SERUM FERRITIN AS AN EARLY INDICATOR OF SEVERITY OF DENGUE …

SERUM FERRITIN AS AN EARLY INDICATOR OF

SEVERITY OF DENGUE

DISSERTATION SUBMITTED FOR THE DEGREE OF

M.D BRANCH VII

(PAEDIATRIC MEDICINE)

REG. NO: 201717102

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 entitled “SERUM

FERRITIN AS AN EARLY INDICATOR OF SEVERITY OF

DENGUE” is the bonafide work of Dr. V. MOHAN RAJ 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. J. SANGUMANI, MD.,

Dean, Madurai Medical College,

Government Rajaji Hospital,

Madurai – 625020

BONAFIDE CERTIFICATE

This is to certify that the dissertation entitled “SERUM


DENGUE” submitted by Dr. V MOHAN RAJ 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 him under our direct supervision and guidance.

Dr. S.BALASANKAR MD DCH

Director I/C & Professor of Paediatrics

Institute of Child Health & Research center,

Madurai Medical College,

Madurai.

DECLARATION

I, Dr. V MOHAN RAJ, solemnly declare that the dissertation

titled “SERUM FERRITIN AS AN EARLY INDICATOR OF

SEVERITY OF DENGUE” has been conducted by me at Institute

of Child Health and Research Centre, Madurai under the guidance and

supervision of Prof. Dr. S. BALASANKAR 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. V MOHAN RAJ

Date:

CERTIFICATE - II

This is to certify that this dissertation work titled “SERUM


DENGUE” of the candidate Dr. V MOHAN RAJ with registration

Number 201717102 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. J. SANGUMANI, M.D.,

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 M.D., DCH., 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 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.M.S.

Rajarajeshwaran, M.D., DCH., Prof. Dr. M. Kulandaivel, M.D.,

DCH., Prof. Dr. S. Shanmugasundaram, M.D., DCH., and

Prof. Dr. Nandhini, M.D., DCH., Prof. Dr. M. Balasubramanian,

M.D., DCH., Prof Dr. D Raj Kumar, M.D., and

Prof. Dr. J Ashokaraja, M.D., DM., 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.P.Ramasubramaniam, M.D., DCH., Dr. Vanitha, M.D.,

Dr.J.Balasubramanian, M.D., DCH., Dr.R.Suresh, M.D., for their

constant guidance, encouragement and support throughout my study. I also

extend my thanks to Dr. P. Murugalatha, M.D., Dr. P. Kannan, M.D.,

Dr. K. Ramya, M.D., Dr. P. Guna, M.D., DMRD.,

Dr. S. Murugesalakshmanan, M.D., Dr. Abu backer siddiq, M.D.,

DCH., Dr. Suganthi, M.D., Dr. A.N.Karthik, M.D.,

Dr. Venkataramanan, M.D., DCH., Dr. Sonia Rosalind Martina,

M.D., Dr. M. K. Lenin, M.D., 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

and Department of Radiology of Government Rajaji hospital, 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. Aims and Objectives 38

3. Review of literature 39

4. Materials and Methods 41

5. Observation and Results 46

6. Discussion 74

7. Conclusion 77

8. Limitations 78

Annexures :

• Bibliography

• Proforma

• Abbreviations

• Consent form

• Master Chart

1

INTRODUCTION

Dengue is a arthropod borne viral infection caused by flavi virus

which causes simple fever to severe complications like dengue shock

syndrome and dengue hemorrhagic syndrome. It is epidemic all over the

world and in recent upsurge.

Dengue epidemic has resulted in significant mortality. There is no

appropriate treatment and only supportive treatment can be given.

Most of the cases recover with no complications and only certain

number of cases can have complications and they require careful

monitoring and assessment. In order to predict the complications and to

find out which case requires monitoring, we need a parameter to assess.

In this study we aimed to predict the severity in early phase of the

disease, using Serum Ferritin.

SERUM FERRITN & DENGUE

In recent times a phenomenon called macrophage activation

syndrome (MAS) or hemophagocytic syndrome (HS) is being frequently

reported in patients with severe dengue. MAS is a severe systemic

inflammatory condition due to excessive activation and proliferation of T

cells and well differentiated macrophage that leads to hyperactivated but

dysregulated immune responses22,23,24. This results in an overwhelming

2

inflammatory response leading to non remitting high rise of temperature,

organomegaly (involving liver and spleen), hemorrhage,

lymphadenopathy, and central nervous system (CNS) dysfunction.

Hyperferritinemia (levels above 10000ug/L) is a flagship sign of MAS;

however hypoalbuminemia, cytopenia, coagulopathy, abnormal liver

function tests, hypertriglyceridemia, hemophagocytosis, and elevated

serum sCD25 and sCD16 levels also serve as adjunct markers of MAS.

Hemophagocytic syndrome (HS) is being increasingly reported in patients

with severe dengue with multiorgan complication and is observed in severe

dengue involving children.25-28

Another mechanism by which ferritin can be used as a predictor is

increased expression of acute phase reactants is observed in patients with

severe dengue infection when compared to non-severe cases. This serves

to prognosticate the dengue infected patients well ahead of the appearance

of clinical warning signs. One such acute phase reactant is ferritin which is

produced by reticuloendothelial cells in response to infection and

inflammation. Ferritin is highly elevated in dengue infected patients than

in patients with other febrile illnesses. Hyperferritinemia seen in these

patients exhibit two opposite functions; Early in the phase of clinical

illness, increased serum ferritin levels exert a protective effect by chelating

the toxic free iron radicals at the site of inflammation15. while in severe

3

cases, raised ferritin may assume a pathogenic role by activating immune

cells resulting in cytokine storm.

GEOGRAPHICAL DISTRIBUTION

Global

Dengue fever is known in the tropical Southeast Asia and Western

Pacific for more than a century. The haemorrhagic form was first

recognized in Philippines in 1953. Subsequently DHF was recognized in

Thailand, India, Singapore, malaysia and Vietnam. In 1978, a big outbreak

was reported from China resulting in 22,122 cases. In 1981, a large

epidemic occurred in Cuba resulting in nearly 0.35 million cases of dengue

fever. Out of these, 24,000 had DHF and 10,000 had DSS. Dengue has also

been noticed in temperate regions of North America, Africa, and

Mediterranean Europe1,2.

As per current estimates, more than 100 countries are endemic for

dengue fever and about 40% of the world populations (2.5 billion people)

are at risk in tropics and subtropics. In 2010, 1.6 million cases of dengue

were reported in the Americas alone, of which 49,000 cases were severe

dengue3,4. Recently, dengue has also been reported from Costa Rica,

France, Mexico, Croatia and Portugal. Incidence of dengue infections

annually has almost doubled from 50 million to 96 million (2010) in last

few years.

4

Figure 1- Global distribution of Dengue

Figure 1.1 Countries/areas at risk of dengue transmission, 2008

10.C

10.C

(As of 1 November 2008)

5

India

India alone accounted for almost 34% of global dengue burden by

2010. The National Dengue Day was observed on 16th May 2016. Disease

is prevalent throughout India in most of the metropolitan cities and towns

and is endemic in 18 out of 35 states. Outbreaks have also been reported

from rural areas of Haryana, Maharashtra, and Karnataka. Recent trends in

transmission have shown occurrence of larger and more frequent

outbreaks, geographic expansion of endemic transmission, spread of the

disease from urban to semiurban and rural areas and an increasing

proportion of severe cases and deaths. An increased propensity to

hyperendemic areas particularly in large urban areas, is also noted5.

During 1996, a severe outbreak of dengue or DHF occurred in Delhi

wherein about 10,252 cases and 423 deaths were reported. In 2006, India

witnessed another outbreak with 12,317 cases and 184 deaths in 21 states.

The initial epidemics in India were due to serotype 2 or 4. The dengue

serotype 1 was seen as predominant serotype in Delhi during 2007-2010.

Concurrent infection of chikungunya and dengue serotype 2 has been

reported from Delhi and vellore.

Cyclic epidemics are increasing in frequency and in-country

geographic expansion is reported in India due to deciduous dry and wet

climatic zone with circulation of multiple virus serotypes. Cyclic

6

epidemics occurs once in every few years, which is usually associated with

good monsoon rains. Sporadic case usually present throughout the year.

However, with improved case management, the case fatality has decreased

from 3.3% in 1996 to 0.4% in 2010.

DENGUE VIRUS

Dengue virus has at least four serotypes.

DENV-1

DENV-2

DENV-3

DENV-4

These are antigenically similar but do not offer a complete cross

protection after infection by any one of them to other serotypes. Infections

in human by a serotype will produce lifelong immunity against reinfection

only for that particular serotype but not aganist other serotype. Subsequent

infection (secondary infection) by another serotype causes severe dengue

along with increased mortality. The severity of epidemics mostly caused

by serotype 1 followed by types 2 and 3.

EPIDEMIOLOGY

Vector

Aedes aegypti is the vector for dengue virus. Female mosquito bites

the man mostly during daytime. After feeding on a person with viremia,

7

the female mosquito can transmit dengue virus immediately or after a

period of 10-14 days (extrinsic incubation period). The extrinsic incubation

period is a important factor in successful transmission of the disease. A

lower environmental temperature increases the extrinsic incubation period,

which in turn, decreases the transmission. Once the mosquito becomes

infective, it remains so till its death

Figure 2- Aedes mosquito

The flight range of an adult A. aegypti mosquito is usually around

25-50 metres in an urban environment. However, the vector can be

transported by water, land, and air travel contributing to the speedy

transmission for vast areas. For dengue transmission, the number of

infected female mosquitoes per house is important. Usually this number is

small, and for an Indian epidemic it was observed to be just 1 per household

(house index). The minimum vector density, below which the dengue

8

transmission ceases, is not known at present. The Aedes aegypti mosquito

breeding is not necessarily related to the ambient temperature. The

mosquito has been found at very high altitudes like 2,200 metres above the

sea level. Vectors must survive longer than the sum of the initial non-

feeding period after birth (usually 2 days) and the extrinsic incubation

period to be able to infect another human. Longevity under natural

conditions ranges from 8 days to 42 days. The eggs of Aedes aegypti can

survive without water for a year.

Host factor

People at all ages are susceptible to dengue. In Asians, disease is

more severe in children. This is in contrast to America where infection

mainly occurs in adults which is usually mild. Severe dengue occurs in

children who have experienced a previous dengue infection. Other factors

which are associated with increase host susceptibilities to severe dengue

include HIV infection, bronchial asthma, certain HLA types like HLA

1(A04, A2, B0, B46), HLA 2 (DQ, DR4, DR1), TNF alpha, TGF beta,

G6PD deficiency, mutation in mannose binding lectin 2 gene.

ENVIRONMENTAL FACTORS

In tropical countries, a positive association between rainfall or larval

density and dengue incidence has been documented. The vector survives at

hot and humid temperature (16-30 degree celsius & 60-80% humidity). But

9

dengue cases are also reported in areas with less rainfall also. The

transmission of disease occurs only, if the ambient temperature is above 16

degree Celsius. In winter season the transmission rate is very low. This is

due to prolongation of extrinsic incubation period beyond the longevity of

mosquito.

TRANSMISSION RISK FACTORS

When a member of house hold is infected with dengue, other family

members who are living with them are at risk. Dengue virus spread is

mainly due to vector infestation.

Place where people gather, like offices, schools, hospitals, factories,

etc… in urban places, the movement of infected people causes the spread

of virus than the movement of aedes mosquitoes.

Figure 3-Transmission cycle of Dengue

10

PATHOGENESIS

Dengue virus infects the peripheral blood mononuclear cells within

a few days of infective mosquito bite.

Two patterns of immune response follow:

(1) Primary and (2) secondary (anamnestic).

Persons never previously infected with a flavivirus, nor immunized

with a flavivirus vaccine (e.g. Yellow Fever, Japanese encephalitis), mount

a primary IgM antibody response when infected with dengue virus,

appearing within 2-3 days of defervescence and peaking at 2 weeks after

the onset of symptoms. Antidengue IgG appears afterwards. Individuals

with immunity due to previous flavivirus infection or immunization mount

a secondary anamnestic antibody response when infected with dengue

virus5,6. In secondary flavivirus infections, which account for most cases

of severe dengue, the dominant immunoglobulin is IgG; the levels of IgM

being much lower. A mechanism of immune enhancement or antibody

dependent enhancement (ADE) is observed in dengue due to heterologous

non-neutralizing antibodies. This is responsible for serious organ

dysfunction and haemorrhagic disturbances which can occur during

secondary infection by a different serotype. This mechanism promotes

binding of dengue virus to surface expressed Fc gamma receptors on

monocytes and macrophages, further promoting viral replication and

11

spread. Thus, sequential rather than simultaneous exposure of different

serotypes of dengue virus carry a higher chance of ADE resulting in serious

disease. Thus, antibody against a strain of dengue virus does not protect

from a different strain of virus. Rather, it may increase its capacity to

multiply in human monocytes. The infected monocytes result in activation

of cross-reactive CD4+ and CD8+ cytotoxic lymphocytes. Cytotoxic

lymphocytes mediate release of cytokines resulting in plasma leakage and

haemorrhage and are primarily responsible for host defense in dengue

mediated via interferon-gamma. Recent studies have highlighted the role

of HLA linked protective role of CD8 lymphocytes. Researchers have

found that certain phenotypes of HLA may cause hyporesponsiveness of

interferon -7 response, thereby weekening the host response.7

12

PATHOPHYSIOLOGY

Two main pathophysiological changes occur in dengue,

These are

(1) Increased vascular permeability, resulting in loss of plasma from

the vascular compartment to third space accumulation, hemoconcentration,

low pulse pressure, and other signs of shock8 and

(2) Disorder in the hemostasis involving thrombocytopenia,

coagulopathy and vascular changes.

Secondary dengue infection results in formation of immune

complexes and activation of complement system. TNF-alpha, interferon,

and interleukin-2 are elevated, and C1q, C3-C8, are depressed. As a result,

vasoactive amines are released from the platelets, These cause massive

release of water, electrolytes, and plasma proteins from the blood vessels

and lead to hypovolemic shock. Increased vascular permeability is

mediated through the nitric oxide pathway

Platelet defects are both quantitative and qualitative. Thus, a patient

with a normal platelet count may still have a prolonged bleeding time,

maculopapular and petechial rashes are present. In these lesions, dengue

antigen, IgM, and complement (C3) have been observed.

13

It may be noted that virus is usually not detectable in blood once

shock manifests, though viral replication occurs in various organs.

COURSE OF DENGUE FEVER

Figure 4- Course of Dengue illness

FEBRILE PHASE

Febrile phase lasts from 2-5 days. During the febrile phase child

presents with fever, headache, retro orbital pain, myalgia and vomiting.

14

It is difficult to distinguish dengue fever from non dengue illnesses

at this stage. Child may present with progressive decrease in leucocyte

count with tender hepatomegaly.

CRITICAL PHASE

Following the febrile phase, child may enter into the critical phase,

which usually lasts from 4 to 7 days. Patients with increased capillary

permeability may manifest with the warning signs, mostly as a result of

plasma leakage.

The warning signs are persistent vomiting and severe abdominal

pain which are the early indications of plasma leakage and become

increasingly worse and the patient progresses to the shock state.

The patient becomes increasingly lethargic but usually remains

mentally alert. These symptoms may persist into the shock stage.

Weakness, dizziness or postural hypotension, spontaneous mucosal

bleeding or bleeding at previous venepuncture sites are important

haemorrhagic manifestations. Increasing liver size and a tender liver is

frequently observed.

Appropriate management at this stage prevents the complication and

mortality.

15

RECOVERY PHASE

As the patient survives the critical period, enters into the recovery

phase, which occurs due to reabsorption of fluid from the extravascular

compartment. At this stage patient develops bradycardia, electrocardiac

abnormalities and pruritis.

Figure 5- Case classification of dengue

DIAGNOSIS

Hematological Tests

The clinical diagnosis is corroborated by raised hematocrit (Hct) and

thrombocytopenia.

16

An Hct level rise of greater than 20% is a sign of hemo concentration

and precedes shock. The Hct level should be monitored at least every

24 h to facilitate early recognition of warning signs and every 3-4 h

in severe dengue.

Thrombocytopenia occurs in up to 50% of children with dengue.

Platelet counts of less than 100,000 cells/uL indicate onset of critical

phase and typically occur before defervescence and the onset of

shock. The platelet count should be monitored at least every 24 h

initially.

The white blood cell count can be normal or show leukocytosis

during initial phase. Leukopenia, often with lymphopenia, precedes

thrombocytopenia and is observed near the end of the febrile phase

of illness.

Electrolyte abnormalities are seen in critical phase. Metabolic

acidosis and elevated blood urea are observed in those with shock.

Serum glutamic pyruvic transaminase (SGPT) levels are elevated.

Low serum albumin levels are a sign of hemoconcentration10

17

TABLE 1. DIAGNOSTIC TESTS FOR DENGUE FEVER

Diagnostic method

Timing of test (after fever

onset)

Validity

Virus isolation(culture) 1-5 days ++++

Genome detection (PCR) 1-5 days ++++

Antigen detection (NS1) 1-5 days +++

Antibody detection (IgM) After 5 days* ++

IgG (Paired sera) **

Acute sera 1-5 days;

convalescent sera after 15

days

+

(PCR: Polymerase chain reaction; Ig: Immunoglobulin).

*IgM positivity rates: by 3-5 days (50%), 5-7 days (80%), and 10

days (90%). IgM appears between 3 days and 10 days and disappears by

2–3 months9.

**IgG appears after 1-2 weeks and may persist for life.

18

Serodiagnosis

Serum specimens should be sent to the laboratory for serodiagnosis,

polymerase chain reaction (PCR), and viral isolation. Because the signs

and symptoms of dengue fever are nonspecific, attempting laboratory

confirmation of dengue infection is important.

Serodiagnosis is based on (1) detection of viral nonstructural protein

1 (NS1) during initial illness; (2) detection of IgM antibodies to dengue; or

(3) fourfold rise in dengue IgG in paired samples11. Table 1 outlines the

desired timing of these tests for confirming the diagnosis. Laboratory

criteria for definitive diagnosis include one or more of the following:

Isolation of the dengue virus from serum, plasma, leukocytes, or

autopsy samples

Demonstration of dengue virus antigen in serum samples via enzyme

immunoassay or in autopsy tissue via immunohistochemistry or

immunofluorescence can be done. NS1 is a glycoprotein produced by the

virus which can be detected early, between 1st to 4th day of illness. It is

specific and has a high sensitivity.

Demonstration of a fourfold or greater change in reciprocal IgG or

IgM antibody titers to one or more dengue virus antigens in paired serum

samples. IgM antibody appears early in disease course, requires single

sample and is less cross-reactive to other flaviviruses12.

19

Thus, measurement of raised IgM appears to be most prudent when

done after 5th day of illness. As per the NVBDCP, the laboratory test being

followed is the IgM antibody-capture enzyme-linked immunosorbent assay

(MAC-ELISA) for dengue virus, which captures the dengue specific IgM

by using anti-human IgM. There are quicker and cheaper rapid diagnostic

test (RDT) kits available which test the presence of anti-IgM or IgG or NS1

antigen. However, these kits carry a high false positive rate and are not

recommended by the WHO or under NVBDCP.

Detection of viral genomic sequences in autopsy tissue, serum, or

cerebral spinal fluid (CSF) samples via PCR.

Diagnostic kits for NS1 and MAC-ELISA can be procured from

National Institute of Virology, Pune under NVBDCP. The Government of

India has set up surveillance hospitals and apex referral laboratories for

improved disease surveillance.

Dengue diagnostics for clinicians

The objectives of dengue laboratory diagnosis are

(i) to confirm the clinical diagnosis and

(ii) to provide information for epidemiological surveillance.

Laboratory diagnosis is not necessary for clinical management except in

atypical cases or when carrying out differential diagnosis with other

infectious diseases.

20

Laboratory diagnosis of dengue is made by detecting the virus and/or

any of its components (infective virus, virus genome, dengue antigen) or

by investigating the serological responses present after infection

(specifically IgM and IgG levels)

Table 2. Dengue diagnostics and sample characteristics

Clinical sample Diagnostic

method Methodology

Time to

result

Virus

detection

and its

components

Acute serum

(1–5 days of

fever)

and

necropsy tissues

Viral isolation

Mosquito or

mosquito cell

culture

inoculation

One week

or more

Nucleic acid

detection

RT-PCR and

real time

RTPCR

1 or 2

days

Antigen

detection

NS1 Ag rapid

tests Minutes

NS1 Ag ELISA 1 day

Immuno-

histochemistry 2-5 days

Serological

response

Paired sera

(acute serum

from1–5 days

and second

serum 15–21

days after)

IgM or IgG

seroconversion

ELISA

HIA 1–2 days

Neutralization

Test

Minimum

7 days

Serum after day

5 of fever

IgM detection

(recent

infection)

ELISA 1 or 2

days

Rapid tests Minutes

IgG detection

IgG ELISA

HIA

1 or 2

days

ELISA = enzyme-linked immunosorbent assay; HIA = haemagglutination

inhibition assay; IgG = immunoglobulinG; IgM = immunoglobulin M;

NS1 Ag = non-structural protein 1 antigen; RT-PCR = reverse transcriptase

polymerase chain reaction

21

Dengue viruses are RNA viruses belonging to the family

flaviviridae, genus flavivirus. The four dengue viruses (DEN-[1–4]) are

serologically related but antigenically and genetically distinctive 13.

Three main aspects should be considered for an adequate dengue

diagnosis:

● virological and serological markers in relation to the time of dengue

infection;

● type of diagnostic method in relation to clinical illness;

● characteristics of the clinical samples.

Virological and serological markers in relation to time of dengue

infection

An incubation period of 4–10 days occurs after the mosquito bites,

resulting in an asymptomatic or symptomatic dengue infection. During this

period the virus replicates and an antibody response is developed. In

general, viraemia is detectable in most dengue cases at the same time that

symptoms appear, and is no longer detectable at the time of defervescence.

The development of IgM antibody is coincident with the disappearance of

fever and viraemia. Virological and serological markers differ in time

evolution and titre response and according to whether the infection is

primary or secondary.

22

In a primary infection (i.e. when an individual is infected for the first

time with a flavivirus), viraemia develops from 1–2 days before the onset

of fever until 4–5 days after. Accordingly, anti-dengue IgM specific

antibodies can be detected 3−6 days after fever onset. On average, IgM is

detected in 50% of cases by days 3–5 after the onset of illness, this figure

increasing to 95–98% for days 6−10. Low levels of IgM are still detectable

around one to three months after fever. In addition, the primary infection

is characterized by slowly increasing but low levels of dengue-specific

IgG, becoming elevated at days 9−10. Low IgG levels persist for decades,

an indication of a past dengue infection. A totally different picture is

observed during a secondary infection, with a rapid and higher increase of

anti-dengue specific IgG antibodies and slower and lower levels of IgM.

High IgG levels remain for 30–40 days. A short-lasting but higher viraemia

level characterizes the secondary infection compared to the primary

infection.

23

Figure 6. Virological and serological markers of dengue infection

according to time of illness

IgG = immunoglobulin G; IgM = immunoglobulin M

Type of dengue diagnostic method in relation to time of clinical illness

The diagnostic method to confirm an acute infection depends on the

time of clinical illness: the febrile phase is coincident with the presence of

viraemia, some viral components and replication products in blood; the

critical and convalescent phases coincide with the development of

antibodies, as summarized in Table 3.

Febrile phase (day 1 to days 4–5 of fever)

The infective virus can be isolated in serum by inoculation in tissue

culture (mosquito cell cultures) and mosquitoes. This method allows for

identification of the viral serotype.

24

Virus genome detection using reverse transcriptase polymerase

chain reaction (RT-PCR) and real-time RT-PCR confirms an acute dengue

infection. Both methods have a high sensitivity and allow serotype

identification and quantification of genome copies. Some studies suggest

the presence of a higher number of copies in severe dengue cases.

NS1 Ag is a marker of acute dengue infection. Both enzyme-linked

immunosorbent assay (ELISA) and rapid commercial tests are available for

NS1 Ag detection. The sensitivity and specificity of commercial kits in

different serotype infections and days of illness are being evaluated.14

Critical and convalescent phases (after days 4–5 of illness)

Specific IgM is the best marker of a recent dengue infection. MAC-

ELISA and rapid tests are the most frequent methods for IgM detection;

however a recent evaluation of four rapid tests demonstrated a low

sensitivity. In addition to IgM, high levels of specific IgG in sera collected

early after fever onset as detected by ELISA and haemagglutination

inhibition assay (HIA) also suggest a recent dengue infection.

Primary infections are characterized by high levels of IgM and low

levels of IgG, while low levels of IgM with high levels of IgG characterize

secondary infections.

25

A single serum sample collected after day 5 of fever onset is useful

for IgM determination. Depending on the IgG level in the sample,

classification into primary or secondary infection can also be determined

using the IgM/IgG optical density ratio. Ratios greater than 1.2 (using the

patient’s sera at 1/100 serum dilution) or 1.4 (using serum dilution of 1/20)

suggest a primary infection. In addition, IgG titres higher than 1/1280 by

HIA or ELISA are also suggestive of a secondary infection.

As IgM antibodies persist for almost three months after fever onset,

the detection in samples collected late after the acute phase of illness

suggests a recent infection. In dengue endemic countries, acute clinical

cases with a positive IgM are classified as probable dengue cases.

The study of paired sera (acute and convalescent serum samples with

the second sample being collected 15–21 days after the first sample),

allows for serological confirmation of dengue infection. The diagnosis

depends upon the demonstration of rising titres of dengue antibodies

between acute and convalescent sera

A broad cross-reactivity of ELISA and HIA with other flaviviruses

has been observed. Neutralization Test is the method of choice for

determination of specific serotype15.

26

Table 3. Confirmed and probable dengue diagnosis, interpretation

of results and sample characteristics

Method Interpretation Sample

characteristics

Confirmed

dengue

infection

Viral isolation Virus isolated Serum (collected

at 1–5 days of

fever)

Necropsy tissues

Genome

detection

Positive RT-PCR or positive

real-time RT-PCR

Antigen

detection

Positive NS1 Ag

Positive

immunohistochemical Necropsy tissues

IgM

seroconversion

From negative IgM to

positive IgM in

paired sera Acute serum (days

1–5) and convalescent

serum (15–21 days

after first serum) IgG

seroconversion

From negative IgG to

positive IgG in

paired sera or 4-fold increase

IgG levels among paired

sera

Probable

dengue

infection

Positive IgM Positive IgM Single serum

collected after day

5

High IgG

levels

High IgG levels by ELISA

or HI (≥ 1280)

ELISA = enzyme-linked immunosorbent assay; IgG = immunoglobulin G; IgM = immunoglobulin M; NS1 Ag = non-structural protein 1 antigen; RT-PCR = reverse transcriptase polymerase chain reaction

27

TREATMENT OF DENGUE FEVER

Supportive therapy is the mainstay of treatment. Increased oral fluid

intake is recommended to prevent dehydration. Supplementation with IV

fluids may be necessary to prevent dehydration and significant hemo

concentration. But unnecessary iv fluid without dehydration may lead to

fluid overload. Fever is managed with paracetamol and tepid sponging.

Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDS) should

be avoided as these drugs may worsen the bleeding tendency associated

with some of these infections. Shock is managed with isotonic fluids.

Packed cell (PRBC) transfusion is indicated in refractory shock or if there

is significant bleeding.

Patients with known or suspected dengue fever should have their

platelet count and hematocrit measured daily from the third day of illness

until 1-2 days after defervescence. Patients with a rising Hct level or falling

platelet count should be monitored more frequently.

Management of dengue illness can be discussed in three steps:

Step 1: Overall assessment of signs and symptoms

Step 2: Diagnosis and severity assessment

Step 3: Categorizing the patients into mild, moderate or severe

dengue and treating accordingly.

28

Overall Assessment

History and Examination

Emphasis on history should be on assessment of warning signs.

Physical examination should concentrate on hemodynamic assessment, to

determine the presence and extent of shock, confirming or finding the new

warning signs, and checking for bleeding and shock manifestations,

abdominal tenderness, mental state, and hydration. Tourniquet test should

be done compulsorily.

Investigations

Initial investigations should include Hematocrit, WBC count,

platelet count and tests to confirm the diagnosis, as described in the sec.

Laboratory diagnosis. In critical phase, additional tests need to be and

include liver function test, renal function test, chest X-ray, serum

electrolytes, ultrasound abdomen and neuroimaging as necessary.

Diagnosis and Severity Assessment

Determine the phase of disease (febrile, critical, and recovery) and

severity (non-severe, severe) of dengue, as per criteria explained earlier.

The child will need admission if any of the following criteria is fulfilled.

● Presence of any of warning signs

● Bleeding from any site.

29

● Signs and symptoms suggestive of hypotension

● Renal, hepatic, or/and central nervous system (CNS) involvement

● Pleural effusion or pericardial effusion, ascites

● Rising Hematocrit

● Platelet countless than 50,000/mm3

● High-risk age group-infants and old age

Categorize Patients in Mild, Moderate, and Severe Dengue (Table 2)

This step is aimed to place the patient in an appropriate Group (mild,

moderate and severe) to decide on future course of treatment, which is as

follows:

● Mild: Patients, who may be sent home with advised to come for

follow up.

● Moderate: Patients needing continuous monitoring and

hospitalization

● Severe: Patients requiring tertiary level care.

Mild Dengue: Home Management

All children who are tolerating oral fluids, passing urine at least once

in 6 hr, and not having any of the warning signs can be sent home.

Following managements need to be advised:

30

Encourage fluid intake; can give oral rehydration salt (ORS), fresh

fruit juice, tender coconut, etc. The parents should be advised to increase

the amount of oral fluids in any form to be given (e.g. 3-10 kg: 100 ml/kg

and 10-20 kg: 75 ml/kg) Paracetamol (15 mg/kg/dose) if the child is

uncomfortable because of fever once in six hours if required. Avoid aspirin,

ibuprofen, mefenamic acid, nimesulide and other NSAIDS.

Monitor at home for fluid intake, urine output, fever, obvious

bleeding, and altered sensorium. Bring back if any of the above is present

or the child develops any of the warning signs.

Parents of infants should be explained the danger signs before

sending them home. Tepid sponging for fever may be done as febrile

convulsions may occur commonly in this age group. Breastfeeding should

be encouraged and continued.

Moderate Dengue:

Close Monitoring and Hospital Management is needed. Any patient

who fulfill the admission criteria should be admitted and monitered (as

mentioned above). They may or may not have warning signs. A baseline

Hematocrit is measured and continuous monitoring is started. In cases

where no warning signs are there, patients should be started on

maintenance fluids with isotonic fluid. If patient shows signs of mild

dehydration, a correction of 50 mL/kg (<12 months) and 30 ml/kg (>12

31

months) is added to maintainence fluid. At all the times clinical parameters

are monitored closely and correlated with Hematocrit to guide further fluid

therapy. For those who present with warning signs, the following is

advised.

• Start isotonic IV fluids (normal saline or Ringer's lactate) at 6

ml/kg/hr for 1-2hr

• Reassess Hematocrit and clinical status:

• If improvement occurs, decrease to 3 ml/kg/hr for 2-4 h, and then

continue with 1.5 ml/kg/hr for 2-4 hr,

• If clinical status worsens or Hematocrit rises, increase rate of fluids

to 10ml/kg/hr for 1-2 hr

• Reassess clinical status, repeat Hematocrit, and review fluid

infusion rates, till the child is better

• If the child improves, maintain minimum IV fluids at 1 ml/kg/hr

for 24-48 hr. Stop fluids when child demands and accepts adequate oral

fluids, and food along with adequate urine output.

• Those who worsen or develop profound shock bleeding or

multisystem involvement manage as severe dengue.

32

Severe Dengue:

Tertiary Level Care

Emergency treatment is required in children with severe dengue or

those in critical phase as follows:

Obtain Hematocrit, blood count and other organ function tests, as

indicated.

Compensated shock: This stage is characterized by low systolic

blood pressure, narrow pulse pressure (<20 mmHg), and rise in hematocrit

(>20%). In these children, fluid resuscitation is started at 10-20 ml kg/hr,

and further directed as per flowchart below.

33

Hypotensive shock: Administer isotonic fluid bolus 20 ml/kg in 15

min. For further management, follow the algorithm depicted in Flowchart

below.

Colloids may be needed in refractory shock. Colloids can be gelatine

or starch based. They carry theoretical risk of allergic reactions and

impaired coagulation. They should be infused slowly with strict monitoring

for signs of fluid overload.

Haemorrhagic complications: Suspect severe bleeding if there is

an unexplained fall in Hematocrit, refractory shock not responding to 40-

60 mL/kg of fluid, and persistent or worsening metabolic acidosis. Packed

cell transfusion 10 ml/kg over 2-3 hr can be lifesaving in these children.

34

There is not much evidence for platelet transfusion or fresh frozen

plasma (FFP) for severe bleeding. Platelet transfusions should not be used

prophylactically. Its use has neither shown to prevent progression to severe

bleeding nor does it shorten the bleeding time, but may be associated with

severe side effects. Platelet transfusion should be restricted to cases with

severe bleeding or when platelet counts are below 10,000/mm3. Platelets

obtained by single donor apheresis are preferred as they raise the platelet

count by 30,000-50,000 as compared to random donor platelets which

result in rise by 5,000-10,000 per unit.

Monitoring: This essentially remains the basic prerequisite for

treating children with severe dengue, in an emergency setting

• Monitor vital signs and peripheral perfusion, urine output 1-4

hourly till patient is out of critical phase. Monitor Hematocrit before and

after fluid replacement, then 6-12 hourly.

• Monitor blood glucose and other organ dysfunction both clinically

and biochemically.

• A typical monitoring chart for dengue fever should record the

following: body temperature, heart rate, BP, pulse volume, capillary refill

time, abdominal pain, appetite, abdominal pain, vomiting, bleeding, and

sensorium.

35

Treatment of Fluid Overload

A child with dengue can have fluid overload due to excessive or

rapidly transfused IV fluids, use of hypotonic fluids, and inappropriate use

of fresh frozen plasma (FFP) or platelets. Another important reason is

continuation of IV fluids even during the phase of plasma reabsorption and

recovery phase.

These children may present with features of respiratory distress,

pulmonary edema or congestive heart failure.

Following management is suggested:

● Oxygen therapy

● Discontinuation of IV fluids

● Frusemide 0.1-0.5 mg/kg/dose once or twice daily

● Correction of electrolyte imbalance

● Look for occult hemorrhage and transfuse packed cells (PRBC)

Management of Other Complications

Encephalopathy in dengue may result due to dengue encephalitis,

intracranial bleeding, electrolyte disturbances, occlusion due to DIVC or

hepatic failure (hepatic encephalopathy). Appropriate diagnosis for cause

and specific management should be instituted. Cardiac involvement may

36

be seen during shock or during convalescence, which may manifest as

arrhythmias or heart failure.

Criteria for Discharge

Patient should be discharged only if he has been

Afebrile for at least 48 hours

Passing urine normally and adequately

Having improved appetite

And has no respiratory distress

Laboratory parameters should show a stable Hematocrit and platelet

count of more than 50,000/mm3 and in rising trend.

37

VACCINES FOR DENGUE

A live attenuated tetravalent dengue vaccine (CYD-TDV) has been

developed which contains four dengue virus with expression of dengue pre

membrane and envelope protein and non-structural and capsid protein of

yellow fever strain (YF-17D)

Dosage : 3 dose schedule against serotypes 1,3 and 4

The vaccine is still under multicentric phase III trials.

The Chimeri Vax TM system, originally developed to construct JE

vaccine, has now been applied to dengue viruses. This vaccine was shown

to be safe and immunogenic in a monkey study.

38

AIM AND OBJECTIVES

To find out whether Serum Ferritin level during the febrile phase of

dengue fever can be used as a predictor of complications.

39

REVIEW OF LITERATURE

Chaiyaratana w. et al (2008)16, 172 children with dengue infection

between 2002 to 2005 were included in their study. Their study showed

median duration of febrile period is 5 days. Their data showed that both

patients with DF and DHF had higher serum ferritin levels during the

febrile, toxic and convalescent stages than they did at follow-up. Patients

with DHF had higher levels of serum ferritin than those with DF

throughout the course of the illness. Those with DHF grades III and IV had

higher serum ferritin levels than those with DF and DHF grades I and II.

Cornelia A.M.Van de wag (2014), conducted a study on the

carribean island aruba, during the epidemic between sep 2011 to april 2012.

It stated that ferritin can be used as a clinical marker to discriminate

between dengue and other febrile illnesses. The occurrence of

hyperferritinaemia in dengue virus infected patients is indicative for highly

active disease resulting in immune activation and coagulation disturbances.

Therefore, they recommended that patients with hyperferritinaemia should

be monitered carefully, as there is a high risk for them to develop severe

disease.

R.Soundravally et al (2014)17, conducted a study on 96 patients

with dengue fever and other febrile illness. A steady increase in the level

of serum ferritin was recorded throughout the course of illness. The

40

elevated ferritin level could predict the disease severity with highest

sensitivity and specificity of 76.9 and 83.3 %, respectively, on the day of

admission and the same was found to be 90 and 91.6% around

defervescence. On the basis on its diagnostic efficiency, they proposed that

ferritin may serve as a potential biomarker for an early prediction of disease

severity.

Muhammed Nadeem et al (2016)18, conducted a study on 104

patients with dengue infection. In that study they concluded 1. Raised

serum ferritin levels are significantly associated with severe dengue. Mean

ferritin levels are also high in patients with severe dengue as compared to

dengue fever. 2. Serum ferritin levels on the day of admission may serve

as biomarker for an early prediction of disease severity in dengue virus

infection.

Soumyabatra Roy Chaudri et al (2017)19 conducted a study on 358

cases of serological proven cases and selected only 30 cases of confirmed

dengue and compared them with other febrile illness. Ferritin was

evaluated as an adjunct marker for the diagnosis of dengue which could

possibly aid their clinical judgment and prompt early fluid resuscitation

which in turn could be useful in avoiding undue complications. Ferritin, as

evaluated in the present study may serve as a significant marker for

differentiating between dengue fever and fever of other etiology, even in

the absence of a positive NS1 antigen or a positive IgM antibody for

dengue.

41

MATERIALS AND METHODS

STUDY POPULATION:

The study was conducted on 80 dengue patients admitted in

Institute of Child Health & Research Centre, Madurai during the study

period of 6 months from February 2019 to august 2019.

INCLUSION CRITERIA:

All fever cases which are dengue NS1 antigen positive or which

show classical signs and symptoms of dengue.

Children from 1 month to 12 years of age

EXCLUSION CRITERIA:

Patients who do not have serological evidence of NS1 positivity or

IgM antibody.

Patient with anemia (Hb <11g/dl) and transfusion dependent chronic

disease.

DATA COLLECTION:

A previously designed proforma was used to collect the

demographic and clinical details of the patients. All the patients

underwent detailed clinical evaluation and appropriate investigations

are noted.

42

STUDY PROTOCOL:

DESIGN OF STUDY:

Prospective study

PERIOD OF STUDY:

February 2019 to august 2019.

METHODOLOGY:

History was taken in detail and duration of fever and other

associated signs and symptoms noted. Necessary investigations were done.

Child was then followed throughout the clinical course of the disease.

LABORATORY INVESTIGATIONS:

Total count, Platelet count, serum ferritin, USG abdomen, LFT,

RFT, neuroimaging and other investigations if necessary.

SERUM FERRITIN ESTIMATION20

Ferritin is a globular protein found mainly in the liver, which can

store about 2250 iron (Fe3+) ions. The ferritin molecule consists of a

protein shell (Apoferritin) composed of heavy and light subunits, which

surrounds a crystalline core containing iron oxide and phosphate. Ferritin

is synthesized in the liver, spleen and numerous other body tissues, with

major concentrations found in the liver, spleen, bone marrow, and intestinal

mucosa. The ferritin levels measured have a direct correlation with the total

43

amount of iron stored in the body. If ferritin is high there is iron in excess,

which would be excreted in the stool. If ferritin is low there is a risk for

lack in iron, which sooner or later could lead to anaemia. In the setting of

anaemia, serum ferritin is the most sensitive lab test for iron deficiency

anaemia. In contrast, serum ferritin levels are normal or increased in

anemia associated with chronic disease. Elevated serum ferritin levels

have been observed in acute and chronic liver disease and lymphoid

malignancy (leukemia and Hodgkin lymphoma).

Ferritin is an acute-phase reactant, it is often elevated in the course

of disease.

In dengue serum ferritin is elevated because of macrophage

activation.

METHOD AND PRINCIPLE OF SERUM FERRITIN

ESTIMATION:

Immuno enzymatic colorimetric method for quantitative

determination of Ferritin concentration in human serum or plasma.

Ferritin ELISA test is based on simultaneous binding of human

Ferritin to two monoclonal antibodies, one immobilized on microwell

plates and the other conjugated with horseradish peroxidase (HRP). After

incubation the bound/free separation is performed by a simple solid-phase

washing. Then the enzyme HRP in the bound-fraction reacts with the

44

Substrate (H2O2) and the TMB Substrate and develops a blue colour that

changes into yellow when the Stop Solution (H2SO4) is added. The colour

intensity is proportional to the Ferritin concentration in the sample. The

Ferritin concentration in the sample is calculated based on a standard curve.

Typical reference interval

Children 1-9 years, 10-60 ng/ml

10-18 years male 10-300 ng/ml

10-18 years female 10-70 ng/ml

Men, 18 - 60 years: 30–400 ng/ml

Women, 18 - 60 years: 15‐150 ng/ml

Men and women, 60–90 years: 15–650 ng/ml

COLLABORATING DEPARTMENTS:

Department of Microbiology

Department of Biochemistry

Department of Radiology

ETHICAL CLEARANCE: Clearance obtained

CONSENT: Individual written and informed consent obtained from the

parents of their children who were included in this study.

45

STATISTICAL ANALYSIS:

All data were entered in Excel 2007 and statistical analysis was

performed using the statistical software SPSS 1.6.0. Data were

expressed as frequency (with percentages), median values (with range

(min, max). For continuous variables, Mann Whitney U-test was

performed to find the differences between two groups and for

categorical variables Pearson’s chi-square test was performed. Results

were defined as statistically significant when the P value (2-sided) was

less than 0.05.

46

OBSERVATION AND RESULTS

Table 4

AGE DISTRIBUTION

Out of 80 cases 23 were less than 5 years and 57 cases were more than 5

years of age.

Age in years No. of cases Percentage

< 5 (1 - 5) 23 28.75

> 5 (5 - 12) 57 71.25

Total 80 100

47

Figure 7 - Age distribution

23

57

28.75

71.25

0

10

20

30

40

50

60

70

80

< 5 (1 - 5) > 5 (5.5 - 12)

AGE DISTRIBUTION

No.of cases Percentage

(5-12) 5

48

Table 5

GENDER DISTRIBUTION

Gender No.of cases Percentage

Male 43 53.75

Female 37 46.25

Total 80 100

Among 80 cases, 43 were male and 37 cases were female.

49

53.75

46.25

Figure 8 - Gender distribution

Male Female

50

Table 6

Age & Gender

Age in years Male Female

< 5 (1 - 5) 11 12

> 5 (5 - 12) 32 25

Total 43 37

51

0

5

10

15

20

25

30

35

< 5 (1 - 5) > 5 (5.5 - 12)

11

32

12

25

Figure 9 Age vs Gender

Male Female

(5-12) 5

52

Table 7

DENGUE CASE CLASSIFICATION

Out of 80 cases, 63 cases were non severe dengue and 17 were severe

dengue.

Dengue No. of cases Percentage

Non severe 63 78.75

Severe 17 21.25

Total 80 100

53

78.75

21.25

Figure 10 - Dengue Case Classification

Non severe Severe

54

Table 8

SEVERE DENGUE MANIFESTATIONS

Out of 17 severe dengue cases, 1 case had fluid accumulation with

respiratory distress, 11 cases had shock, 3 cases had bleeding, 2 cases had

organ involvement (1 case had liver involvement with elevated liver

enzymes and 1 other case had CNS involvement in the form of

encephalitis).

Complications No. of cases Percentage

Fluid Accumulation With

Respiratory Distress 1 1.25

Shock 11 13.75

Bleeding 3 3.75

Organ Involvement 2 2.5

55

0

2

4

6

8

10

12

FLUID

ACCUMULAT

ION WITH

RESPIRATOR

Y DISTRESS

SHOCK BLEEDING ORGAN

INVOLVEME

NT

No.of cases 1 11 3 2

1

11

3

2

Figure 11 - Severe Dengue Manifestations

No.of cases

56

Table 9

SERUM FERRITIN ON DAY 3 OF ILLNESS

Out of 80 cases, 5 cases had serum ferritin < 300, 54 cases had 301-600

cases, 8 cases had serum ferritin 601 - 800 and 13 cases had serum ferritin

levels >800.

Sr Ferritin Day 3

(ng/ml) No. of cases Percentage

< 300 5 6.25

301 - 600 54 67.5

601 - 800 8 10

> 800 13 16.25

Total 80 100

57

0

10

20

30

40

50

60

70

< 300 301 - 600 601 - 800 > 800

6.25

67.5

10

16.25

Figure 12 - SERUM FERRITIN - DAY 3

Percentage

(ng/ml)

58

Table 10

SERUM FERRITIN ON DAY 4 OF ILLNESS

Sr Ferritin Day 4

(ng/ml) No. of cases Percentage

< 300 0 0

301 - 600 47 58.75

601 - 800 13 16.25

801 - 1200 2 2.5

> 1200 18 22.5

Total 80 100

47 cases had serum ferritin levels 301-600, 13 cases had levels between

601-800 and 2 cases had levels between 801-1200. 18 cases had serum

ferritin levels of more than 1200.

59

0

10

20

30

40

50

60

< 300 301 - 600 601 - 800 801 - 1200 > 1200

0

58.75

16.25

2.5

22.5

Figure 13 - SERUM FERRITIN - DAY 4

PERCENTAGE

(ng/ml)

60

Table 11

SERUM FERRITIN ON DAY 3 & 4 OF ILLNESS

Serum ferritin levels is elevated in 18 cases to levels more than 1200 on

day 4.

Sr Ferritin

(ng/ml) Sr Ferritin Day 3 Sr Ferritin Day 4

< 300 5 0

301 - 600 54 47

601 - 800 8 13

801 - 1200 13 2

> 1200 0 18

Total 80 80

61

0

10

20

30

40

50

60

< 300 301 - 600 601 - 800 801 - 1200 > 1200

5

54

8

13

00

47

13

2

18

Figure 14 -SERUM FERRITIN on Day 3 & 4

Sr Ferritin Day 3 Sr Ferritin Day 4

(ng/ml)

62

Table 12

FEVER DAYS

Majority of cases had fever for 4 to 5 days. Mean fever day is 4.78.

Fever no.of cases Percentage

3 days 2 2.5

4 days 25 31.25

5 days 43 53.75

6 days 9 11.25

7 days 1 1.25

Total 80 100

Mean fever days 4.78

63

2

25

43

9

12.5

31.25

53.75

11.25

1.250

10

20

30

40

50

60

3 days 4 days 5 days 6 days 7 days

Figure 15 - FEVER DAYS

no.of cases Percentage

64

Table 13

MEAN & SD FOR SERUM FERRITIN ON DAY 3 OF ILLNESS

The mean value of serum ferritin in non severe dengue on day 3 of

illness is 404.43 with standard deviation 102.82. The mean value of serum

ferritin in severe dengue on day 3 of illness is 830.006 with standard

deviation 160.064.

Dengue Mean SD

Non severe 404.43 102.82

Severe 830.006 160.064

65

404.43

102.82

830.006

160.064

0

100

200

300

400

500

600

700

800

900

Mean SD

Figure 16 - Day 3 Serum Ferritin Mean and SD

Non severe Severe

Serum Ferritin (ng/ml)

66

Table 14

MEAN & SD FOR SERUM FERRITIN ON DAY 4 OF ILLNESS

Dengue Mean SD

Non severe 585.905 193.082

Severe 1259.824 192.461

The mean value of serum ferritin in non severe dengue on day 4 of illness

is 585.905 with standard deviation 193.082. The mean value of serum

ferritin in severe dengue on day 4 of illness is 1259.824 with standard

deviation 192.461.

67

585.905

193.082

1259.824

192.461

0

200

400

600

800

1000

1200

1400

Mean SD

Sr. Ferritin

Figure 17 - Day 4 Serum Ferritin Mean and SD

Non severe Severe

Serum Ferritin (ng/ml)

68

Table 15

CLINICAL DIAGNOSIS VS SERUM FERRITIN DAY 3

p value < 0.001 Significant

In severe dengue 12 cases out of 17 cases had serum ferritin levels of more

than 800 which is statistically significant compared to non severe cases

where only 1 had high ferritin levels.

Day 3 Ferritin > 800 (13)

(ng/ml)

Ferritin < 800 (67)

(ng/ml)

Severe dengue 12 5

Non severe dengue 1 62

69

12

5

1

62

0

10

20

30

40

50

60

70

Ferritin > 800 (13) Ferritin < 800 (67)

Figure 18 Clinical Diagnosis vs Serum Ferritin on Day 3

severe dengue non severe dengue

(ng/ml)

70

Table 16

SENSITIVITY & SPECIFICITY OF SERUM FERRITIN DAY 3

Value

Sensitivity 92.31%

Specificity 92.54%

Positive predictive value 70.59%

Negative predictive value 98.41%

Accuracy 92.50%

Over all the sensitivity and specificity of serum ferritin in predicting the

severity on Day 3 is 92.31% and 92.54%

71

Table 17

CLINICAL DIAGNOSIS VS SERUM FERRITIN DAY 4

In severe dengue 14 cases out of 17 cases had serum ferritin levels of more

than 1200 which is statistically significant compared to non severe cases

where only 4 had high ferritin levels.

Ferritin > 1200 (18)

(ng/ml)

Ferritin < 1200 (62)

(ng/ml)

Severe Dengue 14 3

Non severe Dengue 4 59

p value < 0.001 Significant

72

0

10

20

30

40

50

60

Ferritin > 1200 (18) Ferritin < 1200 (62)

14

34

59

Figure 19. Clinical Diagnosis vs Serum Ferritin on Day 4

severe dengue non severe dengue

(ng/ml)

73

Table 18- Sensitivity & Specificity of Serum Ferritin on Day 4

Serum ferritin Value

Sensitivity 77.78%

Specificity 95.16%



Accuracy 91.25%

Over all the sensitivity and specificity of serum ferritin in predicting the

severity on Day 4 is 77.78% and 95.16%.

74

DISCUSSION

Dengue hemorrhagic fever (DHF) and dengue shock syndrome

(DSS) causes more mortality if not diagnosed earlier and treated promptly.

Many studies are available in adult patients to predict the severity but very

few studies are available in pediatric patients to predict the severity.

Our study was done to co-relate the elevation in serum ferritin in

dengue patients during febrile phase with severity of the disease during

critical period. By predicting the severity earlier, we can improve the

outcome and can decrease the mortality.

Among the 80 dengue cases, 43 cases were male children and 37

cases were female children. (Table 5)

In gender predilection, male children were affected mostly (Table

5), however it doesn’t affect the serum ferritin levels.

23 cases belong to the age group less than 5 years and 57 belong to

the age group between 5-12 years. (Table 6)

Duration of fever was counted and the mean febrile period was 4.78

days. (Table 12)

The mean value of serum ferritin on day 3 for non-severe dengue is

404.43 with standard deviation 102.82 and the mean value of serum ferritin

on day 3 for severe dengue is 830.006 with standard deviation 160.064.

(Table 13)

75

The mean value of serum ferritin on day 4 for non-severe dengue is

585.905 with standard deviation 193.082 and the mean value of serum

ferritin on day 4 for severe dengue is 1259.824 with standard deviation

192.461. (Table 14)

We had taken a cut-off value of 800 ng/ml for serum ferritin on day

3 and calculated its significance in predicting severe dengue.

We calculated sensitivity, specificity, positive predictive value,

negative predictive value for serum ferritin on Day 3 with the above

mentioned cut off value for severe dengue.

Value

Sensitivity 92.31%

Specificity 92.54%



Accuracy 92.50%

We had taken a cut-off value of 1200ng/ml for serum ferritin on day

4 and calculated its significance in predicting severe dengue.

76

We calculated sensitivity, specificity, positive predictive value,

negative predictive value, for serum ferritin on day 4 with the above

mentioned cut off value, for severe dengue

The values are

Value

Sensitivity 77.78%

Specificity 95.16%



Accuracy 91.25%

The p value is significant for both day 3 (p<0.001) and day 4

(p<0.001) of serum ferritin with disease severity. (Table 15 & 17)

Serum ferritin is elevated in severe dengue cases compared to the

non severe dengue cases on day 3 and 4.

77

CONCLUSION

Dengue is a serious infection which can range from mild illness to

severe life threatening complications like DSS/DHF. In this period of

dilemma in the clinicians’ front, ferritin was evaluated as an adjunct marker

for the diagnosis of dengue which could possibly aid their clinical

judgement and prompt early fluid resuscitation which in turn could be

useful in avoiding undue complications. Ferritin, as evaluated in the

present study may serve as a significant marker for differentiating between

non severe dengue cases and severe dengue cases.

78

LIMITATIONS

The spectrum of dengue infection is wide and this study only looked

at the hospitalized patients with severe dengue and also at admitted patients

with non severe dengue having warning signs positive.

However, a large set of patients with non severe dengue treated on

an OPD basis are not captured in this study.

A major limitation is that we have conducted the study with a

relatively small number of samples. However this data adds to the existing

data of medical literature and recommends extending our study during

future dengue outbreaks.

BIBLIOGRAPHY

1) Nelson textbook of pediatrics, 1st south Asian edition, vol 2, chapter

269, page 1629; scott B. halstead

2) Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J, et al. (2010)

Dengue: a continuing global threat. Nat Rev Microbiology 8: S7–

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PROFORMA

Name :

Age/ Sex :

Ip / Op no :

Address:

Symptoms:

Fever : yes / no duration :

Nausea and vomiting yes /no duration :

Aches and pain : yes /no

Abdominal pain : yes /no duration :

Persistent vomiting : yes/no duration :

Clinical fluid accumulation :yes/no

Mucousal bleed : yes/no

Lethargy /restlessness : yes/no

Past history :

Birth history :

Developmental history :

Immunization history :

General Examination :

Vitals :

PR : RR: BP:

TEMP : CRT : PERIPHERIES:

Anthropometry :

Height : Weight : MAC :

HC: AG:

Abdomen :

Cardio vascular system :

Respiratory system :

Central nervous system:

Diagnosis : Non severe dengue / Severe dengue

Severe dengue manifestations :

- Shock

- Bleeding

- Fluid Accumulation with respiratory distress

- Organ Involvement

Investigations :

Complete heamogram :

RFT :

LFT :

USG abdomen & pelvis :

IgM dengue / NS1 antigen:

SERUM FERRITIN : Day 3

Day 4

OTHER TESTS:

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




DF - Dengue fever

DHF - Dengue haemorrhagic fever

DIVC - Disseminated intravascular coagulopathy

DSS - Dengue shock syndrome

ECG - Electrocardiogram

ELISA - Enzyme-linked immunosorbent assay

FBC - Full blood count

FFP - Fresh frozen plasma

FWB - Fresh whole blood

GP - General practitioner

Hb - Haemoglobulin

HCO3 - Bicarbonate

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

IBW - Ideal body weight

ICU - Intensive care unit

IgM - Immunoglobulin M

IgG - Immunoglobulin G

IHA - Indirect haemagglutination

INR - International normalized ratio

JVP - Jugular venous pressure

NS1 Ag - Non-structural protein 1 antigen

NSAID - Non-steroidal anti-inflammatory agent

NT - Neutralization test

ORS - Oral rehydration solution

PaCO2 - Partial pressure of carbon dioxide

PCR - Polymerase chain reaction

PEEP - Positive end-expiratory pressure

PICU - Paediatric intensive care unit

PLT - Platelets

PR - Pulse rate

PT - Prothrombin time

PTT - Partial thromboplastin time

RBC - Red blood cell

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

URTI - Upper respiratory tract infection

WBC - White blood cell

WHO - World Health Organization

CONSENT FORM

I hereby give consent to

participate in the study being conducted by Dr.V.MOHANRAJ

postgraduate in the Institute of Child Health & Research Centre, Madurai

medical college, Madurai and to use my personal clinical data and result of

investigations for the purpose of analysis and to study serum ferritin as a

predictor of dengue severity. I also give consent for further investigations.

Place:

Date:

Signature of the parents/guardian

MASTER CHART

S.No Age Sex Fever

Non

Severe

Dengue

Fluid

Accumu

lation With

Respiratory

Distress

Shock Bleeding Organ

Involvement

Severe

Dengue

IgM/ Ns1

Sr

Frt

Day

3

Sr

Frt

Day

4

Fever

Days

1 3 M present Y Y 347 479 5

2 10 M present Y Y 345 467 5

3 6 M present Y Y 297 362 4

4 5 F present Y Y 295 342 4

5 11 F present Y Y 484 686 3

6 6 M present Y Y 394 664 4

7 7 M present Y Y 454 578 5

8 8 F present Y Y 444 576 4

9 3.5 F present N CNS Y Y 922 1294 5

10 11 M present N Y Y Y 826 1222 4

11 6 M present Y Y 332 512 4

12 2 M present Y Y 634 1208 5

13 6.6 F present Y Y 445 578 4

S.No Age Sex Fever

Non

Severe

Dengue

Fluid

Accumu

lation With

Respiratory

Distress


Involvement

Severe

Dengue

IgM/ Ns1

Sr

Frt

Day

3

Sr

Frt

Day

4

Fever

Days

14 7 M present Y Y 432 543 5

15 1 M present Y Y 342 532 6

16 8.8 M present N Y Y Y 832 1375 5

17 9 F present N LIVER Y Y 856 1278 6

18 10 F present Y Y 312 567 4

19 2.5 M present Y Y 367 578 3

20 4.5 F present Y Y 324 546 4

21 6 M present Y Y 635 1224 5

22 7.5 F present N Y Y Y 602 952 6

23 8 M present Y Y 487 678 5

24 5 F present Y Y 356 487 5

25 11 F present Y Y 312 432 6

26 10 M present Y Y 423 567 5

27 4 M present Y Y 478 555 5

28 4 M present N Y Y Y 760 941 5

S.No Age Sex Fever

Non

Severe

Dengue

Fluid

Accumu

lation With

Respiratory

Distress


Involvement

Severe

Dengue

IgM/ Ns1

Sr

Frt

Day

3

Sr

Frt

Day

4

Fever

Days

29 11 F present N Y Y Y 937 1331 5

30 10 F present Y Y 327 634 4

31 10 F present Y Y 317 465 5

32 3 M present Y Y 376 453 4

33 5.5 M present Y y Y Y 389 778 7

34 6 F present Y Y 512 760 4

35 2.5 F present Y Y 498 512 5

36 2 M present N Y Y 846 1294 5

37 7 F present N Y Y Y 878 1432 6

38 8 F present Y Y 447 623 5

39 7 M present Y Y 371 557 5

40 1.5 F present N Y Y Y 936 1390 4

41 8 F present Y Y 290 444 6

42 7 M present Y Y 333 523 5

43 6 M present Y Y 344 543 5

S.No Age Sex Fever

Non

Severe

Dengue

Fluid

Accumu

lation With

Respiratory

Distress


Involvement

Severe

Dengue

IgM/ Ns1

Sr

Frt

Day

3

Sr

Frt

Day

4

Fever

Days

44 11 M present Y Y 421 624 4

45 3 F present Y Y 465 613 5

46 11 F present Y Y 324 467 5

47 10 M present N Y Y Y 1015 1442 5

48 9 M present N Y Y Y 789 1378 4

49 9 F present Y Y 354 512 5

50 5 F present Y Y 355 423 4

51 9 F present Y Y 356 478 4

52 9 F present Y Y 323 534 4

53 8 M present Y Y 444 578 6

54 4 F present Y Y 412 632 5

55 6 M present Y Y 423 676 5

56 7 M present Y Y 434 569 5

57 6.5 F present Y Y 323 555 4

58 4.5 F present N Y Y Y 812 1267 5

S.No Age Sex Fever

Non

Severe

Dengue

Fluid

Accumu

lation With

Respiratory

Distress


Involvement

Severe

Dengue

IgM/ Ns1

Sr

Frt

Day

3

Sr

Frt

Day

4

Fever

Days

59 8 M present Y Y 640 1234 5

60 8.5 M present N Y Y Y 1053 1455 5

61 3 F present Y Y 411 534 5

62 9 F present Y Y 441 656 4

63 11 M present Y Y 625 790 5

64 5.5 M present Y Y 376 443 4

65 6 M present Y Y 336 478 5

66 8 M present N Y Y Y 883 1234 4

67 2 M present N Y Y Y 942 1356 5

68 7 F present Y Y 337 576 4

69 7 M present Y Y 332 498 5

70 4 F present Y Y 331 555 5

71 7 M present Y Y 443 598 5

72 8 F present Y Y 445 600 5

73 6 M present Y Y 299 378 4

S.No Age Sex Fever

Non

Severe

Dengue

Fluid

Accumu

lation With

Respiratory

Distress


Involvement

Severe

Dengue

IgM/ Ns1

Sr

Frt

Day

3

Sr

Frt

Day

4

Fever

Days

74 4 M present Y Y 298 398 4

75 9 F present N Y Y Y 678 1292 5

76 10 F present Y Y 465 579 5

77 11 M present Y Y 441 578 6

78 10 F present Y Y 326 489 6

79 3 M present Y Y 348 442 5

80 10 M present Y Y 351 456 5

Documents

SERUM FERRITIN AS AN EARLY INDICATOR OF SEVERITY OF DENGUE …