared to
the other two groups.
The difference might imply the possibility of lower vitamin C levels being
associated with less severe dengue again contradictory to the original
hypotheses in this study.
The reason for increased vitamin C levels in severe dengue children may again
be explained by the need for high anti-oxidant levels. Moreover the increased
capillary permeability demands the need for increased ascorbic acid levels.
74
Strengths and Limitations
This is the first study to estimate vitamin C levels in dengue and compare and
correlate the levels in mild, moderate and severe dengue.
The laboratory technician was blinded for Vitamin C analysis. Samples were
labelled and sent so that it would not be possible to distinguish if it was from
controls or mild, moderate or severe dengue.
The estimation method was a standard protocol for estimation of vitamin C. We
had adequate resources and logistics required for storage and transportation of
samples in our institution. Our study was institution funded. Ethical
considerations were strictly adhered.
Our study had a few limitations.
The diet, multivitamin supplements and herbal preparation intake was not
documented which could have impacted on the vitamin C levels.
The controls were from in-patients with minor illnesses which could have
affected vitamin C levels.
Vitamin C sample collection at fasting was not always possible especially in
severe dengue children requiring ICU admission.
HPLC method for estimation of vitamin C levels would have been more
accurate, but was not economically feasible.
CONCLUSION
75
Conclusion:
With the available data in the study, it has been shown that there is a
significantly higher level of serum vitamin C in dengue children when
compared with controls. And also, severe dengue children have higher levels
when compared to mild dengue cases. These findings are contradictory to the
original hypothesis and may be explained by multivitamin and herbal
preparation supplementation in these children. The study suggests that though
vitamin C levels can be increased by enteral supplementation, it does not alter
the course or severity of the illness.
Recommendations:
1. To complete the study to achieve adequate sample size, while including
data about multivitamin supplements, diet etc.,
2. A RCT maybe done comparing oral supplementation and parenteral
supplementation with placebo in children with moderate and severe
dengue.
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ANNEXURES
ANNEXURE A: Consent Forms
SOP 03-V 3.0 / ANX 10-V 3.0
Institutional Human Ethics Committee
PSG Institute of Medical Sciences and Research, Coimbatore
Parental Consent Form
Title of Study: Association between Serum ascorbic acid levels and severity of dengue in children – a
cross sectional observation study.
Name of the Principal Investigator: Dr. Vikram.S
Department: Paediatrics
Your (son/daughter/child/infant/adolescent youth) is invited to participate in a study of association
between serum ascorbic acid and severity of dengue.
My name is Dr. Vikram.S and I am a Post-graduate at PSG Institute of Medical Sciences and
Research, Coimbatore. This study is to know if there could be any correlation between serum ascorbic
acid levels and severity of dengue in children.
I am asking for permission to include your (son/daughter/child/infant/adolescent youth) in this study
because I expect to have 160 participants in the study.
If you allow your child to participate, I (Dr. Vikram.S) will take your child in the study, assess the
severity of dengue clinically and take 2ml of blood to ascertain his serum ascorbic acid level.
Any information that is obtained in connection with this study and that can be identified with your
(son/daughter/child/infant/adolescent youth) will remain confidential and will be disclosed only with
your permission. His or her responses will not be linked to his or her name or your name in any
written or verbal report of this research project.
Your decision to allow your (son/daughter/child/infant/adolescent youth) to participate will not affect
your or his or her present or future relationship with PSGIMS&R or PSG Hospitals or (include the
name of any other institution connected with this project). If you have any questions about the study,
please ask me. If you have any questions later, call me at 9444708589. If you have any questions or
concerns about your (son/daughter/child/infant/adolescent youth)’s participation in this study, call
9444708589.
You may keep a copy of this consent form.
You are making a decision about allowing your (son/daughter/child/infant/adolescent youth) to
participate in this study. Your signature below indicates that you have read the information provided
above and have decided to allow him or her to participate in the study. If you later decide that you
wish to withdraw your permission for your (son/daughter/child/infant/adolescent youth) to participate
in the study, simply tell me.
You may discontinue his or her participation at any time. This will not affect in any way your future
treatment in this hospital.
Printed Name of (son/daughter/child/infant/adolescent youth)
Signature of Parent(s) or Legal Guardian with Date Signature of Investigator with Date
SOP 03-V 3.0 / ANX 09-V 2.0
Institutional Human Ethics Committee
PSG Institute of Medical Sciences and Research, Coimbatore
Assent to be in a Research Study
For children between 7-18 years old
Why are we meeting with you?
We want to tell you about something we are doing called a research study. A research study is
when doctors collect a lot of information to learn more about something related to health and
disease. Dr. Vikram.S and some other doctors are doing a study to learn more about ‘Association
between Serum ascorbic acid levels and severity of dengue in children – a cross-sectional
observation study’. After we tell you about it, we will ask if you’d like to be in this study or not.
Why are we doing this study?
We want to find out if there is any correlation of serum ascorbic acid levels with severity of
dengue.
So we are getting information from lots of boys and girls like you.
In the whole study, there will be about 160 children.
What will happen to you if you are in this study?
Only if you agree, two things will happen:
1. A small amount of your blood will be drawn. That means it will be taken by a needle in
your arm. This will happen once.
[If some or all of blood draws would be done anyway as part of child’s clinical care,
emphasize here what will be done extra for the study.]
2. The doctors will do some tests on your blood
3. You will need to answer some questions about your symptoms and let us examine you
Will this study hurt?
The stick from the needle to draw your blood will hurt, but the hurt will go away after awhile.
Will you get better if you are in this study? No, this study won’t make you feel better or get well. But the doctors might find out something
that will help other children like you later.
Will everybody come to know about my condition? (Confidentiality) We will not tell other people that you are in this research and we won't share information about
you to anyone who does not work in the research study
Is this bad or dangerous for me? (Risks involved) This has minimal risks associated with blood sampling.
Do I get anything for being in the research? All the doctors would appreciate you for your courage and goodwill to participate in this study.
Will you tell me the results? The results will take sometime and will be disclosed at the end of the study. But your information
would remain confidential. The results will be made available to others through my thesis report
and journal publications.
Do you have any questions? You can ask questions any time. You can ask now. You can ask later. You can talk to me or
you can talk to someone else.
Do you have to be in this study? No, you don’t. No one will be mad at you if you don’t want to do this. If you don’t want to be in
this study, just tell us. Or if you do want to be in the study, tell us that. And, remember, you can
say yes now and change your mind later. It’s up to you. This will not affect in any way your
future treatment in this hospital.
Who can I talk to or ask questions to? List and give contact information for those people who the child can contact easily (a local person
who can actually be contacted). Tell the child that they can also talk to anyone they want to about
this (their own doctor, a family friend, a teacher).
If you don’t want to be in this study, just tell us. If you want to be in this study, just tell us. This
will not affect in any way your future treatment in this hospital.
The doctor will give you a copy of this form to keep.
SIGNATURE OF PERSON CONDUCTING ASSENT DISCUSSION I have explained the study to ______________________ in language he/she can understand, and
the child has agreed to be in the study.
__________________________________ _______________
Signature of Person Conducting Assent Discussion Date
_______________________________
Name of Person Conducting Assent Discussion (print)
Part 2: Certificate of Assent
I have read this information (or had the information read to me) I have had my questions
answered and know that I can ask questions later if I have them.
I agree to take part in the research.
OR
I do not wish to take part in the research and I have not signed the assent below.___________
(initialed by child/minor)
Only if child assents:
Print name of child ___________________
Signature of child: ____________________
Date:________________
day/month/year
If illiterate: A literate witness must sign (if possible, this person should be selected by the participant, not be
a parent, and should have no connection to the research team). Participants who are illiterate
should include their thumb print as well.
I have witnessed the accurate reading of the assent form to the child, and the individual has had
the opportunity to ask questions. I confirm that the individual has given consent freely.
Print name of witness (not a parent)_________________ AND Thumb print of participant
Signature of witness ______________________
Date ________________________
Day/month/year
I have accurately read or witnessed the accurate reading of the assent form to the potential
participant, and the individual has had the opportunity to ask questions. I confirm that the
individual has given assent freely.
Print name of researcher_________________
*Modified from the Informed Assent form template for children/minors –World Health
organization
ANNEXURE B: PROFORMA
ANNEXURE C: DETERMINATION OF VITAMIN C (Volumetric method)
Principle:
Ascorbic acid reduces the 2, 6 –dichlorophenol indo phenol dye to a colourless leuco-
base. Ascorbic acid gets oxidized to dehydroascorbic acid.
Materials Required:
• Oxalic acid 4%
• Dye solution(42mg NaHCO3 + 2,6-dichorophenol indophenol and make upto 200ml)
• Stock Std Ascorbic acid solution(100mg in 100ml 4% oxalic acid)
• Working Std Solution(10ml Stock Std made upto 100ml with 4% oxalic acid)
Procedure:
• Pipette out 5ml of working std into 100ml conical flask
• Add 10ml of 4%oxalic acid and titrate against the dye. End point is appearance of
pink colour which persists for few min.
• Extract the sample (0.5-5g depending on sample) in 4% oxalic acid and make upto
known volume and centrifuge
• Pipette out 5ml of supernatant add 10ml of 4% oxalic acid and titrate against the
dye.
Calculation:
Amt of ascorbic acid, mg/100g of sample= 0.5/V1 X V2/5 X 100/ W X 100
V1 – Volume of dye used for STD ascorbic acid solution
V2- Volume of dye used for sample.
W- Weight of sample taken for test.
Reference:
1. Harris. L J and Ray, S.N (1935) Lancet 1 462.
2. Sadasivam.S and Theymoli Balasubramaniam(1987) In: Practical Manual in
Biochemistry Tamil Nadu Agricultural University
Titration of Dye against standard solution:
Titration of Dye against test solution:
ANNEXURE D: Master Chart
S.No Sample IP No Age Sex Vit. C level Severity Primary or not
1 V1 I18028877 7.5 M 0.9 MOD 2ᵒ
2 V3 I18033739 12 M 3.6 MILD 2ᵒ
3 V4 I18034403 8 F 1.8 MOD 2ᵒ
4 V11 I18035837 8 F 1.8 SD 2ᵒ
5 V12 I18036468 10 M 3.6 MOD 2ᵒ
6 V14 I18036679 7 F 3.6 MOD 1ᵒ
7 V15 I18038081 1 M 2.25 MILD 1ᵒ
8 V16 I18038057 2 M 3.6 MILD 2ᵒ
9 V19 I18038997 3.8 F 6 MILD 2ᵒ
10 V22 I18039100 11 F 4 MILD 1ᵒ
11 V23 I18038889 3.25 F 4 MOD 2ᵒ
12 V24 I18039572 3 M 4 MILD 2ᵒ
13 V25 I18040600 2 F 2 MOD 1ᵒ
14 V26 I18041198 7 F 6 MOD 2ᵒ
15 V27 I18041762 12 M 4 MOD 2ᵒ
16 V28 I18041681 1 F 2 MILD 2ᵒ
17 V30 I19000923 12 M 6 SD 2ᵒ
18 V31 I19000765 2 M 6 MOD 1ᵒ
19 V32 I19002796 5.8 M 4 MOD 2ᵒ
20 V33 I19004362 11 M 4.9 MILD 2ᵒ
21 V34 I19004515 7.3 F 1.96 MOD 2ᵒ
22 V35 I19010493 13 M 3.92 MOD 2ᵒ
23 V36 I19013702 0.6 M 4 SD 1ᵒ
24 V37 I19016334 8 M 5 SD 1ᵒ
25 V38 I19019166 10 M 4 MOD 2ᵒ
26 V39 I19019410 6 F 3 SD 2ᵒ
27 V41 I19019909 1 M 2 MILD 2ᵒ
28 V42 I19024202 10 M 1.5 MILD 2ᵒ
29 V43 I19024633 5.2 F 1 SD 2ᵒ
30 V44 I19024878 6.8 F 1 MOD 2ᵒ
31 V45 I19026829 7.2 M 0.5 MOD 2ᵒ
32 V46 I19026918 4.1 M 0.5 MOD 2ᵒ
33 V47 I19027177 12 M 1.67 MOD 1ᵒ
34 V48 I19028345 2 F 2 MILD 1ᵒ
35 V49 I19029564 6.9 M 0.67 MOD 1ᵒ
36 V50 I19029549 9 F UNDETECTABLE MILD 2ᵒ
37 V51 I19030159 11 M 1 MILD 1ᵒ
38 V52 I19031088 6 F 3 MILD 2ᵒ
39 V53 I19031019 0.9 M 1.11 MOD 1ᵒ
40 V56 I19031091 14 M 1 MILD 1ᵒ
41 V58 I19030960 7 M 1 MOD 2ᵒ
42 V61 I19032490 14 M 1.96 MOD 2ᵒ
43 V62 I19032743 10 F 2.94 MILD 2ᵒ
44 V63 I19032844 2 F 2.94 SD 1ᵒ
45 V65 I19032768 1 M 1.47 MOD 2ᵒ
46 V66 I19033007 8 M 1.96 MILD 2ᵒ
47 V67 I19033136 0.7 F 0.98 MOD 1ᵒ
48 V68 I19033307 14 M 2.94 MILD 1ᵒ
49 V69 I19033804 6.7 F 1.47 MOD 2ᵒ
50 V70 I19033805 3 M 1.84 MILD 1ᵒ
51 V71 I19033744 11 M 1.47 MOD 2ᵒ
52 V75 I19034448 5 M 1.96 MILD 1ᵒ
53 V76 I19034225 12 M 1.96 SD 2ᵒ
54 V77 I19034706 14 M 1.96 MILD 2ᵒ
55 V78 I19035034 14 F 2.94 SD 2ᵒ
56 V79 I19035107 2 M 1.47 MILD 1ᵒ
57 V80 I19035124 5 M 1.96 MILD 1ᵒ
58 V82 I19035456 0.7 F 1.47 MOD 1ᵒ
59 V83 I19035584 12 M 1.23 SD 2ᵒ
60 V85 I19035709 12 F 1.96 MOD 2ᵒ
61 V86 I19035975 2 F 0.98 MOD 2ᵒ
62 V87 I19035745 11 M 1.23 MILD 2ᵒ
63 V88 I19036352 10 M 0.98 MILD 2ᵒ
64 V89 I19036502 5 M 1.96 MILD 1ᵒ
65 V90 I19036651 15 F 0.98 MOD 1ᵒ
66 V92 I19036733 11 M 1.23 MOD 1ᵒ
67 V93 I19036805 7 M 1.84 MILD 2ᵒ
68 V94 I19036846 14 M 0.98 MOD 1ᵒ
69 CV18 I19034806 4 F 1.96 MILD 1ᵒ
70 V2 I18030067 9 F 2.7 CONTROL
71 V5 I18034450 3 M 3.6 CONTROL
72 V6 I18034567 9 M 1.8 CONTROL
73 V7 I18034586 1 M 3.6 CONTROL
74 V9 I18034992 2 F 3.6 CONTROL
75 V13 I18036365 17 M 3.6 CONTROL
76 V54 I19030938 12 M 1.25 CONTROL
77 V55 I19030534 11 F 1 CONTROL
78 V57 I19031187 11 M 2 CONTROL
79 V59 I19031739 1 M 0.98 CONTROL
80 V60 I19032175 2 M 1.47 CONTROL
81 V64 I19032744 6 M 1.96 CONTROL
82 V72 I19034138 3 M 2.94 CONTROL
83 V73 I19034571 11 F 2.18 CONTROL
84 V81 I19035180 14 M 1.09 CONTROL
85 CV1 I19031123 13 F 1 CONTROL
86 CV3 I19031826 11 F 1.47 CONTROL
87 CV5 I19032252 1 M 0.49 CONTROL
88 CV6 I19032422 2 F 1.23 CONTROL
89 CV7 I19032758 4 M 0.98 CONTROL
90 CV8 I19032641 5 F 1.23 CONTROL
91 CV9 I19032875 4 M 0 CONTROL
92 CV10 I19032882 2 M 0.98 CONTROL
93 CV12 I19033134 8 M 2.94 CONTROL
94 CV13 I19033303 5 M 1.47 CONTROL
95 CV14 I19033956 3 M 1.47 CONTROL
96 CV15 I19034100 1 M 2.94 CONTROL
97 CV16 I19033923 5 M 0.98 CONTROL
98 CV17 I19034580 6 M 2.94 CONTROL
99 CV19 I19034719 9 M 0.49 CONTROL
100 CV20 I19035027 7 M 1.96 CONTROL
101 CV21 I19034971 1 M 0.98 CONTROL
102 CV23 I19035120 5 F 2.94 CONTROL
103 CV24 I19035136 13 M 1.96 CONTROL
104 CV25 I19035104 2 M 0.98 CONTROL
105 CV26 I19035166 3 M 1.96 CONTROL
106 CV27 I19035167 5 F 0.98 CONTROL
107 CV28 I19035290 12 M 0.49 CONTROL
108 CV29 I19035455 6 F 1.96 CONTROL
109 CV31 I19035452 1 M 0.61 CONTROL
ANNEXURE E –Abbreviations
ANOVA – Analysis of Varience
CD – Cluster of Differentiation
CDC – Centre for Disease Control
DALY – Disability Adjusted Life Years
DCPIP – Dichlorophenolindophenol
DENV – Dengue Virus
DF – Dengue Fever
Etc – et cetera
GOI – Government Of India
HLA – Human Leukocyte Antigen
HPLC – High Pressure Liquid Chromatography
IAP – Indian Academy of Paediatrics
IL – Interleukin
MoHFW – Ministry of Health and Family Welfare
NS – Non- Structural
NVBDCP – National Vector Borne Disease Control Programme
PICU – Pediatric Intensive Care Unit
PSGIMS&R – PSG Institute of Medical Sciences and Research
RCT – Randomized controlled trial
TNF – Tumor Necrosis Factor
USD – US Dollar
WBC – White Blood Cells
WHO – World Health Organisation