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Chapter - 1

INTRODUCTION

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1.1 Dengue Virus Structure and types of infection caused by the virus 2

1.2 WHO Classification of Dengue 4

1.3 Laboratory Diagnosis of Dengue 5

1.4 Aims and Objectives 9

1.5 Presentation of the thesis 10

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INTRODUCTION

1.1 Dengue Virus Structure and types of infection caused by the virus

Dengue virus (DV) is a mosquito-borne flavivirus and the most prevalent arbovirus (Gubler,

etal.,1997) affecting the tropical and subtropical regions of the world, mostly in urban and

semi-urban areas. Dengue is transmitted mainly by Aedes aegypti mosquito and also by Aedes

albopictus.

The global incidence of dengue has grown dramatically in recent decades and as per WHO

about half of the world's population is now at risk of acquiring Dengue. (WHO, Fact sheet,

March, 2014).

Dengue viruses (DENV) belong to the genus Flavivirus of the family Flaviviridae and there

are four serotypes of the virus referred to as DENV-1, DENV-2, DENV-3 and DENV-4.

DENV is a positive-stranded encapsulated RNA virus and is composed of three structural

protein genes, which encode the nucleocapsid or core (C) protein, a membrane-associated

(M) protein, an enveloped (E) glycoprotein and seven non-structural (NS) proteins; NS1,

NS2a, NS2b, NS3, NS4a, NS4b, NS5 (Henchal and Putnak, 1990). Based on the antigenic

differences of the E protein, DENV can be subgrouped into four different serotypes: DENV

1, 2, 3 and 4 (Guzmanet al., 2010).

Marked genetic variation is observed among different serotypes of dengue virus. Despite this

genetic variation the serotypes are more genetically stable than many other RNA viruses,

with an estimated 1 nucleotide change per year (Dunham et al., 2007). Based on intra-

serotype genetic variation DENV are classified in to various genotypes. Reports have been

documented that certain genotypes are more virulent and has potential to cause more fatal

epidemics (Twiddy et al., 2002). All four serotypes can cause the full spectrum of disease

from a subclinical infection to a mild self limiting disease, the dengue fever (DF) and a

severe disease that may be fatal, the dengue haemorrhagic fever/dengue shock syndrome

(DHF/DSS).

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Severe dengue is a leading cause of serious illness and death among children in some Asian

and Latin American countries. Recovery from infection by one serotype provides lifelong

immunity against that particular serotype. However, cross-immunity to the other serotypes

after recovery is only partial and temporary. Subsequent infections by other serotypes

increase the risk of developing severe dengue. DHF or severe dengue usually affects children

younger than 15 years, although it can occur in adults (Gubler et al., 1998). DHF is

characterized by a transient increase in vascular permeability resulting in plasma leakage,

with high fever, bleeding, thrombocytopenia and haemoconcentration, which can lead to

shock termed dengue shock syndrome (DSS) (Guzman et al., 2002). However, it can be

difficult to differentiate DHF from DF and other viral diseases, e.g. typhoid fever,

particularly during the acute phase of the illness (Gubler et al., 1998).

Secondary dengue infection is considered to be the principal risk factor for DHF, but the

interaction of virus, host and epidemiological risk factors are determinants of the occurrence

of DHF epidemics. There is no specific treatment for dengue/severe dengue, but early

detection and access to proper medical care lowers fatality rates below 1%.

Flavivirus NS1 is a relatively conserved glycoprotein with a molecular weight of 46–55 kDa,

depending on its glycosylation status, which exists in different forms at different cellular

locations (Muller et al., 2013). Immature NS1 exists as a monomer in the endoplasmic

reticulum, and it is processed into a stable homodimer that can be covalently linked to the

surface membrane via a glycosyl-phosphatidylinositol anchor (Jacobs et al., 2000). Mature

DENV NS1 contains 352 amino acid residues with two N-linked glycosylation sites at

residues 130 and 207. NS1 antigen circulates in dengue patients from the first day after the

onset of fever up to day 9, when the clinical phase of the disease is over (Alcon et al., 2002).

Therefore, DENV NS1 antigen detection has been successfully used for the early diagnosis of

DENV infection (Young et al., 2000; Peeling et al., 2010). Confirmation of dengue infection

may be possible during the acute phase by testing the serum for presence of the non-structural

protein (NS1) antigen (Chuansumrit et al., 2008).

1.2 WHO Classification of Dengue

The 2009 WHO criteria classify dengue according to levels of severity: dengue without

warning signs; dengue with warning signs (abdominal pain, persistent vomiting, fluid

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accumulation, mucosal bleeding, lethargy, liver enlargement, increasing haematocrit with

decreasing platelets); and severe dengue (dengue with severe plasma leakage, severe bleeding

or organ failure). Patients who recover following defervescence are considered to have non-

severe dengue, but those who deteriorate tend to manifest warning signs. These individuals

are likely to recover with intravenous rehydration. However, further deterioration is classified

as severe dengue, though recovery is possible if appropriate and timely treatment is given.

(WHO, 2009).

Figure 1.1. 2009 WHO criteria for classification of dengue according to levels of severity.

In 2011 WHO classification and guidelines for dengue was revised and dengue was divided

into DF, DHF without shock or with shock (DSS) and expanded dengue syndrome.

According to the severity DHF is divided in to 4 grades: DHF grade I, DHF grade II – DHF,

DHF grade III and DHF grade IV – DSS (WHO, SEARO., 2011). Expanded dengue

syndrome is a new entity added to the classification system to incorporate a wide spectrum of

unusual manifestations of dengue infection affecting various organ systems including

gastrointestinal, hepatic, neurological (Solomon et al., 2000), pulmonary and renal systems.

Unusual manifestations of patients with severe organ involvement such as liver, kidneys,

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brain or heart-associated with dengue infection have been increasingly reported in DHF and

also in dengue patients who do not have evidence of plasma leakage. These unusual

manifestations may be associated with coinfections, comorbidities or complications of

prolonged shock. Exhaustive investigations should be done in these cases. Most DHF patients

who have unusual manifestations are the result of prolonged shock with organ failure or

patients with comorbidities or coinfections (WHO, SEARO., 2011). Very few studies have

documented the unusual and rare manifestations of dengue, especially from South India.

In view of the high mortality rate associated with the infection and to reduce the disease

burden, it is imperative to diagnose the infection early in the course using a sensitive

laboratory assay.

Diagnosis of dengue virus infection on the basis of clinical syndromes is not reliable, and the

diagnosis should be confirmed by laboratory studies, because more than half of infected

individuals either are asymptomatic or have a mild undifferentiated fever (Burke et al., 1988;

Endy etal., 2002). No effective vaccine has been developed against DENV infection and

attempt to eradicate Aedes in endemic area is not yet successful. Therefore, there is an urgent

need for the rapid detection and differentiation of DENV infection in the acute phase of

illness in order to provide timely clinical treatment, etiologic investigation and control of

epidemics in future.

1.3 Laboratory Diagnosis of Dengue

The laboratory diagnosis of dengue is based on the stage of the infection.

1. Serology for

- NS 1 Antigen

- Anti-dengue immunoglobulin M (IgM) antibodies, which are produced 3 to 5 days

after the onset of the disease (Bundo et al., 1985; Innis et al., 1989). (Figure 1.2).

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Figure 1.2. Diagnostic markers of dengue based on stage of illness

Unfortunately, anti-dengue IgG and IgM antibodies in human sera cross-react with all four

dengue virus serotypes (Nawa et al., 2000) and even with other flaviviruses. The detection of

dengue specific secretory NS1 (non-structural protein 1), a highly conserved glycoprotein

represents a new approach to the diagnosis of acute DV infection, in recent times. NS1 is one

of the seven Dengue Virus non-structural proteins which are thought to be involved in viral

replication. NS1 exists as a monomer in its immature form but is rapidly processed in the

endoplasmic reticulum to form a stable dimer. A small amount of NS1 remains associated

with intracellular organelles where it is thought to be involved in viral replication. The rest of

NS1 is found either associated with the plasma membrane or secreted as a soluble hexadimer.

NS1 is essential for viral viability but its precise biological function is unknown. Antibodies

raised in response to NS1 in viral infection can cross react with cell surface antigens on

epithelial cells and platelets and this has been implicated in the development of Dengue

hemorrhagic fever.

Enzyme-linked immunosorbent assay (ELISA) directed against NS1 antigen (NS1 Ag) have

demonstrated its presence at high concentrations in the sera of DV infected patients during

the early clinical phase of the disease (Dussart et al., 2006). Assays based on the detection of

nonstructural protein 1 (NS1) tend to be specific for DENV infection, but the sensitivity of

these assays ranges widely in published reports, from 24–93%, and varies based on the

specific assay used and the infecting serotype (Tricou etal., 2010; Guzman etal., 2010) NS1

assays also show a roughly 20% decrease in sensitivity in the setting of secondary infection

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compared to primary infection (Tricou et al., 2010; Chaterji et al., 2011). NS1 antigen levels

correlate well with viremia and it circulates at high levels during first few days of illness

especially in patients with DHF. NS1 antigen remains circulating in patient’s blood for longer

period than does viral RNA and is reported to be detectable even up to 14th day of illness

(Huhtamo et al., 2010; Dongmei et al., 2011).

Among the other available methods, virus isolation followed by type-specific monoclonal

antibody immunofluorescence staining, the neutralization test, and RT-PCR are widely used

by many laboratories studying dengue virus (Lanciotti et al., 1992; Russell et al., 1967;

WHO Dengue., 1997).

Nucleic acid detection tests for dengue diagnosis were first developed over 20 years ago and

have been generally used for research, but not validated for diagnostic use globally.

Although the most effective method to diagnose dengue in the acute phase of the illness

recommended by the WHO (WHO dengue guidelines 2009) is detection of DENV RNA,

widespread use of dengue molecular diagnostics has been hampered by lack of validated

tests and testing capability, perceptions that molecular diagnostics are cost prohibitive

compared to immunoassays and lack of recommendations for their use.

Molecular diagnosis based on reverse transcription (RT)-PCR, 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 methods as the new standard for the detection of dengue

virus in acute-phase serum samples.

Serotype differentiation has been possible only by testing immune responses by

neutralization tests or type-specific reverse transcription (RT)-PCR (Deubel, et al., 1990;

Laue et al., 1999). Successful dengue virus serotyping based on a real-time one-step RT-PCR

and an NS1 serotype-specific IgG ELISA was reported (Shu, et al., 2002, 2003). Numerous

other RT-PCR assays have been reported in the literature and are used around the world,

including real-time RT-PCR (rRT-PCR) and isothermal amplification techniques (Das et al.,

2008; Harris et al., 1998). However, an international external quality control assessment,

published in 2010, involving 37 laboratories performing 46 tests showed that 80% of these

tests lacked sensitivity, specificity or both (Domingo et al., 2010). Although several

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diagnostic methods are available for dengue diagnosis, proper comparative data on their

performance are lacking.

In this study, we established and compared in house developed single step NS1 serotype-

specific RT PCR assay in the differentiation of dengue virus serotypes with samples collected

from patients with Dengue virus infections. “Single-step” denotes the inclusion of primers in

one reaction tube that accomplishes both reverse transcription and cDNA amplification. The

single-step format was selected to improve workflow and decrease the opportunity for

contamination.This study targets NS1 region of DENV by designing 4 sets of primers for

differentiation of serotypes of dengue.

The first isothermal amplification methods introduced in 1990s included transcription

mediated amplification (TMA), nucleic acid sequence based amplification (NASBA) and

strand displacement amplification (SDA). Loop-mediated isothermal amplification is a novel

method for amplifying DNA and RNA (LAMP and RT-LAMP respectively) with high

specificity, sensitivity, and simplicity (Notomi et al., 2000). The method consists of

incubating a mixture of the target gene, four or six different primers, Bst DNA polymerase,

AMV reverse transcriptase (for RNA), and substrates. It is a one-step, isothermal reaction

with a rapid outcome and no need for expensive equipment. In addition, visual detection is

possible due to an increase in turbidity from the formation of magnesium pyrophosphate, a

by-product of the reaction. This makes the LAMP method an attractive alternative for

traditional PCR which requires dedicated equipment and can be time-consuming. The RT-

LAMP assay is being increasingly used by various investigators for rapid detection and

typing of emerging viruses (Chan et al., 1999; Mori et al., 2001; Parida et al., 2005). These

earlier reports, however, evaluated their RT-LAMP assays for the detection of DENV

infection with a small clinical sample size (<100) and using the C-prM gene (Lu et al.,2012)

or serotype-specific regions of the 3′ untranslated region (UTR) ( Parida et al.,2005; Li et

al.,2011; Sahni et al., 2013). The C-prM gene, however, was relatively less conserved among

all four DENV serotypes (inter-serotype) in comparison to the 3′UTR (Teoh et al., 2013).

However, we have targeted a highly conserved region of NS1, revealing >90% sequence

identity among various genotypes within each serotype. As such genotyping of dengue

serotypes can be performed employing many gene including NS3 and NS5 (Klungthong et

al., 2008).

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In the present study, RT-LAMP assay was developed for the detection and serotyping of

DENV infection targeting the serotype specific regions of the NS1 gene using a real-time

flourometer (Genie® II from Optigene, U.K.). The detection sensitivity and specificity of the

reported DENV NS1 serotype specific RT-LAMP in in freshly obtained dengue-suspected

patient samples in actual clinical laboratory setting of a hospital in dengue endemic

environment and is compared with available test system for suitable algorithm.To the best of

our knowledge this is the first report of detection and differentiation of Dengue using NS1

RT-LAMP with real time fluorometer (Genie® II from Optigene, U.K.) from South India.

This prospective study was also undertaken to document unusual and rare manifestations of

dengue fever in hospitalized patients in Hyderabad, Telengana State of South India, and

compare them with the WHO dengue classification (WHO, SEARO: 2011) .

To the best of our knowledge this is the first NS1 serotype specific RT-PCR and RT-LAMP

assay which can be potentially used for seroepidmiological studies of dengue in the early

stages of infection in dengue endemic regions like India.

1.4 AIMS & Objectives

AIM: The work presented in this thesis is to study Perspectives of NS1 Antigen Detection

for Early Clinical Diagnosis of Dengue. The study is undertaken with the following

objectives:

1) To design, develop, standardize NS1 serotype specific Reverse Transcription Polymerase

Chain Reaction (RT-PCR) for early diagnosis of Dengue in acute phase of illness using

NS1 region of Dengue genome.

2) Comparative evaluation of NS1 antigen detection with antibody detection and

NS1serotype specific RT- PCR.

3) To design, develop, standardize the Dengue serotype specific RT-LAMP (Real Time

Loop Mediated Isothermal Amplification), using NS1 region of Dengue genome and

explore the potential of RT LAMP assay as a point of care testing.

4) To document Unusual and rare manifestations of Dengue in patients suffering with

Dengue fever/Dengue hemorrhagic fever/Dengue shock syndrome.

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1.5 Presentation of thesis

The thesis is presented in 6 chapters followed by references.

Chapter 1- Introduction ; Chapter 2 - Review of literature; Chapter 3 - Materials and

Methods; Chapter 4 - Results; Chapter 5 - Discussion; Chapter 6 - Summary and

Conclusions.

At the end, the references of the literature are arranged in alphabetical order according to

names of the authors, year of publication, full title of the article, Journal title, volume

number, first and last page numbers according to guidelines of American Society of

Microbiologists (ASM). References by same authors are arranged chronologically and if

more than one publication of the author is published in the same year, then they are

distinguished in the text and reference by the letters a, b, c, d, after year of publication.

A list of paper presentations/awards at various conferences and symposiums are given at the

end of the thesis.

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Chapter – 2

REVIEW OF LITERATURE

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2.1 History and origin of Dengue 13

2.2 Global Scenario 15

2.3 South East Asian Scenario 16

2.4 Indian Scenario 18

2.5 Dengue Serotypes 26

2.6 Dengue Virus Genome and Structure 28

2.7 Dengue Virus Replication and Infectious Cycle 35

2.8 Dengue Pathogenesis 36

2.8.1 Pathogenesis of DF/DHF 37

2.9 Transmission 39

2.10 Clinical features of Dengue (WHO Criteria) 42

2.11 Case definition of Dengue (WHO, 2011) 48

2.12 Complications and unusual manifestations of Dengue infections 52

2.13 Differential diagnosis of Dengue infection 55

2.14 Management and treatment of patients with Dengue infection 56

2.15 Approaches used for detection of Dengue 57

2.15.1 Virus Isolation 60

2.15.2 Nucleic acid-based methods 61

(a) Reverse Transcription PCR 61

(b) Real time PCR 64

(c) Transcription Mediated Amplification (TMA) & Nucleic

Acid Sequence-Based Amplification (NASBA) 67

(d) DNA Sequencing 68

2.16 Real-Time Reverse transcription loop-mediated isothermal amplification 69

(RT-LAMP) assay

2.16.1 LAMP cycling 72

2.16.2 Monitoring of amplification by the RT-LAMP assay 76

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2.17 Limitations of available Molecular assay 78

2.17.1 Factors affecting NAAT performance 79

2.18 NS1 antigen Detection 79

2.19 Immunohistochemistry 81

2.20 Serological Methods 81

2.21 Detection of Dengue specific IgG and IgM antibodies 81

2.21.1 IgM-based assays 82

2.21.2 IgG-based assays 85

2.21.3 IgM/IgG ratio 86

2.21.4 IgA 86

2.22 Haemagglutination-inhibition tests 87

2.23 Haematological tests 88

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REVIEW OF LITERATURE

2.1 History and origin of Dengue

The origin of dengue is unclear, but scientists have recently proposed that dengue originated

in Asian forests in an infectious cycle involving mosquitoes and primates. As early as 992, a

dengue-like outbreak in humans was recorded in a Chinese medical encyclopedia. The

earliest known documentation of dengue fever–like illness was in the Chinese Encyclopedia

of Symptoms during the Chin Dynasty (CE 265-420). The illness was called "the water

poison" and was associated with flying insects near water. Epidemics of dengue-like illnesses

were reported in the French West Indies in 1635 and in Panama in 1699.

In 1771, Dr. Jose Sabater, a physician at the military hospital in San Juan, Puerto Rico,

recommended treating dengue with small quantities of rum. At the time, the disease was

called "break-bone fever." As, patients with the disease experienced a high fever

accompanied by such severe bone and joint pains that they felt their bones were breaking.

In 1780, Dr. Benjamin Rush recorded an epidemic of the disease later known as dengue in

Philadelphia, Pennsylvania. Rush described the symptoms of patients with the disease, which

he called "bilious remitting fever." These symptoms include fever, joint and muscle pain,

headache, rash, weakness, nausea, vomiting, and bleeding.

As early as 1801, people called the disease "dengue." The word dengue is Spanish for

"affectation," "careful," or "fastidious." The term probably described the cautious, stiff

movements of patients suffering from the muscle, bone, and joint pain caused by dengue

fever. Some researchers believe that the name came from a Swahili phrase Ka dinga pepo, or

a disease caused by an evil spirit.

In 1818, there was a serious dengue epidemic in Peru - 50,000 people were stricken with the

illness. The first recorded dengue pandemic occurred between 1827 and 1828, and it affected

the Virgin Islands, Jamaica, Cuba, Venezuela, United States, and Mexico. Pandemics are

epidemics that affect a very large region, multiple countries, or even the entire globe. Later in

the 1800s, epidemics plagued Brazil, the southern United States, and the Caribbean. Dengue