ASSOCIATION BETWEEN SERUM ASCORBIC ACID LEVELS AND

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


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.


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



The increase in incidence of infection and deaths related to

has been attributed to unplanned urbanization,


lack of access to proper health-care facilities (1).


predilections. The severity of the disease had bimodal distribution, with



morbidities in elderly complement the disease severit

3

fold in last 50 years with geographic



and deaths related to

has been attributed to unplanned urbanization,



imodal distribution, with



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


0.6-2 >2.1

0.245 Moderate Dengue 22 09

Total 22 09


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


0.6-2 >2.1

0.126 Severe Dengue 04 06

Total 04 06


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.

BIBLIOGRAPHY

BIBLIOGRAPHY

1. Special Programme for Research and Training in Tropical Diseases, World Health

Organization, editors. Dengue: guidelines for diagnosis, treatment, prevention, and

control. New ed. Geneva: TDR : World Health Organization; 2009. 147 p.

2. Dengue-National-Guidelines-2014 by GOI - NVBCDP.

3. Sharma K, Angel B, Singh H, Purohit A, Joshi V. Entomological studies for

surveillance and prevention of dengue in arid and semi-arid districts of Rajasthan,

India. J Vector Borne Dis. 2008 Jun;45(2):124–32.

4. Reiter P. Yellow fever and dengue: a threat to Europe.

5. DENGUE/DHF SITUATION IN INDIA :: National Vector Borne Disease Control

Programme (NVBDCP) [Internet]. [cited 2019 Oct 13]. Available from:

https://nvbdcp.gov.in/index4.php?lang=1&level=0&linkid=431&lid=3715

6. Ganeshkumar P, Murhekar MV, Poornima V, Saravanakumar V, Sukumaran K,

Anandaselvasankar A, et al. Dengue infection in India: A systematic review and meta-

analysis. Rodriguez-Barraquer I, editor. PLoS Negl Trop Dis. 2018 Jul

16;12(7):e0006618.

7. Shepard DS, Halasa YA, Tyagi BK, Adhish SV, Nandan D, Karthiga KS, et al.

Economic and Disease Burden of Dengue Illness in India. Am J Trop Med Hyg. 2014

Dec 3;91(6):1235–42.

8. Mutheneni SR, Morse AP, Caminade C, Upadhyayula SM. Dengue burden in India:

recent trends and importance of climatic parameters. Emerging Microbes &

Infections. 2017 Jan;6(1):1–10.

9. Nimmannitya S. Clinical spectrum and management of dengue haemorrhagic fever.

Southeast Asian J Trop Med Public Health. 1987 Sep;18(3):392–7.

10. Zabet MH, Mohammadi M, Ramezani M, Khalili H. Effect of high-dose Ascorbic

acid on vasopressor’s requirement in septic shock. J Res Pharm Pract. 2016

Jun;5(2):94–100.

11. Matsuda T, Tanaka H, Shimazaki S, Matsuda H, Abcarian H, Reyes H, et al.

High-dose vitamin C therapy for extensive deep dermal burns. Burns. 1992

Apr;18(2):127–31.

12. Kremer T, Harenberg P, Hernekamp F, Riedel K, Gebhardt MM, Germann G, et

al. High-Dose Vitamin C Treatment Reduces Capillary Leakage After Burn Plasma

Transfer in Rats. J Burn Care Res. 2010 May 1;31(3):470–9.

13. Tanaka H, Matsuda T, Miyagantani Y, Yukioka T, Matsuda H, Shimazaki S.

Reduction of resuscitation fluid volumes in severely burned patients using ascorbic

acid administration: a randomized, prospective study. Arch Surg. 2000

Mar;135(3):326–31.

14. Vitamin C deficiency - Symptoms, diagnosis and treatment | BMJ Best Practice

[Internet]. [cited 2019 Sep 30]. Available from:

https://bestpractice.bmj.com/topics/en-us/632

15. Office of Dietary Supplements - Vitamin C [Internet]. [cited 2019 Oct 14].

Available from: https://ods.od.nih.gov/factsheets/VitaminC-HealthProfessional/

16. Gupta N, Srivastava S, Jain A, Chaturvedi UC. Dengue in India. Indian J Med

Res. 2012 Sep;136(3):373–90.

17. Fulmali PV, Walimbe A, Mahadev PVM. Spread, establishment & prevalence of

dengue vector Aedes aegypti (L.) in Konkan region, Maharashtra, India. Indian J Med

Res. 2008 Jun;127(6):589–601.

18. Tikar SN, Mendki MJ, Chandel K, Parashar BD, Prakash S. Susceptibility of

immature stages of Aedes (Stegomyia) aegypti; vector of dengue and chikungunya to

insecticides from India. Parasitol Res. 2008 Apr;102(5):907–13.

19. Ashok Kumar V, Rajendran R, Manavalan R, Tewari SC, Arunachalam N, Ayanar

K, et al. Studies on community knowledge and behavior following a dengue epidemic

in Chennai city, Tamil Nadu, India. Trop Biomed. 2010 Aug;27(2):330–6.

20. Research C for BE and. DENGVAXIA. FDA [Internet]. 2019 Sep 18 [cited 2019

Oct 14]; Available from: http://www.fda.gov/vaccines-blood-biologics/dengvaxia

21. Libraty DH, Young PR, Pickering D, Endy TP, Kalayanarooj S, Green S, et al.

High circulating levels of the dengue virus nonstructural protein NS1 early in dengue

illness correlate with the development of dengue hemorrhagic fever. J Infect Dis. 2002

Oct 15;186(8):1165–8.

22. Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S,

et al. Dengue viremia titer, antibody response pattern, and virus serotype correlate

with disease severity. J Infect Dis. 2000 Jan;181(1):2–9.

23. Jaenisch T, Tam DTH, Kieu NTT, Van Ngoc T, Nam NT, Van Kinh N, et al.

Clinical evaluation of dengue and identification of risk factors for severe disease:

protocol for a multicentre study in 8 countries. BMC Infect Dis [Internet]. 2016 Mar

11 [cited 2019 Oct 14];16. Available from:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788847/

24. Phakhounthong K, Chaovalit P, Jittamala P, Blacksell SD, Carter MJ, Turner P, et

al. Predicting the severity of dengue fever in children on admission based on clinical

features and laboratory indicators: application of classification tree analysis. BMC

Pediatr. 2018 Dec;18(1):109.

25. Kedia N, Mahadevan S, Kumar RR. Abstract PD-011: RISK FACTORS FOR

SEVERE DENGUE: RETROSPECTIVE OBSERVATIONAL STUDY FROM A

PICU IN SOUTHERN INDIA. Pediatric Critical Care Medicine. 2018 Jun;19(6S):31.

26. Fernández‐Mestre MT, Gendzekhadze K, Rivas‐Vetencourt P, Layrisse Z. TNF-

α-308A allele, a possible severity risk factor of hemorrhagic manifestation in dengue

fever patients. Tissue Antigens. 2004;64(4):469–72.

27. Yacoub S, Mongkolsapaya J, Screaton G. Recent advances in understanding

dengue. F1000Res [Internet]. 2016 Jan 19 [cited 2019 Oct 15];5. Available from:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754027/

28. Nguyen NM, Tran CNB, Phung LK, Duong KTH, Huynh H le A, Farrar J, et al. A

Randomized, Double-Blind Placebo Controlled Trial of Balapiravir, a Polymerase

Inhibitor, in Adult Dengue Patients. J Infect Dis. 2013 May 1;207(9):1442–50.

29. Tricou V, Minh NN, Van TP, Lee SJ, Farrar J, Wills B, et al. A Randomized

Controlled Trial of Chloroquine for the Treatment of Dengue in Vietnamese Adults.

PLoS Negl Trop Dis [Internet]. 2010 Aug 10 [cited 2019 Oct 15];4(8). Available

from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2919376/

30. Tam DTH, Ngoc TV, Tien NTH, Kieu NTT, Thuy TTT, Thanh LTC, et al. Effects

of Short-Course Oral Corticosteroid Therapy in Early Dengue Infection in Vietnamese

Patients: A Randomized, Placebo-Controlled Trial. Clin Infect Dis. 2012 Nov

1;55(9):1216–24.

31. Tassniyom S, Vasanawathana S, Chirawatkul A, Rojanasuphot S. Failure of high-

dose methylprednisolone in established dengue shock syndrome: a placebo-controlled,

double-blind study. Pediatrics. 1993 Jul;92(1):111–5.

32. Whitehorn J, Nguyen CVV, Khanh LP, Kien DTH, Quyen NTH, Tran NTT, et al.

Lovastatin for the Treatment of Adult Patients With Dengue: A Randomized, Double-

Blind, Placebo-Controlled Trial. Clin Infect Dis. 2016 Feb 15;62(4):468–76.

33. Dimaano EM, Saito M, Honda S, Miranda EA, Alonzo MTG, Valerio MD, et al.

Lack of efficacy of high-dose intravenous immunoglobulin treatment of severe

thrombocytopenia in patients with secondary dengue virus infection. Am J Trop Med

Hyg. 2007 Dec;77(6):1135–8.

34. Ahmed S, Finkelstein JL, Stewart AM, Kenneth J, Polhemus ME, Endy TP, et al.

Micronutrients and Dengue. Am J Trop Med Hyg. 2014 Nov 5;91(5):1049–56.

35. Marón GM, Clará AW, Diddle JW, Pleités EB, Miller L, MacDonald G, et al.

Association between Nutritional Status and Severity of Dengue Infection in Children

in El Salvador. Am J Trop Med Hyg. 2010 Feb;82(2):324–9.

36. Alagarasu K, Bachal RV, Bhagat AB, Shah PS, Dayaraj C. Elevated levels of

vitamin D and deficiency of mannose binding lectin in dengue hemorrhagic fever.

Virol J. 2012 May 4;9:86.

37. Fatima H, Riaz M, Mahmood Z, Yousaf F, Shahid M. Dengue viral infection

deteriorates vitamin D3, K, thrombopoietin, and angiotensinogen levels in humans.

Eur J Inflamm. 2018 Jan 1;16:2058739218791100.

38. Villamor E, Villar LA, Lozano A, Herrera VM, Herrán OF. Vitamin D serostatus

and dengue fever progression to dengue hemorrhagic fever/dengue shock syndrome.

Epidemiol Infect. 2017;145(14):2961–70.

39. Paediatrica Indonesiana Serum zinc levels and clinical severity of dengue infection

in children [Internet]. [cited 2019 Oct 18]. Available from:

https://scholar.googleusercontent.com/scholar?q=cache:vwHBI4W9rbIJ:scholar.googl

e.com/&hl=en&as_sdt=0,5

40. Klassen P, Biesalski HK, Mazariegos M, Solomons NW, Fürst P. Classic dengue

fever affects levels of circulating antioxidants. Nutrition. 2004 Jun;20(6):542–7.

41. Chaiyaratana W, Chuansumrit A, Atamasirikul K, Tangnararatchakit K. Serum

ferritin levels in children with dengue infection. Southeast Asian J Trop Med Public

Health. 2008 Sep;39(5):832–6.

42. Shrivastava R, Nagar R, Ravishankar GA, Upreti RK, Chaturvedi UC. Effect of

pretreatment with chromium picolinate on haematological parameters during dengue

virus infection in mice. Indian J Med Res. 2007 Nov;126(5):440–6.

43. Rubeena Mattummal DKG, Rubeena Mattummal DKG. Bioactive molecules in

Siddha Polyherbal Nilavembu Kudineer alleviating symptoms of Dengue/Chikugunya.

Traditional Medicine Research. 2018 Sep 4;3(5):215–29.

44. Jain J, Kumar A, Narayanan V, Ramaswamy RS, Sathiyarajeswaran P, Shree Devi

MS, et al. Antiviral activity of ethanolic extract of Nilavembu Kudineer against

dengue and chikungunya virus through in vitro evaluation. J Ayurveda Integr Med.

2019 Jan 23;

45. G.J Christina, M. Subramanian, D.Periyasamy, K. Manickavasakam, P.

Gunasekaran, S. Sivasubramaniam et al. Protective effect of polyherbal siddha

formulation - Nilavembu kudineer against common viral fevers including dengue - A

case control approach. IJPSR. 6(4).

46. Charan J, Saxena D, Goyal JP, Yasobant S. Efficacy and safety of Carica papaya

leaf extract in the dengue: A systematic review and meta-analysis. Int J Appl Basic

Med Res. 2016;6(4):249–54.

47. Mishra S, Agrahari K, Shah DK. Prevention and control of dengue by diet therapy.

48. Sánchez-Valdéz E, Delgado-Aradillas M, Torres-Martínez JA, Torres-Benítez JM.

Clinical response in patients with dengue fever to oral calcium plus vitamin D

administration: study of 5 cases. Proc West Pharmacol Soc. 2009;52:14–7.

49. Vaish A, Verma S, Agarwal A, Gupta L, Gutch M. Effect of vitamin E on

thrombocytopenia in dengue fever. Annals of Tropical Medicine and Public Health.

2012 Jul 1;5(4):282.

50. Gershoff SN. Vitamin C (ascorbic acid): new roles, new requirements? Nutr Rev.

1993 Nov;51(11):313–26.

51. Vitamin C Infusion for Treatment in Sepsis Induced Acute Lung Injury - Full Text

View - ClinicalTrials.gov [Internet]. [cited 2019 Nov 7]. Available from:

https://clinicaltrials.gov/ct2/show/NCT02106975

52. HIGH-DOSE VITAMIN C INFUSION REDUCES FLUID REQUIREMENTS IN

THE RESUSCITATION OF BURN-INJURED SHEEP [Internet]. [cited 2019 Nov

7]. Available from: https://insights.ovid.com/crossref?an=00024382-200508000-

00007

53. Zhang M, Jativa DF. Vitamin C supplementation in the critically ill: A systematic

review and meta-analysis. SAGE Open Med [Internet]. 2018 Oct 19 [cited 2019 Nov

9];6. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6196621/

54. Patak P, Willenberg HS, Bornstein SR. Vitamin C is an important cofactor for

both adrenal cortex and adrenal medulla. Endocr Res. 2004 Nov;30(4):871–5.

55. May JM, Qu Z, Meredith ME. Mechanisms of Ascorbic Acid Stimulation of

Norepinephrine Synthesis in Neuronal Cells. Biochem Biophys Res Commun. 2012

Sep 14;426(1):148–52.

56. Carr AC, Shaw GM, Fowler AA, Natarajan R. Ascorbate-dependent vasopressor

synthesis: a rationale for vitamin C administration in severe sepsis and septic shock?

Crit Care. 2015 Nov 27;19:418.

57. Rajapakse S, Rodrigo C, Rajapakse A. Treatment of dengue fever. Infect Drug

Resist. 2012 Jul 23;5:103–12.

58. Mallhi TH, Khan AH, Sarriff A, Adnan AS, Khan YH. Determinants of mortality

and prolonged hospital stay among dengue patients attending tertiary care hospital: a

cross-sectional retrospective analysis. BMJ Open. 2017 Aug 1;7(7):e016805.

59. Said HM. Intestinal absorption of water-soluble vitamins in health and disease.

Biochem J. 2011 Aug 1;437(3):357–72.

60. Society (DGE) GN. New Reference Values for Vitamin C Intake. ANM.

2015;67(1):13–20.

61. Pisoschi AM, Danet AF, Kalinowski S. Ascorbic Acid Determination in

Commercial Fruit Juice Samples by Cyclic Voltammetry [Internet]. Journal of

Analytical Methods in Chemistry. 2008 [cited 2019 Nov 9]. Available from:

https://www.hindawi.com/journals/jamc/2008/937651/

62. S. S, Balasubramanian T. Practical Manual in Biochemistry. Tamil Nadu

Agricultural University, Coimbatore. (1987).

63. VITC - Clinical: Ascorbic Acid (Vitamin C), Plasma [Internet]. [cited 2019 Nov

9]. Available from: https://www.mayocliniclabs.com/test-

catalog/Clinical+and+Interpretive/42362

64. Nelson Textbook of Pediatrics 21th Edition 2020.

65. handbook_for_clinical_management_of_dengue.pdf [Internet]. [cited 2019 Nov

9]. Available from:

http://www.wpro.who.int/mvp/documents/handbook_for_clinical_management_of_de

ngue.pdf

66. Ranjit S, Ramanathan G, Ramakrishnan B, Kissoon N. Targeted Interventions in

Critically Ill Children with Severe Dengue. Indian J Crit Care Med. 2018

Mar;22(3):154–61.

67. Upper Respiratory Tract Infection Symptoms, Treatment & Causes [Internet].

MedicineNet. [cited 2019 Nov 9]. Available from:

https://www.medicinenet.com/upper_respiratory_infection/article.htm

68. Long CL, Maull KI, Krishnan RS, Laws HL, Geiger JW, Borghesi L, et al.

Ascorbic acid dynamics in the seriously ill and injured. J Surg Res. 2003

Feb;109(2):144–8.

69. Total vitamin C, ascorbic acid, and dehydroascorbic acid concentrations in plasma

of critically ill patients. - PubMed - NCBI [Internet]. [cited 2019 Nov 9]. Available

from: https://www.ncbi.nlm.nih.gov/pubmed/8615361

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

Documents

ASSOCIATION BETWEEN SERUM ASCORBIC ACID LEVELS AND