THE TAMILNADU DR. M.G.R MEDICAL UNIVERSITY

TO ESTABLISH URINE PROTEIN CREATININE RATIO AS A PREDICTOR OF DISEASE SEVERITY IN

PEDIATRIC DENGUE FEVER

DISSERTATION SUBMITTED FOR THE DEGREE OF M.D BRANCH VII

(PAEDIATRIC MEDICINE) REG.NO: 201717103

MAY 2020

MADURAI MEDICAL COLLEGE, MADURAI

THE TAMILNADU DR. M.G.R MEDICAL UNIVERSITY

CHENNAI, TAMIL NADU

CERTIFICATE

This is to certify that the dissertation “TO ESTABLISH URINE PROTEIN CREATININE RATIO AS A PREDICTOR OF DISEASE SEVERITY IN PEDIATRIC DENGUE FEVER” is bonafide work of Dr. N.J.NIROSHINI in partial fulfilment of the university regulations of the Tamil Nadu Dr. M.G.R Medical University, Chennai, for M.D Degree Branch VII – PAEDIATRIC MEDICINE examination to be held in May 2020.

Dr. K.VANITHA MD DCH Dean, Madurai Medical College, Government Rajaji Hospital, Madurai – 625020

BONAFIDE CERTIFICATE

This is to certify that the dissertation “TO ESTABLISH URINE PROTEIN CREATININE RATIO AS A PREDICTOR OF DISEASE SEVERITY IN PEDIATRIC DENGUE FEVER” submitted by

Dr.N.J.NIROSHINI to the faculty of Pediatrics, The Tamil Nadu Dr. M.G.R Medical University, Chennai in partial fulfillment of the

requirement for the award of M.D Degree Branch VII (PAEDIATRIC MEDICINE) is a bonafide research work carried out by her under our direct supervision and guidance.

Dr. S.BALASANKAR, MD., DCH Director I/C & Professor of Paediatrics Institute of child health & research centre, Madurai medical college,

Madurai.

CERTIFICATE

This is to certify that the dissertation “TO ESTABLISH URINE PROTEIN CREATININE RATIO AS A PREDICTOR OF DISEASE SEVERITY IN PEDIATRIC DENGUE FEVER” submitted by

Dr.N.J.NIROSHINI to the faculty of Pediatrics, The Tamil Nadu Dr. M.G.R Medical University, Chennai in partial fulfillment of the

requirement for the award of M.D Degree Branch VII (PAEDIATRIC MEDICINE) is a bonafide research work carried out by her under our direct supervision and guidance.

Dr. M. BALASUBRAMANIAN, MD., DCH., Professor of pediatrics

Institute of Child Health & Research Centre Madurai Medical College

DECLARATION

I, Dr. N.J.NIROSHINI, solemnly declare that the dissertation titled

“TO ESTABLISH URINE PROTEIN CREATININE RATIO AS A PREDICTOR OF DISEASE SEVERITY IN PEDIATRIC DENGUE FEVER” has been conducted by me at Institute of Child Health and

Research Centre, Madurai under the guidance and supervision of Prof. Dr.M.BALASUBRAMANIAM, M.D., DCH..

This is submitted in part of fulfillment of the regulations for the award of M.D Degree Branch VII (Paediatric Medicine) for the May 2020 examination to be held under The Tamil Nadu Dr. M.G.R Medical University, Chennai. This has not been submitted previously by me for any Degree or Diploma from any other University.

Place : Madurai Dr. N.J.NIROSHINI Date :

CERTIFICATE - II

This is to certify that this dissertation work “TO ESTABLISH URINE PROTEIN CREATININE RATIO AS A PREDICTOR OF DISEASE SEVERITY IN PEDIATRIC DENGUE FEVER” of the candidate Dr.N.J.NIROSHINI with registration Number 201717103 for the award of M.D., in the branch of PAEDIATRICS 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 result shows 16 percentage of plagiarism in the dissertation.

Guide & Supervisor sign with Seal.

ACKNOWLEDGEMENT

First, I would like to thank the almighty for giving me this opportunity. My sincere thanks to Prof. Dr.K.VANITHA MD., DCH, Dean, Government Rajaji Hospital and Madurai Medical College for permitting me to do this study and utilize the Institutional facilities.

I express my sincere thanks and gratitude to Prof. Dr. S.Balasankar, Professor and Director I/C, Institute of Child

Health & Research Centre, Madurai, for his able supervision, encouragement, valuable suggestions and support for this study.

I am also greatly thankful to Prof.Dr.M.Balasubramaniam, for his able guidance, critical review, constant encouragement and full support rendered in every aspect of this study.

I would extend my sincere thanks to Prof. Dr.Nandhini kuppusamy, Prof.Dr.D.Rajkumar, for their valuable advice and encouragement at every stage of this study.

I wish to express my sincere thanks to my Assistant Professors of Pediatrics, Dr. J. Balasubramanian, Dr.R.Venkatramanan for their

constant guidance, encouragement and support throughout my study.

I also extend my thanks to Dr.P.Ramasubramanian, Dr.B.Vanitha, Dr.A.Abubackar siddiq, Dr.T.Suganthi, Dr.S.Murgesa Lakshmanan,

Dr.S.Sonia Rosalind Martina, Dr.M.R.Lenin for their guidance, supervision, valuable suggestions and support throughout this study.

I thank the Institutional Ethical Committee for granting me permission to conduct the study. I also express my gratitude to all my fellow Postgraduates for their kind cooperation in carrying out this study and for their critical analysis.

I thank the Institute of Microbiology, Department of Biochemistry of Madurai Medical College, Madurai for their co-operation throughout my study.

Last but not the least, I submit my heartfelt thanks to the children and their parents for extending full co –operation to complete my study successfully.

CONTENTS

Sl. No Title Page no

1. INTRODUCTION 1

2. AIM 4

3. REVIEW OF LITERATURE 43

4. MATERIALS AND METHODS 46

5. OBSERVATION AND RESULTS 50

6. DISCUSSION 74

7. CONCLUSION 78

8. LIMITATIONS 79

ANNEXURES

 BIBLIOGRAPHY

 PROFORMA

 ABBREVIATIONS

 CONSENT FORM

 MASTER CHART

INTRODUCTION

Dengue fever is caused by arthropod borne virus and has typical characters like fever, hemorrhage, shock and even death. It affects all age group and recent years has faced outbreak in various places with significant mortality rates.¹

Epidemics are common in Australia, Asia, Europe and America which are all temperate areas in the 20^th century. In tropical Asia, it is endemic now.²

Outbreaks of dengue fever (DF)/DHF are recurrent now and have been reported in India from various states/UTs - Kerala, Karnataka, Punjab, Andhra Chandigarh, Delhi, Goa, Haryana, Tamil Nadu and West Bengal³ . There is a peak in the occurrence of dengue and DHF cases during July to November. There is also a seasonal variation where there is upsurge of cases following monsoon and not throughout the year.

Figure 1-Distribution of Dengue cases in India

Dengue mainly affects the pediatric age group and causes mortality because of increased capillary permeability, abnormalities of hemostasis and in severe cases dengue shock syndrome

Initiating management after occurrence of shock or hemorrhage results in higher mortality rate. The risk factors for development of severe disease are poorly characterized and consequently uncomplicated cases are frequently hospitalized for observation during the critical phase for capillary leakage syndrome, thereby increasing the financial burden to patients. Therefore improvements in early diagnosis and risk prediction for severe disease are urgently needed, particularly with respect to identification of simple clinical and/ or laboratory indicators that are practical and affordable for use in resource poor countries. This would enable appropriate and early intervention. Ideally, the test should be cheap, fast, easy to perform, highly sensitive and specific. This study was undertaken to establish urine protein creatinine ratio as early predictor of disease severity.

AIM

To assess whether urine protein creatinine ratio could be used as a predictor of disease severity in children with dengue

DENGUE VIRUS

Dengue is caused by Dengue virus which belong to flavivirus family^4,5. It is a single stranded DNA virus of size 50nm. Dengue virus has 4 serotypes

Figure 2- Aedes mosquito

DENV-1 DENV-2 DENV-3 and DENV4

All four serotypes are similar antigenically, infection by one serotypes elicit cross protection only for a few months. Infection with any one serotype confers lifelong immunity to that virus serotype.

In dengue viruses, antibody response varies among each individual.

Secondary infections can have increased risks of severe disease. Based on their antibody responses, primary and secondary infection can be differentiated.

Dengue virus has structural protein genes – 3, which encodes the nucleocapsid of core protein ©, a membrane associated protein (M), an envelope protein (E) and seven non-structural (NS) proteins – NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. NS protein reacts with host immune system and evokes T cell response. This NS antigen is detectable in the blood and used in diagnosis of dengue infection.

VECTOR

Dengue virus is transmitted by the bite of female Ades mosquito from an infected person. The female mosquito lays single eggs on damp surfaces .In seven days, the adult emerges out. It may take several weeks for the eggs to emerge out and it can remain in a viable dry condition for more than a year. When it comes in contact with water, it can emerge within 24 hours. This creates a major difficulty in prevention and control of dengue.

The life cycle of the vector and transmission of the disease is affected mainly by the temperature and rainfall. The average survival ranges between 30 days to eight weeks. During the rainy season, when survival is longer, the risk of virus transmission is greater. Aedes bite from dawn to dusk and can fly up to a distance of 400metres. Aedes mosquitoes breeds in manmade water receptables in and around the residence like water reservoirs, disposable cups, unused tyres and vessels, grinders and old fridges, coconut shells. They also breed in tree holes, leaf axils, bamboo stumps etc.

Figure 3 – Breeding sources of Aedes mosquitoes

TRANSMISSION CYCLE

The female mosquito bites the infected person during the acute febrile (viremia) phase of illness and takes up the dengue viruses. The mosquito becomes infected after an extrinsic incubation period of eight to ten days.

When the infected female mosquito bites and injects its saliva into the wound of the person bitten, the virus gets transmitted. This process continues and there by spreading the dengue infection. Dengue begins abruptly after an intrinsic incubation period of 4 to 7 days (range 3–14 days).

Vertical transmission from infected female mosquitoes to the next generation can also occur.

Though transmission is mainly by bite of the mosquitoes, there are case of dengue acquired through blood transfusion and organ transplantation. congenital infection has also been reported in late pregnancy.

Figure4 – Transmission of Dengue

PATHOPHYSIOLOGY IN DENGUE FEVER

Pathogenesis of dengue mainly depends on the host immune response. The exact pathogenesis is still not clearly understood. Various mechanisms such as -Tcell mediated antibodies cross reactivity with vascular endothelium, complex immune mechanism, enhancing antibodies, various soluble mediators including cytokines and chemokines and complement and its products are said to be the causative pathogenesis in causing dengue infection⁵.

The most important mechanism is “Cytokine Tsunami” which is caused by the virus strains enhancing antibodies and memory T-cells resulting in a secondary infection.

All these mechanism ultimately target vascular endothelium, platelets and various organs leading to vasculopathy and coagulopathy leading to hemorrhage and shock.

CAPILLARY LEAK IN DENGUE

Hypotension is due to plasma leakage^6,7. This plasma leakage can be mild and transient and can lead to profound shock with undetectable pulse and blood pressure. A transient disturbance in the function of the endothelial glycocalyx layer may be involved during dengue infection and alter temporarily the characteristics of the fibre matrix of the endothelium.

Anti-NS1 antibody acts as autoantibodies that cross-react with platelets and endothelial cells which leads to alteration in capillary permeability. Plasma leakage is due to this altered capillary permeability resulting in ascites, pleural effusion and hemoconcentration. This phase is during 4-7 days of illness. This capillary leak is because of the cytokines released from the dengue virus infected monocytes and thrombocytes.

Proteinuria and hypoalbuminemia are said to occur in dengue infection. This is because the dengue virus and NS1 antigen binds to the heparan sulphate present in the glycocalyx of the basement membrane, there by altering the filtration of the kidneys and resulting in proteinuria.^8,9

Causes of Bleeding in DF/DHF

• Abnormal coagulogram

• Thrombocytopenia

• Increase level of fibrinogen degradation product(FDP)

• Increased level of D-Dimer

• Platelet dysfunction

• DIC and Prolonged aPTT

• Decreased fibrinogen level

• Consumptive coagulopathy (activation of mononuclear phagocytes)

• Prothombin complex deficiency secondary to Liver involvement

• Endothelial injury

CAUSES OF THROMBOCYTOPENIA IN DENGUE

Figure5- Causes of thrombocytopenia in Dengue

CLINICAL MANIFESTATIONS OF DENGUE

• The incubation period for dengue infection is 4-7 days (range 3-14).

• The incubation period is followed by the febrile phased, critical phase and recovery phase.

• Symptomatic dengue infection is a systemic and dynamic disease with clinical, haematological and serological profiles changing from day to day. ^10,11

• These changes accelerate within hour or even minutes during the critical phase, particularly in those with plasma leakage.

Figure6 – Course of Dengue illness

FEBRILE PHASE

 Patients has sudden onset of high grade fever which usually lasts for 2-7 days

 It is associated with vomiting, myalgia, rash and facial flushing

 Sore throat, injected pharynx and conjunctival injection.

 Complete hemogram shows decrease in total white blood cell count followed by decrease in the platelet which is the earliest abnormality to occur in dengue illness.

CRITICAL PHASE

 Following the febrile phase, oh the third to fourth day or following defervescence the patient enter into the critical phase which is characterised by plasma leakage and bleeding.

 In other viral infections, the patient’s condition improves as the temperature subsides, but the contrary happens in severe dengue infection wherein the patient may deteriorate and manifest third space plasma leakage or organ dysfunction

RECOVERY PHASE

 Plasma leakage stops followed by reabsorption of extravascular fluid

 General well being improves, appetite returns, gastrointestinal symptoms improve, haemodynamic status stabilises and diuresis ensues

 “isles of white in the sea of red” with generalised pruritus

 The recovery of platelet count is typically preceded by recovery of white cell count.

Case classification of dengue is as follow

EXPANDED DENGUE SHOCK SYNDROME:

There are certain conditions who can have sever manifestations of dengue and require close monitoring. These are the following high risk groups who need intensive monitoring and care.

Chronic diseases: diabetes, COPD, bronchial asthma, hypertension • Patients on steroid, antiplatelet, anticoagulant drugs • HIV infected persons/Immuno-compromised persons

• Pregnancy • Infant • Elderly • Obesity • Peptic ulcer diseases • G6PDdeficiency • Thalassemia • Coronary Artery Disease

CATEGORIES OF DENGUE

Figure7 – Categories of Dengue

CASE DEFINITION PROBABLE DF

A case compatible with clinical description of dengue Fever during outbreak.: OR Non-ELISA based NS1antigen/IgM positive.

(A positive test by RDT will be considered as probable due to poor sensitivity and Specificity of currently available RDTs.)

CONFIRMED DENGUE FEVER

A case compatible with the clinical description of dengue fever with at least one of the following

a. Isolation of the dengue virus (Virus culture+VE) from serum, plasma, leucocytes.

b. Demonstration of IgM antibody titre by ELISA positive in single serum sample.

c. Demonstration of dengue virus antigen in serum sample by NS1- ELISA.

d. IgG seroconversion inpaired sera after 2weeks with Four fold increase of IgG titre.

e. Detection of viral nucleic acid by polymerase chain reaction (PCR)

CLINICAL CRITERIA FOR DF/DHF/DSS DENGUE FEVER

An acute febrile illness of 2-7 days duration with two or more of the following manifestations:

Headache

Retroorbital pain Myalgia

Arthralgia Rash

Hemorrhagic manifestations.¹¹ DENGUE HEMORRHAGIC FEVER

A. A case with clinical criteria of dengue Fever plus

B. Hemorrhagic tendencies evidenced by one or more of the following 1.Positive tourniquet test

2.Petechiae,ecchymoses or purpura

3.Bleeding from mucosa, gastrointestinal tract, injection sites or other sites Plus

c). Thrombocytopenia(<100000cellspercumm) plus

d). Evidence of plasma leakage due to increased vascular permeability,

1.A rise in average hematocrit for age and sex _> 20%

2.A more than 20% drop in hematocrit following volume replacement treatment compared to baseline

3.Signs of plasma leakage (pleural effusion, ascites, hypoproteinemia) DENGUE SHOCK SYNDROME

All the above criteria for DHF with evidence of circulatory failure manifested by rapid and weak pulse and narrow pulse pressure( mmHg) or hypotension for age, cold and clammy skin and restlessness.

Tourniquet test: The tourniquet test is performed by inflating a blood pressure between systolic and diastolic for 5minutes¹²

10 or more petechiae per 2.5sq cm gives positive result, in DHF it is more than 20.

LABORATORY DIAGNOSIS OF DENGUE

A glycoprotein named NS1 antigen is present in both membrane associated and secretion form and it is present in early stages of DENV infection. It is a useful tool in the diagnosis of acute dengue infections. It is more specific and has high sensitivity.^13,14

Figure8- Diagnostic tests in Dengue

SUMMARY OF CHARACTERISTICS AND COSTS OF DENGUE DIAGNOSTIC METHOD

IgM-capture enzyme-linked immunosorbent assay (MAC-ELISA) MAC ELISA is a simple test that doesn’t requires more sophisticated equipment. MAC-ELISA is based on detecting the dengue-specific IgM antibodies in the test serum by capturing them using anti-human IgM that was previously bound to the solid phase.

This is followed by addition of dengue antigen if the IgM antibody from the patient's serum is anti-dengue, it will bind to the dengue antigen.

The anti-dengue IgM antibody is detectable before IgG and by day 5 of the illness. On day 2 to 4 of illness, few cases develop detectable IgM while others may not have detectable IgM for seven to eight days. It is very useful for patients, who are admitted late in the illness after detectable IgM is already present in the blood.

Figure 9- MAC ELIZA

ISOLATION OF DENGUE VIRUS

Isolation of viruses from the clinical specimen can be done within 5 days of illness. Specimens for virus isolation include acute phase serum, plasma or washed buffy coat from the patient, autopsy tissues from fatal cases, especially liver, spleen, lymph nodes and thymus and mosquitoes collected in nature. Isolation takes 7–10days, hence cannot be used in managing the cases.

POLYMERASE CHAIN REACTION

Molecular diagnosis based on reverse transcription polymerase chain reaction (RT-PCR)¹⁵, such as one-step or nested RT-PCR, nucleic acid sequence-based amplification (NASBA) or real-time RT-PCR has gradually replaced the virus isolation method as the new standard for the detection of dengue virus in acute-phase serum samples.

RDT’S

A number of commercial RDT kits for anti-dengue IgM/IgG antibodies and NS1 antigen are available, which give the results within 15 to 25 minutes. According to WHO guidelines, these kits should not be used in clinical settings to guide management of DF/DHF cases because many serum samples taken in the first five days after the onset of illness

will not have detectable IgM antibodies. The tests would thus give a false negative result.

Diagnosis of dengue depends on proper collection, processing, storage and shipment of the specimens. All the universal precautions to be taken before collecting sample. While sending the samples for lab confirmation, the day of onset of fever and day of sample collection should be mentioned to guide the laboratory for the type of test to be performed (NS1 for samples collected from day 1 to day 5 and IgM after day5).

For dengue infection confirmation, Government of India (GoI) recommends use of ELISA-based antigen detection test (NS1) for diagnosis of cases from day 1 onwards and antibody detection test IgM capture ELISA (MAC-ELISA) for diagnosis of the cases after the fifth day of onset of disease.

URINE PROTEINURIA

Hypoalbuminemia and proteinuria are well recognized in dengue infection. This is caused due to altered filtration of the glycocalyx , as dengue virus and NS1 attaches directly to heparan sulphate¹⁶, which is part of the glycocalyx.

Increased urinary excretion of proteins is said to be due to the defect

hallmark of complication. This passage of proteins in urine can be used as a prognostic marker.

MANAGEMENT OF DENGUE/DHS/DSS

DF

Fever of 2-7 days with two or more of following- Headache, Retro orbital pain, Myalgia, Arthralgia with or without leukopenia, thrombocytopenia and no evidence of plasma leakage.

DHFI

Above criteria plus positive tourniquet test and evidence of plasma leakage. Thrombocytopenia with platelet count less than 100000/cu.mm and Hct rise more than 20% over baseline.

DHFII

Above plus some evidence of spontaneous bleeding in skin or other organs (black tarry stool, epistaxis, gum bleeds) and abdominal pain.

Thrombocytopenia with platelet count less than 100000/cu.mm and Hct rise more than 20% over baseline.

DHFIII (DSS)

Above plus circulatory failure (weak rapid pulse, narrow pulse

Thrombocytopenia with platelet count less than 100000/cu.mm and Hct rise more than 20% over baseline.

DHFIV (DSS)

Profound shock with undetectable blood pressure or pulse.

Thrombocytopenia with platelet count less than 100000/cu.mm and Hct rise more than 20% over baseline.^18,19

Figure 10 – Dengue case management

GROUP A MANAGEMENT (CATEGORY A)

Patients who do not have warning signs and able to take orally can be sent home¹⁹.

They should pass urine atleast once in 6 hours Investigations

Complete blood count, hematocrit Treatment:

Management is mainly Symptomatic

• Paracetamol according to body weight

• Adequate Oral rehydration using ORS, water and home based fluids

• Look for warning signs

Patients with more than 3 days of illness should be monitored daily for disease progression (which is monitored by decreasing white blood cell and platelet counts and increasing hematocrit, defervescence and warning signs) until they are out of the critical period.

Caregivers should be advised to bring the patient immediately if the following occurs

Deterioration after defervescence,

Severe abdominal pain and persistent vomiting

Cold and clammy peripheries lethargy or irritability/restlessness, Bleeding (e.g. black stools or coffee ground vomiting),

Shortness of breath, not passing urine for more than 4−6 hours.

GROUP B MANAGEMENT (CATEGORY C)

Patients with high risk factors (pregnancy, infants, old age), those coming far from hospital, living alone and those with warning signs should be admitted^20.

LABORATORY INVESTIGATIONS – CBC, Hematocrit

No warning signs – encourage oral fluids, if not tolerated, start intravenous fluids 0.9% saline or RL

Cases with warning signs or signs of dehydration requires volume replacement by intravenous fluid therapy from the early stage which may modify the course and the severity of disease.

Get a reference hematocrit before intravenous fluid therapy. Give only isotonic fluid. Start the I.V fluid therapy with 5−7 ml/kg/hour for 1−2

Reassess the clinical status of the child and repeat the hematocrit.

If hematocrit is same or rises minimally, continue at the same rate (2−3 ml/kg/hour) for another 2−4 hours.

If the vitals and conditioning of the child is worsening and the hematocrit is rising rapidly, increase the rate to 5−10 ml/kg/hour for 1−2 hours.

Reassess the clinical status, repeat the hematocrit and change fluid infusion rates accordingly.

Give minimum fluids to maintain urine output of about 0.5 ml/kg/hour.

Intravenous fluids are usually needed for only 24−48 hours. Reduce intravenous fluids gradually when the rate of plasma leakage decreases towards the end of the critical phase.

Patients with warning signs requires careful monitoring by health- care providers till the period of risk is over. Vitals and peripheral perfusion (1−4 hourly until the patient is out of the critical phase), urine output (4−6 hourly), hematocrit (before and after fluid replacement, then 6−12 hourly), blood glucose and other organ functions (such as renal profile, liver profile,

Figure 11 – I.V fluid management in DHFI &II

GROUP C MANAGEMENT (CATEGORY C)

Patients with

a. Severe plasma leakage leading to dengue shock and/or fluid accumulation with respiratory distress²⁴

b. Severe hemorrhage

c. Severe organ impairment (hepatic damage, renal impairment, cardiomyopathy, encephalopathy or encephalitis) are included .

LABORATORY PARAMETERS

CBC, hematocrit, other investigations related to organ damage

TREATMENT OF COMPENSATED SHOCK

Figure 12- Fluid management in DHF grade III

TREATMENT OF HYPOTENSIVE SHOCK

Figure13- volume replacement in DHF IV

INDICATION FOR PLATELET TRANSFUSION

1. Platelet count less than 10000/cu.mm in absence of bleeding manifestations (Prophylactic platelet transfusion).

2. Hemorrhage with or without thrombocytopenia.

Packed cell transfusion/FFP along with platelets may be required in cases of severe bleeding with coagulopathy. Whole fresh blood transfusion doesn't have any role in managing thrombocytopenia.

Platelets can be classified as random donor platelets (prepared by buffy coat removal method or by platelet rich plasma method), BCPP (buffy coat pooled platelet) and single donor platelets (SDP) or aphaeretic platelets(AP)

CONTROL OF DENGUE

Figure 14- Control of Dengue

DRUGS & VACCINES

No licensed vaccine available against dengue virus IN 2015 (CYD- TDV)-DENGVAXIA -0/6/12 MONTH, a live attenuated tetravalent vaccine which may be promising in near future.

CPLE -Carica papaya extract which contain increased expression of ALOX 12 and PTAFR GENE which is responsible for platelet production

REVIEW OF LITERATURE

Nguyen Thi Hanh Tien et al in 2013, concluded that UACRs were increased in the 465 confirmed dengue patients, with a significant time trend showing peak values around the critical period, urine albumin excretion was also increased in the comparison group of 391 patients with other febrile illnesses (OFI). The dengue patients had higher UACRs than the OFI patients, but microalbuminuria, using the cutoff of 30 mg albumin/g creatinine discriminated poorly between the two diagnostic groups in the early febrile phase. Secondly UACRs is not useful in predicting either development of warning signs for severe dengue or need for hospitalization. Low-level albuminuria is common, even in relatively mild dengue infections, but is also present in many OFIs. Simple point-of- care UACR tests are unlikely to be useful for early diagnosis or risk prediction in dengue endemic areas.

Priyanka Datla et al²⁶ in 2017 done a study in 76 children hospitalized with dengue fever and categorized as per WHO guidelines.

Of these, 25% had category 1 disease, 36.8% had category 2 and 38.2%

category 3 disease. All children were classified according to UPCR into 4groups. It was observed that 34.2% had UPRC <0.5, 26.3% had 0.5-1.0,

correlation between UPRC and the severity of illness in dengue fever. The association of occurrence of bleeding manifestations, requirement of inotropes and outcome with UPCR was statistically significant. Thus UPCR appear to be useful tools for deciding hospitalization, management as well as prognostication in childhood dengue fever.

Anne-Claire Andries et al²⁷ in 2017 studied 108 patients for urine protein creatine ratio. The Sensitivity of this marker, however was limited as only 16.1% of the patients with warning signs had proteinuria. They concluded that Urine Dipstick and UPCR Do not seem to be very valuable for the triage of the patients at the time of the initial consultation but the observation of a decrease of the UPCR During the course of the illness appears to indicate an evolution towards recovery.

Vasanwala FF et al²⁸ in 2011 studied UPCR ratio in patients admitted to hospital with dengue fever. Starting from admission until discharge, each patient’s daily spot urine protein creatinine ratio (UPCR) was measured. They classified as DF or DHF (including DSS) based on WHO criteria. Peak and day of onset of proteinuria was compared between both groups. Compared to those with DF, patients with DHF had significantly higher median peak proteinuria levels (0.56 versus 0.08 g/day; p < 0.001). Peak UPCR could potentially predict DHF in patients

Sakthi Selva Kumar Et Al²⁹ in 2017 studied prediction of DHF/DSS using proteinuria in 160 adult patients. DHF was diagnosed in cases according to the WHO 1997 guideline. Dengue fever (DF) patients were predominantly younger. Compared to DF, DHF cases had significantly higher peak urine protein creatinine ratio (UPCR) during clinical course (26 vs. 40 mg/mmol; p, 0.001). thus Proteinuria measured by a laboratory-based UPCR test may be sensitive and specific in prognosticating adult dengue patients.

MATERIALS AND METHODS

STUDY DESIGN

It is a Prospective study with study period of 6months from February 2019 to august 2019 done in children from 1 month to 12years.

This Study was done in Institute of Pediatrics, Government Rajaji hospital, Madurai Medical College with the help of Department of biochemistry and Institute of microbiology, Madurai medical college, Madurai after getting informed consent from parents / guardian and suspected participants. The study is approved by the Institute Ethical Committee

Dengue positive cases will be followed up from the day of admission and the subsequent days.

Demographic data including the age, sex, and epidemiological data will be collected on admission. Detailed general and systemic examination will be done .Biochemical analysis and imaging studies will be done.

The patients will be categorized into three categories based on WHO guidelines. Symptoms and signs will be recorded each day.

Complete blood count and Spot urine collection will be done everyday.

Dengue detection

Dengue detection is done using NS1 antigen or IgM antibody depending on the day of admission

Laboratory diagnosis of dengue depends on proper collection, processing, storage and shipment of the specimens. While collecting blood for serological studies from suspected DF/DHF cases, all universal precautions were taken. While sending the samples for lab confirmation, the day of onset of fever and day of sample collection were mentioned to guide the laboratory for the type of test to be performed (NS1for samples collected from day1 to day5 and IgM after day5)

URINE PROTEIN CREATININE RATIO

Urine protein is detected and quantified by pyrogallol red method.

Creatinine by modified Jaffe method

Normal Urine PCR <2yrs - <0.5, >2yrs - <0.2, values lower than this range is considered insignificant.

Peak value of urine protein creatinine ratio in patients with dengue with no warning signs ,dengue with warning signs and DHF /DSS are compared.

URINE PROTEIN ANALYSIS

Quantitative Colorimetric Determination of Total Protein in Urine is done by pyrogallol red method²⁵

It is based on the principle that the pyrogallol red is combined with molybdenum acid, forming a red complex with maximum absorbance at 470 nm. When this complex is combined with protein in acidic conditions, a blue-purple color develops with an increase in absorption to 610 nm.

Specimen Collection and Preparation: Urine samples collected randomly or 24 hour samples were used. Samples are Stored at 2-8°C or freezed until assayed. No special additives or preservatives are required.

URINE CREATININE BY MODIFIED JAFFE METHOD

Quantitative estimation of creatinine in urine is done by modified Jaffe method. The principle behind this method is creatinine in alkaline medium reacts with picric acid to produce a red coloured complex. The rate of this reaction measures creatinine concentration.

INCLUSION CRITERIA

All dengue NS1 or IgM positive case admitted on any day of illness.

Children from 1 month to 12years of age

EXCLUSION CRITERIA

Children with other causes of proteinuria like Nephrotic syndrome and pre existing renal disease.

STATISTICAL ANALYSIS

Data obtained was analysed using SPSS software – version 19.

Outcomes were tested using the Chi-square test. Significant p value is<0.05

OBSERVATION & RESULTS

Total number of cases included in the study is 80. The cases were grouped into 3 categories based on the WHO guidelines.

28 cases belonged to category A, 32 belonged to category B and 20 cases belonged to category C.

Figure 15- Categories of cases

28

32 20

NUMBER OF CASES

category A category B category C

AGE VS UPCR Table 1- AGE VS UPCR

Age in Years

Peak value

Total

< 0.5 0.5 - 1.0 1.0 - 1.5 > 1.5

< 2 years 11 4 3 0 18

> 2 years 30 21 5 6 62

Total 41 25 8 6 80

chi square 3.077

p' value 0.380 Not Significant

Cases are divided into two groups based on the age ,which is found to be statistically insignificant with urine protein creatinine ratio.

Figure 16- Age distribution vs UPCR 11

30

4

21

3 5

0 5 10 15 20 25 30 35

< 2 years > 2 years AGE COMPARISON

< 0.5 0.5 - 1.0

Figure 17- Gender distribution

Gender distribution has no correlation with dengue severity.

However majority of cases affected are male.

50 30

GENDER DISTRIBUTION

MALE FEMALE

Figure 18- Day of illness on admission

Majority of cases are admitted during the 4^th or 5^th day of illness in all categories.

0 5 10 15 20 25 30

2,3 4,5 >6

NO OF PATIENTS

DAY OF ILLNESS

DAY OF ILLNESS ON ADMISSION

CATEGORY A CATEGORY B CATEGORY C

UPCR ON DAY OF ADMISSION

Table 2 – UPCR on day of admission

Spot PCR on admission

Category A

Category B

Category

C Total

< 0.5 16 20 3 39

0.5 - 1.0 10 10 11 31

1.0 - 1.5 2 2 1 5

> 1.5 0 0 5 5

Total 28 32 20 80

chi square 23.298

p' value < 0.001 Significant

16 patients of category A, 20 patients of category B and 3 of category C admitted with less than 0.5 PCR. In that patients with severe dengue had very high PCR on admission which is found to be statistically significant.

Figure19- UPCR on day of admission 0

5 10 15 20

< 0.5 0.5 - 1.0 1.0 - 1.5 > 1.5 16

10

2 0

20

10

2 0

3

11

1

5 SPOT PCR ON ADMISSION

Category A Category B Category C

Figure 20 – Peak UPCR day

Majority of cases had peak UPCR on day 5 which is the critical phase.

0

7

15

26 25

4 3

5

2 3 4 5 6 7 8 9 10

NO OF PATIENTS

DAY OF ILLNESS OF PEAK UPCR

PEAK UPCR DAY

PEAK VALUE OF UPCR VS CATEGORIES

Table 3- Peak UPCR vs Categories

Peak value Category A

Category B

Category

C Total

< 0.5 19 19 3 41

0.5 - 1.0 7 11 7 25

1.0 - 1.5 2 2 4 8

> 1.5 0 0 6 6

Total 28 32 20 80

chi square 25.35

p' value < 0.001 Significant

Out of 80 cases peak UPCR of >1.5 is seen in category C where there is severe dengue. It is statistically significant

Figure21- Peak UPCR vs Categories 0

5 10 15 20

< 0.5 0.5 - 1.0 1.0 - 1.5 > 1.5 19

7

2

0 19

11

2

0 3

7

4

6 PEAK VALUE

Category A Category B Category C

PLATELET VS PEAK SPOT PCR

Table 4- Platelet vs peak UPCR

PLT ON PEAK SPOT

PCR

Peak value

< 0.5 0.5 - 1.0 1.0 - 1.5 > 1.5 Total

< 50000 5 6 3 6 20

50000 - 1 lakh 16 14 1 0 31

> 1 lakh 20 5 4 0 29

Total 41 25 8 6 80

chi square 28.556

p' value <0.001 Significant

Platelet value is comparatively lower when the UPCR value is high which is found to be statistically significant.

Figure 22- Platelet vs UPCR 0

5 10 15 20

< 0.5 0.5 - 1.0 1.0 - 1.5 > 1.5 3

6

3

6

14 13

1 0

14 16

4

0 PLATELET ON PEAK UPCR VS PEAK VALUE

< 50000 50000 - 1 lakh > 1 lakh

BP VS UPCR

Table 5- BP vs peak UPCR

BP

Peak value

Total

< 0.5 0.5 - 1.0 1.0 - 1.5 > 1.5

Normal 40 24 2 1 67

Hypotension 1 2 6 4 13

Total 41 26 8 5 80

chi square 42.3659

p' value 0.00001Significant

Patients with hypotension showed increased UPCR values, which is found to be statistically significant

Figure 23- BP vs peak UPCR 0

5 10 15 20 25 30 35 40

< 0.5 0.5 - 1.0 1.0 - 1.5 > 1.5 Peak value

40

24

2 1

1 2 6 4

BP VS PEAK VALUE

Normal Hypotension

HEMATOCRIT VS UPCR

Table6 – Hematocrit vs peak UPCR

HCT

Peak value

Total

< 0.5 0.5 - 1.0 1.0 - 1.5 > 1.5

<20 1 3 1 2 7

20-30 2 4 3 1 10

30-40 36 15 3 1 55

>40 2 3 1 2 8

Total 41 25 8 6 80

chi square 25.281

p' value <0.05 Significant

Variability in hematocrit is statistically significant with Rise in UPCR.

Figure 24- Hematocrit vs peak UPCR

0 5 10 15 20 25 30 35 40

<20 20-30 30-40 40

HEMATOCRIT VS UPCR

<0.5 0.5-1 1-1.5 >1.5

BLEEDING VS UPCR

Table 7- Bleeding vs UPCR

Bleeding

Peak value

Total

< 0.5 0.5 - 1.0 1.0 - 1.5 > 1.5

Yes 3 5 4 3 15

No 38 20 4 3 65

Total 41 25 8 6 80

chi square 12.518

p' value 0.006 Significant

Patients with bleeding manifestations are compared with the peak value of UPCR. cases those presented with bleeding had high UPCR values which is statistically significant

Figure 25 - Bleeding vs UPCR 0

10 20 30 40

Yes No

3

38

5

20

4 4

BLEEDING DISTRIBUTION

< 0.5 0.5 - 1.0 1.0 - 1.5

3^rd SPACE COLLECTION VS UPCR

Table 8- 3^rd space collection vs UPCR

3rd space collection

Peak value

Total

< 0.5 0.5 - 1.0 1.0 - 1.5 > 1.5

Yes 5 7 3 3 18

No 36 18 5 3 62

Total 41 25 8 6 80

chi square 6.565

p' value 0.087 Not Significant

Free fluid collection and increase in UPCR is not statistically significant

Figure 26 – 3^rd space collection vs UPCR 0

5 10 15 20 25 30 35 40

< 0.5 0.5 - 1.0 1.0 - 1.5 > 1.5 Peak value

5 7

3 3

36

18

5 3

3RD SPACE COLLECTION

Yes No

ORGANOMEGALY VS UPCR

Table 9- Organomegaly vs UPCR

ORGANOMEGLY

Peak value

Total

< 0.5 0.5 - 1.0 1.0 - 1.5 > 1.5

Yes 23 13 3 3 42

No 18 12 5 3 38

Total 41 25 8 6 80

chi square 0.952

p' value 0.813 Not Significant

Patients with organomegaly does not have increased UPCR. It is statistically insignificant

Figure 27- Organomegaly vs UPCR

MEAN UPCR VS CATEGORIES

Table10 – Mean UPCR vs categories

Spot PCR Category A Category B Category C

Mean 0.407 0.461 1.427

SD 0.302 0.285 1.413

p value < 0.001 Significant

Mean UPCR is found to higher in Category C which is statistically significant

Figure 28- Mean UPCR vs categories 0

0.5 1 1.5

Category A Category B Category C

0.407 0.461

1.427 Mean UPCR VS CATEGORY

Mean

DISCUSSION

Dengue hemorrhagic fever and dengue shock syndrome results in significant mortality in pediatric dengue fever. There are many studies available predicting the severity in adult patients and only few available in pediatric patients. Dengue deaths can be prevented by close monitoring.

Hence this study is undertaken to predict the severity of dengue in early stage, thereby preventing the complications like shock and hemorrhage and thereby death.. Increase in vascular permeability is the hallmark of dengue infection. This is because of the damage to the endothelial cells.

Hypoalbuminemia and proteinuria are well recognized in dengue infection.

This is due to altered filtration of the glycocalyx as dengue virus and NS1 are known to attach to heparan sulphate, which is part of the glycocalyx¹⁶. Hence we studied to estimate urine protein creatinine ratio from the day of admission. We studies 80 cases and they were categorized based on the clinical findings and laboratory parameter into 3 categories .Out of 80, 28(35%) belonged to category A, 32 (40%) belonged to category B and 20 (25%) belonged to category C(Fig15)

Out of 80 cases,50(62%) cases are male and 30(38%) cases are female. Though gender distribution doesn’t produce any change in UPCR, majority of cases affected with dengue are male.(fig17)

Majority of admissions were on the 4^th and 5^th day of illness(fig18).

The values of UPCR estimated on day of admission are found to be increased in cases belonging to category C(table2).

We divided the children into two groups based on the age as less than 2years and more than 2 years based on the values of UPCR.(table1) Age doesn’t show any correlation between UPCR which is similar to the study done by Priyanka Datla et al²⁶.

Out of 80 cases, 51 cases (63%) cases showed peak UPCR values between days 4 and 5, which is the critical phase.(fig20)

In our study 30% of category C had UPCR more than 1.5(table3) which is similar to study by Farhad F. Vasanwala et al.³⁰

When Platelet count is compared with UPCR, very low platelet of less than 50,000 which is seen in 25% cases (table4). In that 6 (30%) is associated with significant rise in UPCR of more than 1.5 (fig22). Hence low platelet and rise in UPCR indicates that the cases can develop DHS/DSS.

In our study 13(16%) cases had hypotension (table5), out of which 4 (30%) cases had UPCR value higher than 1.5(fig23). Most of the cases showed the peak value on day 4 and 5 of illness, which is the critical phase.

This is similar to many other studies where cases showed elevated UPCR before cases developing DSS.

When UPCR is compared with hematocrit 55(68%) cases had hematocrit between 30-40(table6), drastic fall of <20 hematocrit and rise in hematocrit values of more than 40 is associated with increase in UPCR of more than 1.5,which is significant, which denotes that the cases develop DHS/DSS.(fig24)

In our study 15(18%) cases had bleeding manifestations(table7), in that 8(53%) showed UPCR values of more than 1.(fig25)

Third space collection is seen in 18(22%) cases, in that only 3(16%) cases had UPCR >1.5,which is statistically insignificant (pvalue0.087), but in study by Priyanka Datla, 58% cases had third space collection who showed significant elevation of UPCR(table8).

In our study organomegaly is present in 42 cases (52%), but it has no correlation with UPCR.(table9)

In our study we calculated the mean UPCR with the categories A,B and C.(table10) It is found to be significantly high in category C with mean UPCR 1.461 which is higher than other categories A and B , which is also similar to many other studies.(fig28)

In our current study , we observed that the peak UPCR could distinguish patients likely to develop DHF from those who did not and that peak UPCR occurred at day 4- 7 of the illness.

A significant increase in UPCR was seen on the day which corresponded to one day before the development of DHF. Patients with uncomplicated DF had significantly lower UPCR than patients with impending DHF and DSS.

Daily follow-up in this prospective study enabled a time course analysis showing that the discriminatory value of UPCR was not evident in the early febrile period but it is discriminatory between days 4 and 7, just before defervescence when maximal plasma leakage classically occurs.

CONCLUSION

Increasing incidence of dengue fever and its associated complications necessitates the need of early predictors of disease severity . Such markers have not been well studied in the paediatric population.

UPCR assessment is easy to perform and inexpensive.

In this study, we found UPCR to be an accurate marker in predicting disease severity, bleeding manifestations, need of inotropes and adverse outcome in children with dengue fever.

We therefore recommend UPCR estimation in all children affected with dengue fever as a screening tests for hospitalization, management and prognostication.

LIMITATIONS

Our study had less number of patients with DHS/DSS.

Another limitation is we were not able to eliminate many confounding factors of proteinuria which can be even cause by other viruses like EBV, CMV etc.

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PROFORMA

NAME DOB&AGE SEX HOSPITAL NO

INFORMANT

RELIABILTY History :

Day of fever Rash

Myalgia

Abdomen pain Vomiting

Bleeding manifestations Lethargic

Clinical examination Heart rate

Peripheral pulse BP

RR CRT

Systemic Examination

RS : BAE , Pleural Effusion +/- P/A : liver – AG -

CNS: level of consciousness Lab values:

HB

Total count Platelet count Hematocrit NS1 antigen USG Abdomen

ABBREVATIONS

ALT - Alanine aminotransferase

ARDS - Acute respiratory distress syndrome AST - Aspartate aminotransferase

BP - Blood pressure

°C - Degree Celsius

CBC - Complete blood count CNS - Central nervous system CRT - Capillary refill time DEN - Dengue

DEN-1 - Dengue virus serotype 1 DEN-2 - Dengue virus serotype 2 DEN-3 - Dengue virus serotype 3 DEN-4 - Dengue virus serotype 4 DF - Dengue fever

DHF - Dengue haemorrhagic fever

DIVC - Disseminated intravascular coagulopathy DSS - Dengue shock syndrome

ELISA - Enzyme-linked immunosorbent assay FFP - Fresh frozen plasma

G6PD - Glucose-6-phosphate dehydrogenase GCS - Glasgow Coma Scale

Hb - Haemoglobulin HCT - Haematocrit

HF - Haemorrhagic fever

HELLP - Haemolysis, elevated liver enzymes and low platelet count

HI - Haemagglutinin inhibition test HIA - Haemagglutination inhibition assay HIV - Human immunodeficiency virus HR - Heart rate

ICU - Intensive care unit IgM - Immunoglobulin M IgG - Immunoglobulin G

IHA - Indirect haemagglutination

IMCI - Integrated management of childhood illness INR - International normalized ratio

JVP - Jugular venous pressure MAP - Mean arterial pressure

NS1 - Ag Non-structural protein 1 antigen

ORS - Oral rehydration solution PCR - Polymerase chain reaction PLT - Platelets

PR - Pulse rate

PT - Prothrombin time

PTT - Partial thromboplastin time RL - Ringer’s lactate

RR - Respiratory rate

RT-PCR - Reverse transcriptase polymerase chain reaction SD - Standard deviation

SpO2 - Oxygen saturation TWBC - Total white blood count

UPCR - Urine Protein Creatinine Ratio WBC - White blood cell

WHO - World Health Organization

WHO/TDR - Special Programme for Research and Training in Tropical Diseases

WHO/NTD - Department of Control of Neglected Tropical Diseases

CONSENT FORM

I hereby give consent to participate in the study being conducted by Dr.N.J.NIROSHINI. postgraduate in the Institute of Pediatrics , Madurai medical college, Madurai and to use my personal clinical data and result of investigations for the purpose of analysis and to study the predictor of disease severity in dengue fever. I also give consent for further investigations.

Place:

Signature of the parents / guardian Date: