ASSOCIATION BETWEEN SERUM ASCORBIC ACID LEVELS AND SEVERITY OF DENGUE IN CHILDREN – A
CROSS SECTIONAL OBSERVATIONAL STUDY
Dissertation Submitted to
THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY, CHENNAI
In the fulfilment of the regulations for the award of the degree Doctor of Medicine in Paediatrics
By
Dr. S. VIKRAM (Reg. No: 201717502) Under the guidance of Dr. A. JAYAVARDHANA,
Professor of Paediatrics.
PSG INSTITUTE OF MEDICAL SCIENCE AND RESEARCH THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY CHENNAI,
TAMILNADU
MAY 2020
ASSOCIATION BETWEEN SERUM ASCORBIC ACID LEVELS AND SEVERITY OF DENGUE IN CHILDREN – A
CROSS SECTIONAL OBSERVATIONAL STUDY
Dissertation Submitted to
THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY, CHENNAI
In the fulfilment of the regulations for the award of the degree Doctor of Medicine in Paediatrics
By
Dr. S. VIKRAM (Reg. No: 201717502) Under the guidance of Dr. A. JAYAVARDHANA,
Professor of Paediatrics.
PSG INSTITUTE OF MEDICAL SCIENCE AND RESEARCH THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY CHENNAI,
TAMILNADU
MAY 2020
CERTIFICATE
This is to certify that the thesis entitled “ASSOCIATION BETWEEN SERUM ASCORBIC ACID LEVELS AND SEVERITY OF DENGUE IN CHILDREN – A CROSS SECTIONAL OBSERVATIONAL STUDY” is a bonafide work of Dr. S. VIKRAM done under the direct guidance and
supervision of Dr. A. JAYAVARDHANA, Professor, Department of Paediatrics, PSG Institute of Medical Sciences and Research, Coimbatore in fulfilment of the regulations of The Tamil Nadu Dr.MGR Medical University for the award of M.D. degree in Paediatrics.
Prof. Dr. NEELAKANDAN Dr. RAMALINGAM
Head of the Department DEAN
Dept. of Paediatrics PSGIMS&R
PSGIMS&R
CERTIFICATE BY THE GUIDE
This is to certify that the thesis entitled “ASSOCIATION BETWEEN SERUM ASCORBIC ACID LEVELS AND SEVERITY OF DENGUE IN CHILDREN – A CROSS SECTIONAL OBSERVATIONAL STUDY” is a bonafide work of Dr. S. VIKRAM done under my direct guidance and supervision in the department of Paediatrics, PSG Institute of Medical Sciences and Research, Coimbatore in the fulfilment of the regulations of The Tamil Nadu Dr.MGR Medical University for the award of MD degree in Paediatrics.
Dr. A. JAYAVARDHANA, Professor
Dept. of Paediatrics, PSGIMS&R.
DECLARATION
I, Dr. S. Vikram hereby declare that this dissertation entitled
“ASSOCIATION BETWEEN SERUM ASCORBIC ACID LEVELS AND SEVERITY OF DENGUE IN CHILDREN – A CROSS SECTIONAL OBSERVATIONAL STUDY” was prepared by me under the direct guidance and supervision of Dr. A. JAYAVARDHANA, Professor, Department of Paediatrics, PSG Institute of Medical Sciences and Research, Coimbatore.
The dissertation is submitted to The Tamil Nadu Dr.MGR Medical University, Chennai, in fulfilment of the University regulations for the award of M.D. degree in Paediatrics. This dissertation has not been submitted for the award of any other Degree or Diploma.
DR. S. VIKRAM
ETHICAL COMMITTEE APPROVAL
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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
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ACKNOWLEDGEMENT
I am extremely grateful to my guide, Dr. A. Jayavardhana, for his timely advice and guidance at various stages of my dissertation right from choosing the topic till analysis of results.
I would like to extend my whole-hearted gratitude to all faculties in the department; First and foremost, to my HOD, Dr. Neelakandan for his supervision and constant encouragement. I also thank my teachers Professor Dr. K. Jothilakshmi, Dr. S. Ramesh, Dr. P. Venkateswaran and Dr. N.T Rajesh for their care and constant support in completion of this thesis.
I thank Dr. Bharathi Elangovan for her unflinching support and timely help while writing the dissertation. I also thank her father Dr. Elangovan for his valuable data analysis.
I cannot forget all the help made by my Assistant Professors Dr.
Nirmala, Dr. Indumathi, Dr. Muruganantham, Dr. Vadivel Vinoth, Dr.
Gayathri, Dr. Sudhakar, Dr. Saranyaa, Dr. Suchitra, Dr. Lavanya, Dr.
Deepthi Shetty and Dr. Sumathi and would like to use this opportunity to profusely thank all of them.
I would like to make a special mention of Dr. Veda Senthil Velan, Dr.
Shruthi Ravikumar, Dr. Jayamkondan and all seniors who were very supportive and friendly. I thank them all for the help they did during the initial stages of my thesis.
I would also like to thank my colleagues Dr. Christina, Dr. Induja, Dr.
Lavanya, Dr. Arun Prasath, Dr. Naveenkumar, Dr. Lingeshwaran and Dr.
Srinidhi, Dr. Aruna Rani and all other juniors for helping me in sample collection and covering up for me during my ward duties.
This study would not be possible without funding and logistics from the institution for which I express my sincere gratitude to the dean Dr.
Ramalingam and Dr. Sudha Ramalingam. A special mention to all the staff at CMMT lab who were always happy to help.
I express my sincere gratitude to Dr. Arun Padmanandan, Assistant Professor, Department of SPM for his valuable time for the data analysis.
I thank Dr. Gayathri, Associate Professor, Department of Biochemistry for helping me procure reagents and for teaching me about vitamin C estimation.
I cannot forget the help of Mr. Sivakumar of Stanes Phytolab and his team who were very co-operative and I take this opportunity to thank them.
I would like to exceptionally thank all the staffs in the Paediatric ward, PICU and special wards for helping me obtain samples for my study. I thank the staffs in the OPD for all their help and support.
I thank my wife, Dr. Janaki. V for always being there for me. Her part in the completion of this thesis was very crucial and valuable.
I would be failing in my duty if I do not immensely thank my beloved parents, Mr.R. Sriram and Mrs. Jaya Sriram and my brother, Mr.
Vaikunth.S and his wife Mrs. V. Suryalakshmi for making me what I am today, and offering constant support all along without which this thesis would not have been completed.
And I thank the almighty for His grace. I also thank my spiritual guru Mr. Anandan for moral support. I thank all my friends and well-wishers.
Last but not the least I thank all my patients without whose consent, I would not have been able to complete this study.
TABLE OF CONTENTS
SL.NO. CONTENT PAGE NO.
1 INTRODUCTION 1 - 14
2 RESEARCH QUESTION 15
3 REVIEW OF LITERATURE 16 - 37
4 MATERIALS AND METHODS 38 - 47
5 RESULTS 48 - 69
6 DISCUSSION 70 - 74
7 CONCLUSION 75
8 BIBLIOGRAPHY
9 ANNEXURES
A)Consent Forms B)Proforma
C)Vitamin C Estimation by DCPIP D)Master chart
E) List of Abbreviations
LIST OF TABLES
Table 1: Agewise distribution of cases and controls...50
Table 2: Genderwise distribution of cases and controls...53
Table 3: Comparison of laboratory parameters between cases and controls...54
Table 4: Comparison of mean Vitamin C levels in Dengue and in controls...57
Table 5: Distribution of cases according to severity of dengue...58
Table 6: Distribution of primary and secondary dengue based on severity...60
Table 7: Comparison of baseline characteristics among mild, moderate and severe dengue...61
Table 8: Comparison of baseline laboratory parameters among mild, moderate and severe dengue...62
Table 9: Comparison of Vitamin C levels in mild, moderate and severe dengue...64
Table 10: Association between severity of dengue and serum ascorbic acid levels...65
Table 11: Association between mild dengue and ascorbic acid levels...67
Table 12: Association between moderate dengue and ascorbic acid levels...68
Table 13: Association between severe dengue and ascorbic acid levels...69
LIST OF FIGURES
Figure 1Dengue around the world (Source: CDC) ... 3
Figure 2 Incidence of dengue in India - From 1998 to 2014 (Source: Dengue burden in India by Mutheneni et al) (4) ... 5
Figure 3 Seasonal Trend of occurrence of cases. (Source: National Guidelines for clinical management of dengue fever 2014 - NVBDCP) ... 6
Figure 4 Course of the illness (Source: CDC) ... 8
Figure 5 Patho-physiology of dengue fever (Source: NVBDCP National guidelines for clinical management of dengue fever) ... 10
Figure 6 Classification of dengue fever (Source: NVBDCP guidelines for clinical management of dengue fever)...43
Figure 7 Flowchart (Proposed methodology)...47
Figure 8 Flowchart of the actual study...49
Figure 9 Agewise distribution of Dengue cases...51
Figure 10 Agewise distribution of Control...52
Figure 11 Genderwise distribution of Cases and Controls...53
Figure 12 Distribution of Vitamin C levels among Cases...55
Figure 13 Distribution of Vitamin C levels among Control...56
Figure 14 Classification of Dengue cases clinically...59
Figure 15 Primary and Secondary Dengue...60
Figure 16 Outcome of all dengue cases...63
Figure 17 Vitamin C levels in mild, moderate and severe dengue – comparison using cut-off of 0.6mg/dl...64
Figure 18 Vitamin C levels in mild dengue – number of cases in normal and above normal range...67
Figure 19 Vitamin C levels in moderate dengue – number of cases in normal and above normal range...68
Figure 20 Vitamin C levels in severe dengue – number of cases in normal and above normal range...67
INTRODUCTION
1
Introduction:
Dengue fever is a viral infection caused by arthropod-borne virus of the flaviviridae family, occurring in countries with temperate climate (1).
There are four identified serotypes of dengue virus (DENV1, DENV2, DENV3, and DENV4). Each serotype has several genotypes – 3 in DENV1, 2 in
DENV2, 4 in DENV3 and 4 in DENV4. The virus has three structural protein genes (coding for core, membrane associated and an envelope protein of the nucleocapsid) and seven non-structural proteins-NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, of which only NS1 antigen is found to interact with host immune system and the function of others is not well-characterised (2).
The disease is spread by the bite of Aedes mosquitoes (1). Aedes aegypti is the predominant vector species in dengue fever although other species like Aedes albinopictus are significant vectors. These mosquitoes are usually day-time biters and breed in clean water. Trans-ovarian transmission of the virus (i.e., the ability of mosquitoes born out of infected mosquitoes to act as vectors) also contribute to the exponential infection occurring during outbreaks (3). The mosquitoes usually fly only over a short distance and this is the reason why there is clustering of cases noted over a small well-defined geographical area during outbreaks (2).
2
The clinical presentation of dengue shows a wide spectrum of disease
manifestation, varying from asymptomatic infection (80%) to severe dengue (less than 5% of total infections) (4). The occurrence of fatality is less than 1%
of all infections and occurs in a fraction of the severe cases who do not receive appropriate and timely treatment(4).
The immune reaction to the virus is implicated in the severity of the disease.
The central hallmark of severe dengue is “capillary leaks” wherein there is increased capillary permeability causing leakage of plasma from the
intravascular compartment to the interstitial and extravascular compartment leading to the manifestations of “Dengue-shock syndrome”.
Background of the problem
The WHO has considered dengue as a global threat in tropic and subtropical nations (1). It is a major public health concern in south-east Asian countries, especially in India over the last decade (1).
Figure 1Dengue around the world (Source: CDC)
The incidence of dengue has increased 30
expansion to new countries and from urban to rural setting. An estimated 50 million infections occur annually and 2.5 million people are at risk in endemic countries (1). The increase in incidence
dengue over the last decade
overcrowding, population growth rate, inefficient control of mosquitoes and lack of access to proper health
The disease was seen in people of all age groups and had no racial or ethnical predilections. The severity of the disease had b
proportionately more mortality in infants and the elderly. In children the risk of severe dengue is more than that in adults probably due to their robust immune response and co-morbidities in elderly complement the disease severit
Dengue around the world (Source: CDC)
The incidence of dengue has increased 30-fold in last 50 years with geographic expansion to new countries and from urban to rural setting. An estimated 50 million infections occur annually and 2.5 million people are at risk in endemic
The increase in incidence of infection and deaths related to has been attributed to unplanned urbanization, overcrowding, population growth rate, inefficient control of mosquitoes and lack of access to proper health-care facilities (1).
The disease was seen in people of all age groups and had no racial or ethnical predilections. The severity of the disease had bimodal distribution, with
proportionately more mortality in infants and the elderly. In children the risk of severe dengue is more than that in adults probably due to their robust immune
morbidities in elderly complement the disease severit
3
fold in last 50 years with geographic expansion to new countries and from urban to rural setting. An estimated 50 million infections occur annually and 2.5 million people are at risk in endemic
and deaths related to has been attributed to unplanned urbanization, overcrowding, population growth rate, inefficient control of mosquitoes and
The disease was seen in people of all age groups and had no racial or ethnical imodal distribution, with
proportionately more mortality in infants and the elderly. In children the risk of severe dengue is more than that in adults probably due to their robust immune
morbidities in elderly complement the disease severity (1).
4
There was no significant difference noted in the number of cases and proportion of severity between urban and rural areas (5).
In developing countries like India, it causes significant economic burden to the country. In the context of dengue vaccine still under trial, it is important to identify a modifiable risk factor for development of severe dengue, so that we can prevent occurrence of severe manifestations (6).
The Problem Statement
South-East Asia including India is declared to be endemic to Dengue by the WHO(1). Dengue is endemic in all states of India except Lakshadweep (figure 2) (2). The economic burden amounts to 548 million USD every year on
medical expenses for dengue and indirect costs taking into account DALYs lost for the disease leads to a whopping 1.11 billion USD every year (7).
5
Figure 2 Incidence of dengue in India - From 1998 to 2014 (Source: Dengue burden in India by Mutheneni et al) (4)
6
Cyclical outbreaks or epidemics are reported in India, with an increase in
infection occurring during monsoon seasons (Figure3). During the rainy season, water is collected in outdoor reservoirs like plastic cups, coconut kernels,
mortars, buckets etc., which acts as a source for mosquito breeding(8). The sources of mosquito breeding in urban areas were refrigerator trays, air-
conditioners, flower vases, pots for storing drinking water etc. In India, seasonal outbreaks has been occurring with increased numbers of symptomatic cases especially with increasing morbidity and mortality in various parts of India including Delhi, Uttar Pradesh, Maharashtra, Karnataka and Tamil Nadu. The latest outbreak in South India was in 2017, mainly in Chennai and surrounding areas (5).
Figure 3 Seasonal Trend of occurrence of cases. (Source: National Guidelines for clinical management of dengue fever 2014 - NVBDCP)
7
Overview of clinical course of dengue
The severity of the disease varies from asymptomatic sero-conversion to symptomatic infection. It is a dynamic infection and systemic disease.
Symptomatic infection again has a wide clinical spectrum which may be undifferentiated fever to severe clinical manifestations.
The diagnosis of the disease in resource-limited countries and management is mainly clinical.
Capillary leakage is the main clinical feature and thrombocytopenia is the usual laboratory finding in dengue fever.
The illness begins abruptly after the incubation period which usually lasts for 4 to 10 days.
There are three phases during the illness viz., febrile phase, critical phase and recovery.
The following diagram explains the course of the disease through each phase along with the timeline and changes in platelet, hematocrit and serological status of patients with dengue.
8
Figure 4 Course of the illness (Source: CDC)
The febrile phase lasts for 2-7 days and is usually associated with non-specific symptoms resembling a flu-like illness. Fever is usually biphasic and is
associated with rash occurring on first day of illness as a generalised
erythematous maculopapular rash which blanches under pressure and disappears in 24-48 hours.
9
Many children have severe retro-orbital pain, backache, headache, myalgia and tiredness. Nausea and vomiting occurs in many children. There is loss of
appetite and poor oral intake. Dehydration maybe manifest in children with high grade fever and poor oral intake. In infants high fever may cause
encephalopathy and febrile seizures.
Following febrile phase, critical phase occurs around the time of defervescence usually between the 3rd and 7th days of illness. This is characterised by capillary leaks (leaky phase) and severe forms like dengue hemorrhagic fever and dengue shock syndrome manifests.
It is essential to monitor children for warning signs and severe manifestations of dengue. Organ damage may occur during this phase. The cornerstone of
management at this stage is optimal fluid resuscitation in order to maintain adequate intravascular volume and to maintain perfusion to vital organs.
Recovery phase occurs after 24 to 48 hours after critical phase and is
characterised by the reabsorption of leaked fluid back into the intravascular compartment.
Hemodynamic stability and diuresis ensues with an improvement in appetite and well-being. If during critical phase, fluids were administered in excess or intravenous fluids administered during recovery phase, fluid overload features like pulmonary edema or congestive cardiac failure maybe seen in recovery
10
phase, contributing to the mortality if not identified promptly and managed appropriately. A recovery rash described as “isles of white in sea of red” (9) maybe seen in this phase. The child usually recovers from the infection after this phase.
Primary and secondary dengue
Infection with any of the dengue serotypes for the first time is termed as primary dengue whereas re-infection with another serotype is called as
secondary dengue. The clinical importance is that secondary dengue infection is usually seen with increasing severity of the disease. This is because of the immunopathogenesis that has been explained in the diagram below (2):
Figure 5 Patho-physiology of dengue fever (Source: NVBDCP National guidelines for clinical management of dengue fever)
11
The increasing severity of the illness with secondary dengue infection is attributed to the infection-enhancing antibodies with absence of cross-reactive neutralising antibodies. There is rapid activation of complementary system in secondary dengue leading on to cytokine storm with increased release of
Tumour necrosis factor, Interferon-γ and interleukin-2 which contributes to the increase in capillary permeability in addition to the viremia, thus resulting in severe forms of dengue. The internal redistribution of fluids together with fluid deficit caused by fasting, vomiting and anorexia leads to hemoconcentration and is the basis of dengue shock syndrome and dengue hemorrhagic fever occurring in secondary dengue.
Dengue case classification
According to 2014 national guidelines of National Vector Borne Disease Control Programme (NVBCDP)of the Ministry of Health and Family Welfare (MoHFW), Government of India (GOI), the classification of clinical dengue is of three types – mild, moderate and severe dengue (2).
Mild dengue is undifferentiated dengue fever without evidence of capillary leakage.
12
Moderate dengue is dengue fever in certain high risk groups like in infants, pregnancy, immune-compromised, chronic illnesses etc., and with capillary leaks as seen by certain warning signs and raising hematocrit.
Severe dengue is dengue hemorrhagic fever with significant bleeds, dengue shock syndrome and expanded dengue syndrome (with severe organ
involvement). The classification is discussed in further details in methodology section.
Risk Factor for severe dengue
There is no proven risk factor for severe dengue except the presence of enhancing antibodies due to previous infection with a different serotype of dengue virus.
There have been studies implicating certain high-risk groups who could develop severe dengue like infants, pregnant women, elderly people, immune-
compromised and people with chronic diseases.
The severity in infants has been attributed to maternal antibodies that enhance the infection. In all other high-risk groups, it is primarily because of the immune-dysregulation.
13
Capillary leaks in burns and septic shock
In burns patients, there is destruction of tissues causing capillary leaks resulting in hypovolemia and shock.
Similarly in sepsis, there is cytokine storm similar to dengue fever causing capillary leaks into extravascular compartment manifesting as shock.
In the above conditions, it is essential to administer iv fluids and vasopressors to prevent organ damage due to hypotensive shock.
Death due to fluid overload contributes to a significant proportion of burns and septic shock patients. Recent studies (10,10–13) have shown that by treatment with high doses of intravenous ascorbic acid, the fluid requirement is
significantly reduced in these patients.
The mechanism of action of vitamin c is not clearly understood but assumed to be probably the function of vitamin c in maintaining capillary permeability.
Vitamin C and capillary integrity
Vitamin C is a water soluble vitamin found in abundance in citric fruits like oranges, lemon, Indian gooseberries, tomatoes etc.(14) Unlike animals, humans cannot synthesize vitamin C in the body due to the absence of the enzyme l- gulonolactone oxidase and must be obtained from diet(14). Vitamin C has
14
antioxidant roles and more importantly, it is required for synthesis of collagen, l-carnitine and certain neurotransmitters. It also helps in protein metabolism.
Non-synthetic functions include role in immune function and absorption of non- heme iron. (15) The antioxidant function prevents free radical injury to
endothelium and in addition to it collagen biosynthesis helps in wound-healing and thus helps in maintaining endothelial integrity by many mechanisms.
Insufficient vitamin C intake causes a deadly disease called scurvy which manifests with tiredness, lassitude, widespread connective tissue weakness and capillary fragility leading on to mucosal bleeds like petechiae, purpurae, gum bleeds etc.(14) In certain scorbutic patients, there was plasma leakage noted (similar to patients with dengue fever) like pleural effusion, joint effusions, pedal edema, clubbing, congestive cardiac failure and various other
manifestations. Vitamin C deficiency occurs in children with inadequate oral supplementation of vitamin C, infants only on cow’s milk, critical illness, burns, sepsis, ARDS, pancreatitis etc.(14)
In all the above conditions, vitamin C deficiency might have an exacerbating effect on the disease severity and by supplementing with vitamin C could improve outcomes as seen in burns patients and sepsis patients treated with high-dose of parenteral vitamin c.
RESEARCH
QUESTION
15
Research question
“Is there any association between serum ascorbic acid levels in children infected with dengue and the severity of the illness?”
The rationale behind the question is that we do not know the levels of serum ascorbic acid in children who are infected with dengue virus. There is a possibility that it could be low because of poor oral intake and vomiting in children with the disease. Low ascorbic acid levels in scorbutic patients has been found to contribute to plasma leaks (14). Hence the capillary leaks in dengue maybe attributed to low levels of ascorbic acid in children with severe disease. So we hypothesized that the ascorbic acid levels in children with severe dengue could be low when compared with healthy children and children with milder forms of the disease.
REVIEW OF
LITERATURE
16
Review of literature
The dengue infection irrespective of primary or secondary dengue starts usually as an undifferentiated fever and later progresses to cause capillary leaks
manifesting as dengue hemorrhagic fever and dengue shock syndrome (severe dengue).
Prediction of whether a child with dengue infection would proceed to have severe dengue will be helpful in management of the case by early intervention and rigorous monitoring. Many studies have been done previously to identify risk factors that could predict progression to severe forms of dengue in children as well as in adult patients.
Although a few studies have given certain clinical and lab parameters that could predict the severity of the disease, a vast majority could not cite out specific parameters that could identify risk factors for developing severe dengue.
Preventing transmission
Dengue management in developed countries starts with dengue prevention and control of outbreak. This is aimed at curbing transmission of the disease by implementing vector control measures. This is done by use of insecticides and destroying potential sources for breeding of aedes mosquitoes.
17
Certain genetic modifications and sterilisation has been carried out in the mosquitoes with much less beneficial effect than expected (16).
In developing countries like India, it is not possible to achieve vector control mainly because of lack of resources. In a study in Rajasthan, it was noted that water storage habits including cement tanks during arid and semi-arid seasons in urban households were notable mosquito breeding sites (3).
Another study in Maharashtra noted that outdoor non-potable water storage were more important breeding sites than indoor potable water storage (17).
The susceptibility pattern of mosquitoes to various insecticides were studied by sampling mosquitoes from Jodhpur, Delhi, Mumbai, Chennai and Coimbatore showed susceptibility to all insecticides, mainly temephos.(18).
But it requires a short course of fogging especially indoor fogging. This again is not always possible due to lack of public awareness of dengue and acceptance of control measures as noted in a study in Chennai by Ashokkumar et al. (19) The lack of adequate vector control in India has caused the rise in geographical distribution of the disease across the country and also the incidence of dengue fever in the last decade (16). This has indirectly caused an increase in
occurrence of severe dengue compared to previous outbreaks for reasons unknown and hence has led to increased morbidity and mortality.
18
Vaccine developed against dengue is currently in Phase 3 trials and approved for use in children above 9 to 16 years with laboratory confirmed previous infection with dengue virus and living in endemic areas (20). It is not available in India currently.
Inadequate vector control and lack of an effective vaccine has hurdled the
prevention of disease transmission. Hence, rigorous monitoring to look for early signs of worsening and aggressive management remains to be the mainstay of prevention of complications in dengue patients (16).
Possible risk factors for severe dengue
Several studies have concurred that children who have secondary dengue infections, infection with DENV2 serotype and increased viral load will have severe dengue infection (21) (22).
In the study by Libraty et al, it was shown that circulating levels of NS-1 antigen had correlation with severity of the disease (21). But in another study done in this institution (PSG Hospitals) by Lavanya et al, there was no
significant correlation between NS-1 titres and severity of the disease although, secondary dengue was frequently associated with severe capillary leaks as observed in other studies.
19
The challenge in prediction of severity in resource limited countries like India is the cost and availability of test to identify the serotype of infection. Moreover, detection of NS-1 antigen may indicate presence of infection but does not correlate with the severity.
Serology to find out of it is a secondary or primary infection will not be useful in the clinical setting to predict severity because the antibodies appear only after the capillary leaks manifest. This makes serological tests practically useless to predict if a child with the infection would progress to severe dengue. Thus although the above studies could point out certain risk factors, it cannot be used in a clinic setting to predict worsening of the disease (23).
Phakhounthong et al., had tried to formulate a decision tree in order to manage children presenting with dengue like illness (24). In his study he has mentioned that in resource limited endemic nations the differentiation of dengue fever from other febrile illnesses require training and education to health care providers.
The revised classification of dengue fever by WHO in the year 2009 to identify if there were warning signs or severe dengue had room for improvement (24).
Hence in this single-centered study among just 198 children with dengue, they had identified 5 risk factors at very early stage of the disease or at admission that could progress to severe dengue (24). The algorithm was not further evaluated or validated.
20
In another study done in this institution (PSG Hospitals) by Bharathi et al, it was shown that severe dengue usually presented with hypotension and cases of severe dengue had low platelets, absolute neutrophil count and ESR at
admission when compared with children who were diagnosed to have scrub typhus.
Kedia et al. in his retrospective in a PICU in a tertiary care centre in South India had mentioned few risk factors for severe dengue (25). Female sex was
associated with severe dengue with an Odds Ratio of 1.6. Bleeding tendency had significant association with severe dengue (OR = 5.7) and hepatomegaly and severe thrombocytopenia (Platelet count < 50,000 cells/mm3) also had significant association. Except for female sex, all the other factors mentioned were already termed as warning signs or defined as moderate dengue as per NVBCDP guidelines of 2014. Hence this study did not yield any extra information to predict severe dengue.
Genetic and immunological risk factors in dengue
Study of genetic factors that could predispose to severe dengue infections have shown mutations of genes coding for certain cytokines and their receptors, mainly TNF-α, IL-6, IFN-γ, TGF-β1 and IL-10.
21
A specific mutation in TNF-α 308A allele has been observed in a study in patients with dengue fever who had manifestations of severe dengue (20). The mutation allows expression of higher levels of TNF-α contributing to increased amounts of vascular permeability, hemorrhage and severe dengue.
Other genetic factors that have been shown to affect disease severity include certain HLA alleles, variations in the vitamin D receptor and Fc gamma receptor IIa, and also CD209 (27).
Other immunological parameters that may have a role in the pathogenesis of severe dengue include mast cell activation and mast-cell-derived mediators, mainly vascular endothelial growth factor, and antibody-immune complexes (27).
Other risk factor
The other risk factors for severe dengue are host factors like age of the host with children likely to develop plasma leakage and shock and adults could
experience significant bleeding and organ impairment (27).
Elderly and people with co-morbidities like Diabetes Mellitus and Hypertension experience severe symptoms due to pre-existing endothelial dysfunction in this group (27).
22
Obesity and pregnancy is also associated with increased risk for severe dengue due to immeasurable extra-vascular volume and the tendency to get dehydrated faster.
In immune-compromised people, the immune-dysregulation could predispose to an unpredictable immune response to dengue and secondary infections that may directly influence on the severity of the disease(27).
All the above studies have identified risk factors for developing severe dengue but the risk factors are not modifiable. The presence of these risk factors would mean stringent monitoring and fluid management in these cases to ameliorate the severity of the illness (27).
The purpose of identifying such risk factors like high viral load or infection with specific serotype etc., are virtually useless to the clinician because of the non-availability of these tests in the general clinical setting and the cost of these tests. Secondary dengue is usually identified by presence of both IgG and IgM to dengue, which usually takes 5 to 7 days to be detectable. But by that time, the disease would have progressed with manifestations of severe capillary leaks.
This has led to further research to identify therapeutic options to prevent dengue infection from progressing to severe disease.
23
Treatment options for mitigating severity of dengue fever
Treatment of dengue is usually supportive with iv fluids, mainly crystalloids and in severe cases, colloids and plasma replacement is indicated (1). The fluid management must be optimized during the critical phase to maintain adequate perfusion and restriction of fluid during recovery phase to prevent fluid
overload (1).
It has been established that minimal fluid support with appropriate inotropic support would drastically reduce mortality and improve clinical outcomes (27).
Therapeutic trials at earlier stage of the disease with antiviral agents have not been shown to be effective in termination of the disease progression (27).
There are two studies that have studied the use of antivirals like celgosivir and balapiravir in dengue infection. Ngyuen et al. studied the use of balapiravir, a protease inhibitor tried in treatment of hepatitis C in dengue patients in Vietnam (28). The drug at dosage appropriate to bring about a reduction in viral load in chronic hepatitis C patients, failed to show a decrease in viral load in dengue patients as measured by NS-1 levels twice daily during the course of treatment (28). The other study by Low et al. in Singapore, studying the effect of a α- glucosidase inhibitor, Celgosivir in dengue patients, showed no effect in virological log reduction when compared to placebo.
24
Adjunctive therapies also have been tried in dengue patients and again no satisfactory results have been obtained.
The anti-malarial drug chloroquine has shown promising in vitro antiviral effects. But in a randomised control trial by Tricou et al. in adult patients with dengue, it has shown no significant reduction in viral load (29).
Corticosteroids which are helpful in immune thrombocytopenic purpura to improve platelet counts have shown no effect on thrombocytopenia in dengue patients. According to a randomized placebo-controlled trial by Tam et al. in Vietnamese patients with dengue, short courses of oral steroids did not increase viremia as expected but did not have any improvement in clinical outcomes also (30). Another study done in pediatric patients with dengue shock syndrome with single dose of intravenous methylprednisolone was not effective in reducing mortality (31).
Statins inhibited dengue virion assembly in vitro and hence lovastatin was studied in a randomised control trial which showed no significant reduction in dengue severity (32).
Intravenous immunoglobulin also seemed to be ineffective in a study in adult patients in Vietnam (33).
Platelet transfusions are obsolete for treatment of thrombocytopenia unless the patient has severe bleeding manifestations (2).
25
Nutritional factors in dengue
The presence of malnutrition in the countries with dengue endemicity has posed a great risk for dengue mortality.
Although there is no clear association between the host nutritional status and the risk of dengue virus infection, malnutrition by itself is a cause of childhood mortality under 5 years of age (34).
Malnourished children have less robust immune response when compared to well-nourished children and since dengue severity depends on host immune response, it was proposed that malnutrition could be a protective factor against severe dengue (34).
Although in previous studies, it was shown that malnourished children had less chances of severe dengue when compared to well-nourished child, recent studies by Marón et al have refuted the theory (35).Based on anthropometric measurements and comparing it with standard charts to classify children as normal nutritional status, malnourished and overweight did not show any significant association with severity of the disease. It means children in either group had equal chances to have severe dengue. (35)
The complex interplay between nutrition and other infections is well-
established, and modulation of nutritional status by community intervention and nutritional rehabilitation often presents a simple low-cost solution to interrupt
26
transmission, reduce susceptibility, and/or ameliorate disease severity to a significant extent.
Ahmed et al. in his study wanted to examine the effects of micronutrients in dengue fever and whether the levels of micronutrients and vitamins in the blood had any significance in reducing transmission of the virus or reducing the
severity of the infection (34).
Alagarasu et al. in his study (36) had investigated for vitamin D levels and dengue severity. It was shown that though there was no significant association between vitamin D levels and primary and secondary dengue or dengue
hemorrhagic fever and undifferentiated dengue. But vitamin D levels in infected people were higher than that of healthy controls. The higher levels in
symptomatic infection when compared to asymptomatic individuals were explained by the role of vitamin D in immunomodulation.
In yet another study, the authors have proved the association of vitamin D receptor gene polymorphisms with the occurrence of dengue and dengue shock syndrome when compared with healthy controls. In another study by Fatima et al. (37), it was shown that infection with dengue causes lower levels of vitamin D, vitamin K, thrombopoietin and angiotensin. Though the study did not
correlate severity of the manifestation with lower levels, it found a significantly low value of vitamin K and vitamin D in children infected with the virus when compared to healthy controls.
27
Villamor et al. in his study (38), has mentioned that low levels of vitamin D could be a possible risk factor for the development of severe dengue.
Zinc is yet another micronutrient that has an important role in immune
regulation. Its use has been proven in pneumonia and diarrhoea in decreasing the severity of the illness and length of hospital stay.
In an Indonesian study (39), serum zinc levels in children infected with dengue was compared in differing severity of the disease. The study had shown that zinc levels were significantly lower in children with dengue shock syndrome and dengue hemorrhagic fever compared to dengue fever. But zinc levels were not proved as a risk factor for manifesting severe dengue.
Studies to find out if there could be a significant association between vitamin A levels (40), iron (41) and chromium (42) between severity of dengue had shown no significant results.
Vitamin A levels were found to be higher in severe dengue due to a turbulence of the anti-oxidant system (40).
Iron levels could not be accurately assessed in dengue patients because serum ferritin is elevated in most patients with all forms of dengue as it would be expected since it is an acute phase reactant. High ferritin levels (>1200ng/ml) had an increased association with dengue hemorrhagic fever (41).
28
Chromium promotes action of insulin and regulates blood sugar and also it influences immune response by its effects on T and B-lymphocytes, antigen presenting cells and cytokine release. Chromium and DENV infection had a possible association in a study done by Shrivatsava et al. in mice population (42). Exposure to chromium in dengue infected mice showed less severe outcomes and a significantly faster and robust increase in platelet counts (42).
Alternative medicine in Dengue
The administration of alternative medicines in dengue fever has also been researched.
“Nilavembu” extract is a siddha preparation made from 8 indigenous herbs, and has been extensively propagated for use in Tamil Nadu in patients affected with dengue. It has antipyretic, anti-inflammatory, analgesic and immune-stimulant action which is used to manage initial symptoms of dengue fever in siddha medicine (43).
It has been shown to have substances that possess anti-viral activity against DENV-2 and chickungunya virus and shown to decrease severity of the disease (44).
29
It has also shown protective effects in uninfected individuals and hence used in prophylaxis. But in females, it has less protective effect probably due to
metabolic pathways not clearly known (45).
Carica Papaya leaf extract was demonstrated to have a significant improvement in platelet counts in patients with dengue according to a systematic review done by Charan et al (46). The platelet counts do not predict severity of the disease.
But its nadir correlates with severity of plasma leakage and its improvement also correlates with clinical improvement.
It was postulated that papaya leaf extract could contain flavonoids, alkaloids, enzymes and minerals that could have immuno-modulatory and anti-oxidant effects. Since data of safety was not available, the study could not suggest use of these herbal preparations without evidence from further trials (46).
Diet therapy in prevention and control of dengue (47) is a naturopathy treatment. It involves careful selection of diet to meet micronutrient and macronutrient demand during dengue fever and to decrease occurrence of gastritis and vomiting. Although no clear scientific evidence was quoted, citrus fruits especially lemon helped in lessening nausea and improving outcomes.
30
Effects of micronutrients supplementation on course of illness
Very few experimental studies have been done in dengue with micronutrient supplementation. Although there is no replacement for an effective vaccine, it must be noted that modulation of nutritional status and micronutrient
supplementation could be a cheaper and cost-effective alternative.
In a study by Sánchez-Valdéz et al in Mexico (48), it was shown that
supplementation with calcium and vitamin D can improve outcomes in dengue infection. It was explained by the platelet aggregatory effect of calcium and the ability of vitamin D to alter IL-12 expression and dendritic cell maturation.
Vitamin E supplementation has been beneficial in improving platelet count in children with dengue as shown by the study done by Vaish et al (49), but did not alter disease severity.
As mentioned in the article by Ahmed et al (34) in his concluding remarks, the role of multivitamin supplementation (Vitamin B and C) needs to be studied in dengue.
Vitamin C especially has proven benefits in treatment of respiratory tract
infections and pneumonia due to its anti-oxidant properties. Vitamin C has been found to play important role in increasing gut iron absorption, folate
metabolism, and essential roles in amino acid and hormone metabolism (50).
31
The role of vitamin C in collagen formation and maintaining capillary integrity in addition shows promise in treatment of dengue though there have been no studies in this regard till date (34)
Therapeutic role of vitamin C in conditions with capillary leaks
Vitamin C has been tried in various scenarios that are associated with conditions associated with loss of capillary integrity leading on to plasma leakage. High dose of ascorbic acid in sepsis and burns patients is a recently emerged therapy in adults.
The underlying mechanisms for the effect of ascorbate on these conditions have been demonstrated in in vitro studies with cultured endothelial cells.
Ascorbate decreases oxidative stress in endothelial cells by reducing the production reactive oxygen species. Reactive Oxygen Species increase
endothelial permeability causing edema and contributing to organ dysfunction.
Ascorbate can tighten the endothelial barrier through several pathways.
There are very few studies regarding the effect of high dose of ascorbic acid to prevent the capillary leak.
32
The role of ascorbic acid in dengue capillary syndrome is never studied. The following studies studied the effect of ascorbic acid in preventing endothelial syndrome and capillary leakage in sepsis and burns patients and in animal models.
Alpha A Fowler III et al has done a phase 1 randomized control safety trial of intravenous ascorbic acid in adult medical intensive care unit patients with severe sepsis and showed positive impact on the extent of multiple organ failure and biomarkers of inflammation and endothelial injury (51). This phase I trial shows that aggressive repletion of plasma ascorbic acid levels in patients with severe sepsis is safe. This early work in septic patients suggests that
pharmacologic ascorbic acid repletion reduces the extent of multiple organ failure and attenuates circulating injury biomarker levels.
Mohadeseh Hosseini Zabet et al have studied the effect of high-dose ascorbic acid on vasopressor drug requirement in surgical critically ill patients with septic shock (10). They suggested that high-dose of ascorbic acid (25 mg/kg intravenously every 6 h for 72 h) with its probable anti-oxidant, anti-
inflammatory, cortisol sparing, nitric oxide synthase inhibitory and increasing catecholamine synthesis in the brain, and adrenal medulla properties may be considered as an effective and safe adjuvant therapy in critically ill surgical patients with septic shock.
33
A randomized, prospective study by Tanaka et al. (13) evaluated the use of continuous ascorbic acid infusion in burn patients using a group of 37 patients with greater than 30% TBSA burns. Investigators compared resuscitation fluid volume requirements and overall edema formation. A significant reduction in fluid volume requirements, weight gain, and wound edema was noted, along with an overall improvement in pulmonary function, demonstrated by a significant reduction in mechanical ventilation days.
Matsuda T et al studied the hemodynamic effects of antioxidant therapy with high-dose administration (170 mg/kg/24 h) in guinea-pigs with 70 per cent body surface area deep dermal burns (11). They have demonstrated that there was significant reduction of plasma leakage and decreased need of fluid requirement with high dose of ascorbic acid.
Kremer et al (12) demonstrated the reduction capillary leakage in burns animal models with high dose of ascorbic acid. They have also concluded that half dose of ascorbic acid is inefficient to reduce the endothelial damage. They have suggested that high-dose ascorbic acid should be considered for parenteral treatment in every burn patient to reduce capillary permeability.
A randomized, double-blinded study by Horton JW (52) demonstrated a significant reduction in net fluid balance and plasma lipid peroxidation among sheep sustaining 40% TBSA burns who were resuscitated with either Lactated
34
Ringer’s solution or hypertonic saline in conjunction with a high-dose infusion of ascorbic acid.
Gonzalez et al had reported the use of high-dose intravenous ascorbic acid in a 54 year lady with chickungunya infection and noted that her symptoms abated early. He had ascribed the effects to the anti-oxidant property and the property of vitamin C to maintain capillary integrity. He noted that treatment with high dose vitamin C could be useful in infections similar to chikungunya and needs to be studied.
Currently, there is an ongoing randomised control trial in Srilanka by Herath et al. with oral liposomal vitamin C in dengue infected patients to find out if it reduces the morbidity of dengue infected patients above 12 years of age.
Vitamin C in critically ill patients
According to a systematic review done by Zhang et al (53), Low plasma levels of vitamin C are associated with adverse outcomes and especially increased mortality in critically ill patients. Though the study was done in adults, the study points out the importance of vitamin C in critically ill patients.
The patients included in the studies in the meta-analysis included septic shock, severe neurological trauma, post cardiac-surgery patients, ARDS etc.
35
Vitamin C administration parenterally decreased the need for inotropes and mechanical ventilation.
The mechanism proposed was that vitamin C is an important co-factor in synthesis of endogenous vasopressors (54,55). The endogenous synthesis of adrenaline, dopamine etc., requires ascorbic acid as a co-factor.
According to Carr et al (56), the parenteral administration of high dose ascorbic acid can improve endogenous vasopressor production especially in states of hypotension like septic shock and thus reduce exogenous vasopressor requirement.
Since severe dengue usually presents with shock, which is similar to septic shock, administration of vitamin C can reduce need of IV fluids and
vasopressors.
The need to identify a modifiable risk factor to prevent severe dengue Dengue poses a great global problem wherein the progress of disease in an infected individual cannot be prevented but it has been shown that the severity can be mitigated to a certain extent by early identification of critical phase, hospitalisation, judicious fluid administration and intensive monitoring (57).
36
In countries like India where there is high incidence of dengue and inadequate trained health personnel to meet the health needs of the population, there is occasional faltering in monitoring patients and resulting in inadvertent fluid overload. It would be wise to devise a treatment protocol with judicious use of intravenous fluids, optimum use of fluids to achieve the best outcome.
The early identification of cases that would have severe capillary leaks and severe disease can be managed well in High Dependency Units (58). If it is possible to identify a risk factor that could predict progression of disease before clinical worsening happens, resources could be allocated with priority to
monitor/ manage these patients in intensive care units and prevent the disease progression thereby preventing prolonged hospital stay and mortality (58).
Distinguishing patients with dengue infection who will develop more severe forms of disease remains a clinical challenge and is an area yet to be explored and researched intensively.
While studies have shown positive results with high dose ascorbic acid and reduced need for IV fluids in burns and sepsis patients, a similar result may be expected in dengue. If by increasing vitamin C levels, the outcome might improve, then it is safe to assume that low vitamin C levels might predispose to severe dengue and hence we would like to explore the possibility of low levels of vitamin C to be a risk factor for severe dengue.
37
Vitamin C estimation:
Vitamin C (Ascorbic acid) is found in higher concentrations in citrus fruits and is absorbed in the intestines by a sodium dependent carrier mediated mechanism (SVCT-1) which is dose dependent. Dehydroascorbate present in the diet is absorbed by sodium independent mechanisms that is competitively inhibited by hexoses (59).
The absorbed ascorbic acid is transported in the blood as ascorbate anion. A normal serum level of 0.6-2 mg/dl is maintained and excess ascorbate is excreted renally. If there is deficiency of ascorbic acid in the plasma, renal reabsorption is increased.
Vitamin C is accumulated in almost all human tissues (60). The plasma levels of ascorbate in fasting state provides a surrogative measure of adequacy of vitamin C stores in tissues. Vitamin C in plasma is also stored in WBCs and platelets.
Measure of Vitamin C levels in WBCs gives a more accurate measure of deficiency but requires expertise.
High Pressure Liquid Chromatography is the gold standard procedure to measure Vitamin C levels (61). Biosensors are latest devices to measure ascorbic acid levels quickly and reliably. Cheaper alternatives like dye
reduction with DCPIP, Bromate etc., are traditional method employed in most research labs.
MATERIALS AND
METHODS
38
Materials and methods:
Aims and objectives:
Primary objective:
To determine ascorbic acid level in children with dengue fever
Secondary objective:
To determine the correlation of serum ascorbic acid level with severity of dengue fever in children.
39
Study method:
Cross-sectional observational study.
Sample size: 160 children
(By using 4pq/d2; with 5% prevalence and d-7%, 40 cases of severe dengue is to be studied)
By quota sampling method, 40 children will be recruited from each category namely mild dengue, moderate dengue and severe dengue. 40 children between the age group of 1-15 years with normal nutritional status (based on age and sex appropriate IAP growth charts and WHO criteria) without chronic illnesses will be taken as a control group.
Study Area: Department of Pediatrics, PSGIMSR
Study Subject: Children and adolescents between 1-15 years of age
Study Period: January 2018 to November 2019
40
Inclusion criteria:
All children aged 6 months to 18 years admitted with the diagnosis of mild dengue, moderate dengue or severe dengue (as per NVBCD guidelines for management of dengue) in PSG Hospitals, Coimbatore, irrespective of any treatment received outside before admission.
Exclusion criteria:
1. Children who are classified as severe acute malnutrition and moderate acute malnutrition as per WHO criteria (weight for height between -2 SD and – 3 SD and weight for height less than -3 SD respectively).
2. Children with pre-existing systemic illness/chronic underlying medical illness.
41
Operational definitions:
Probable Dengue Fever – Any child presenting during dengue outbreak with clinical features of dengue fever, i.e., an acute febrile illness of 2-7 days with two or more of the following manifestations:
o Headache
o Retro-orbital pain o Myalgia
o Arthralgia o Rash
o Hemorrhagic manifestations.
(Or) non-ELISA based NS-1 or IgM positivity.
Dengue Hemorrhagic fever: Clinical criteria of dengue fever with
Hemorrhagic tendencies (Petechiae, ecchymoses, purpura; bleeding from mucosa, GIT, injection sites), thrombocytopenia (platelets <
1lakhs/cu.mm) and evidence of plasma leakage (pleural effusion, ascites, pedal edema, hemoconcentration etc.)
42
Dengue Shock Syndrome: Dengue Hemorrhagic Fever with evidence of circulatory failure.
The definition of confirmed cases of dengue is made when:
Isolation of dengue virus by viral culture from serum, plasma, leucocytes.
Demonstration of IgM antibody by ELISA positive in single serum sample in significant titre (>9IU/L).
Demonstration of dengue virus antigen (NS1) in serum sample by ELISA.
IgG seroconversion in paired sera with four fold increase of IgG titre after 2 weeks.
Detection of viral nucleic acid by PCR (polymerase chain reaction).
The classification of dengue is made based on the above clinical diagnosis as follows:
43
Figure 6 Classification of dengue fever (Source: NVBDCP guidelines for clinical management of dengue fever)
44
Methodology:
It is a cross-sectional observational study. After obtaining informed consent from a parent/guardian and assent (as applicable) from children, 2ml of blood sample was taken for measurement of ascorbic acid in all children admitted in the department of paediatrics, PSGIMSR with the diagnosis of mild dengue/
moderate dengue/ severe dengue (as per NVBDCP guidelines on management of dengue) on Day 4 of illness or day of admission, whichever is earlier. All children were monitored for the evidence of onset, progression or regression of plasma leakage in the form of pedal edema, facial puffiness, ascites, pleural effusion and shock. Patients were managed as per discretion of treating pediatrician. All the parameters were entered in a predesigned proforma.
Controls were selected from healthy well-nourished in-patients other than probable dengue fever with normal nutritional status (as per IAP growth charts and WHO criteria for malnutrition), and must be without chronic illnesses, 2ml blood sample, from all study participants, was obtained in a vaccutainer with heparin, plasma separated and acidified with 2ml of freshly prepared 10%
metaphosphoric acid and stored at -70 °C. The samples were sent in batches transported with dry ice for analysis in Stanes laboratory, Coimbatore.
Vitamin C levels were estimated in the serum samples using DCPIP
(dichlorophenolindophenol) method which is a dye reduction method (62).
45
The principle in this method is that ascorbic acid reduces DCPIP which is usually a blue-colour solution to a colourless base. The sample with unknown concentration of ascorbic acid is titrated against the dye in the presence of oxalic acid and the end-point is appearance of pink colour (as the dye changes into pink colour in acidic medium) and compared against the volume of dye required to titrate a standard solution of ascorbic acid. Then the concentration of ascorbic acid is given by the formula:
Amt of ascorbic acid, mg/100g of sample= 0.5/V1 X V2/5 X 100/ W X 100 (V1 – Volume of dye used for STD ascorbic acid solution
V2- Volume of dye used for sample.
W- Weight of sample taken for test.)
Funding:
Expenses involved in this study for storage, transportation and analysis of Vitamin C levels in the serum samples were met by intramural funding from the institution.
Conflicts of interest:
None
46
Statistical analysis:
All statistical analyses will be performed using SPSS 25.0. The mean values of serum ascorbic acid between dengue children and control group was compared by student T-test and between mild, moderate and severe groups by one-way ANOVA.
Outcome of study:
If ascorbic acid level is found to be low in children with severe dengue or in dengue with warning signs, further study will be on ascorbic acid
supplementation in children with dengue to prevent plasma leakage.
The dose and route of administration of vitamin C will be based on evidence of improvement in patients with other diseases who had received treatment with vitamin C.
47
Figure 7 Flowchart (Proposed Methodology)
Exclusion criteria:
1. Children with malnourishment and pre existing systemic illness
2. Children on chronic medication
Mild dengue 40 cases
Moderate dengue 40 cases
Severe Dengue 40 cases Assess for
eligibility
Collect 2ml blood sample on Day 4 of illness or on admission whichever is earlier.
Control
40 children with normal nutritional status
Store acidified plasma at - 70oC.
Send Samples to Stanes Lab, Coimbatore in batches in dry ice.
Collect reports Collect 2ml
blood sample
Analyze
RESULTS
48
Results:
A total of 73 dengue patients were admitted to the hospital during the study period which was less than the required sample size. Of the 73 children, one child had congenital heart disease and was excluded from the study. Of the remaining 72 children, 3 parents did not give consent and hence were not included. The rest 69 children were enrolled in the study. 2ml of heparinised sample was obtained from each participant in a vacuutainer. The samples were centrifuged immediately and serum was separated and acidified with freshly prepared metaphosphoric acid which was then stored in -70oC.
Of the 69 dengue children included in the study, there were 10 (14.5%) severe cases, 31(44.9%) moderate and 28 (40.6%) of mild dengue cases. There were totally 26 (37.7%) primary dengue and 43 (62.3%) secondary dengue children.
By quota sampling method, 40 controls were chosen from in-patients with minor illnesses. Most of the controls were children with acute respiratory tract illnesses or viral fevers other than dengue admitted in view of parental concern.
These children were carefully selected that they did not have vomiting or diarrhoea before admission. 2ml of heparinised sample was obtained from each patient on day of admission and stored and analysed for Vitamin C levels as mentioned before.
Data was recorded for all children as per protocol and analysed.
49
Figure 8 Flowchart (Proposed Methodology)
Statistical analysis of the results and completion of study.
73 Dengue children 40 Controls
1 child excluded due to Congenital Heart Disease Parents of 3 children did not give consent for study
2ml Heparinised sample obtained from all children after obtaining consent (and assent as needed).
Plasma stored and processed in Stanes Lab in batches and reports obtained.
69 children included in the study
Mild - 28 Moderate - 31 Severe - 10
50
The mean (±SD) age of the dengue was 7.29(±4.34) whereas that in the control group was 6.1(±4.4) in years which was not statistically significant. The least age in the cases group was 8 months and the highest age was 15 years. There were three infants included in the study group. In the control group the lowest and the highest age among the children included were 1 year and 17 years respectively. No infant was included in the control group.
The following table gives distribution of the cases and controls based on their age:
Table 1: Agewise distribution of cases and controls
Age in years
Cases Controls
Frequency Percentage
(%) Frequency Percentage (%)
0-5 24 34.8 22 55
5.1-10 24 34.8 08 20
11.1-15 21 30.4 10 25
Total 69 100 40 100
The table shows that the number of children with dengue in the age group of 0-5 years was less than that in the control group probably because infants and
toddlers with minor illness are usually hospitalised than older children due to parental concern.
51
Figure 9 Agewise distribution of Dengue cases
52
Figure 10 Agewise distribution of controls
53
Table 2: Genderwise distribution of cases and controls
Sex
Cases Controls
Frequency Percentage (%)
Frequency Percentage (%)
Male 43 72.5 29 62.3
Female 26 27.5 11 37.7
Total 69 100 40 100
Both the groups (cases and controls) had male predominance. But there was no statistical difference in the distribution of boys and girls among cases and the controls chosen.
Figure 11 Genderwise distribution of controls
43
29
0 5 10 15 20 25 30 35 40 45 50
Cases Control
Males Females
