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JAMSHEDPUR WOMEN’S COLLEGE

A CONSTITUENT AUTONOMOUS COLLEGE OF


KOLHAN UNIVERSITY

PROJECT WORK
ON
DETECTION OF NS1 ANTIGEN , IGM ANTIBODY,
IGG ANTIBODY IN PATIENTS SUFFERING
FROM DENGUE FEVER BY ELISA TECHNIQUE

Submitted by: Farhat Bano


Subject : Biotechnology
Roll no : 16VBT17259
ACKNOWLEDGEMENT

Project is an integral part of any curriculum and for making my project experience
wonderful I have joined “ MAHATMA GANDHI MEMORIAL MEDICAL
COLLEGE”
I am extremely thankful and pay my gratitude to my guide Dr. Ravinder Singh for
his guidance and useful suggestions which helped me in completing my project
work on time.
I am thankful to Dr. Prashant Kumar Barla, Principal of M.G.M College, for
allowing me to do my project work in the premises M.G.M Medical College,
Jamshedpur.

I am thankful to Dr. N.K. Sinha, Associate Professor of Biochemistry for his


encouragement and support.

I am also grateful to Dr. Sana Khan, Scientist(non-medical) of VRDL, Mrs. N.


Roopa & Mr. Tarun Kar for their constant support.

I am grateful to Dr. Anita Shukla, HOD of Biotechnology Department, Dr.


Vishwaraj Lal, Mrs. Nitya Naina for their valuable help and guidelines. I am
thankful to them for the encouragement that they have given us in completing this
project.

Date: 17.04.2020 Thank You All


Place: Jamshedpur Farhat Bano
B.Sc. Biotechnology

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DECLARATION
I hereby declare that the project report entitled “ DETECTION OF NS1
ANTIGEN, IGM ANTIBODY, IGG ANTIBODY IN PATIENTS SUFFERING
FROM DENGUE FEVER BY ELISA TECHNIQUE" submitted by me in partial
fulfillment of the requirement for the award of the degree of B.Sc in
Biotechnology is a record of bonafide project work carried out by me under the
guidance of Mr. Ravinder Singh , MGM College , Jamshedpur. I further declare
that the work reported in this project has not been submitted and will not be
submitted, either in part or in full, for the award of any other degree or diploma in
this institute or any other institute or university.

Place: Jamshedpur Signature of the candidate:


Date: 17.04.2020 Farhat Bano

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CONTENTS
Topic Page No.
1. Abstract 5
2. Aim & Purpose 6
3. Introduction 7
4. Literature Review 9
• 4.1 History 9
• 4.2 Dengue Virus 10
• 4.3 Transmission 11
• 4.4 Pathogenesis 13
• 4.5 Sign & Symptoms 15
• 4.6 Epidemiology 17
• 4.7 Diagnosis 21
• 4.8 Treatment 36
• 4.9 Prevention & Control 37
5. Conclusion 39
6. References 40

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1. ABSTRACT
Dengue is the most prevalent arthropod-borne virus affecting humans
today. The virus group consists of 4 serotypes that manifest with similar
symptoms. Dengue causes a spectrum of disease, ranging from a mild febrile
illness to a life-threatening dengue hemorrhagic fever. Breeding sites for the
mosquitoes that transmit dengue virus have proliferated, partly because of
population growth and uncontrolled urbanization in tropical and subtropical
countries. Successful vector control programs have also been eliminated, often
because of lack of governmental funding. Dengue viruses have evolved rapidly
as they have spread worldwide, and genotypes associated with increased
virulence have spread across Asia and the Americas. This article describes the
virology, epidemiology, clinical manifestations and outcomes, and
treatments/vaccines associated with dengue infection.

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2. AIM & PURPOSE
Aim: Detection of NS1 Antigen , IGM Antibody and Igg Antibody in
patients suffering from Dengue fever by ELISA TECHNIQUE

Purpose: To detect the dengue virus in patients .Complete


knowledge and prevention of the disease is the main target for
controlling the spread of devastating disease .

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3. INTRODUCTION
Dengue is a mosquito-borne viral disease that has rapidly spread in all
regions of WHO in recent years. Dengue virus is transmitted by female
mosquitoes mainly of the species Aedes aegypti and, to a lesser extent, Aedes
albopictus. These mosquitoes are also vectors of chikungunya, yellow fever
and Zika viruses. Dengue is widespread throughout the tropics, with local
variations in risk influenced by rainfall, temperature, relative humidity and
unplanned rapid urbanization.
Dengue causes a wide spectrum of disease. This can range from subclinical
disease (people may not know they are even infected) to severe flu-like
symptoms in those infected. Although less common, some people develop
severe dengue, which can be any number of complications associated with
severe bleeding, organ impairment and/or plasma leakage. Severe dengue has
a higher risk of death when not managed appropriately. Severe dengue was
first recognized in the 1950s during dengue epidemics in the Philippines and
Thailand. Today, severe dengue affects most Asian and Latin American
countries and has become a leading cause of hospitalization and death among
children and adults in these regions.

Fig1 : Pictorial summary of Dengue fever

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Dengue is caused by a virus of the Flaviviridae family and there are four
distinct, but closely related, serotypes of the virus that cause dengue (DENV-1,
DENV-2, DENV-3 and DENV-4). Recovery from infection is believed to provide
lifelong immunity against that serotype. However, cross-immunity to the
other serotypes after recovery is only partial, and temporary. Subsequent
infections (secondary infection) by other serotypes increase the risk of
developing severe dengue.
Dengue has distinct epidemiological patterns, associated with the four
serotypes of the virus. These can co-circulate within a region, and indeed
many countries are hyper-endemic for all four serotypes. Dengue has an
alarming impact on both human health and the global and national economies.
DENV is frequently transported from one place to another by infected
travellers; when susceptible vectors are present in these new areas, there is
the potential for local transmission to be established.

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4. LITERATURE REVIEW
4.1 HISTORY

The first record of a case of probable dengue fever is in a Chinese


medical encyclopedia from the Jin Dynasty (265–420 AD) which referred to a
"water poison" associated with flying insects. The primary vector, Ae. aegypti,
spread out of Africa in the 15th to 19th centuries due in part to increased
globalization secondary to the slave trade. There have been descriptions of
epidemics in the 17th century, but the most plausible early reports of dengue
epidemics are from 1779 and 1780, when an epidemic swept across Asia,
Africa and North America. From that time until 1940, epidemics were
infrequent.
In 1906, transmission by the Aedes mosquitos was confirmed, and in 1907
dengue was the second disease (after yellow fever) that was shown to be
caused by a virus. Further investigations by John Burton Cleland and Joseph
Franklin Siler completed the basic understanding of dengue transmission.
The marked spread of dengue during and after the Second World War has
been attributed to ecologic disruption. The same trends also led to the spread
of different serotypes of the disease to new areas and the emergence of
dengue hemorrhagic fever. This severe form of the disease was first reported
in the Phillipines in 1953; by the 1970s, it had become a major cause of child
mortality and had emerged in the Pacific and the Americas. Dengue
hemorrhagic fever and dengue shock syndrome were first noted in Central
and South America in 1981, as DENV-2 was contracted by people who had
previously been infected with DENV-1 several years earlier.

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4.2 DENGUE VIRUS

Fig 4.1: A TEM microgrograph showing dengue virus virions (the


cluster of dark dots near the center)

Virus Classification:

Realm Riboviria
Kingdom Orthonavirae
Phylum Kitrinoviricota
Class Flasuviricetes
Order Amarillovirales
Family Flaviviridae
Genus Flavivirus
Species Dengue virus
Table :1

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4.3 TRANSMISSION

Mosquito-to-human transmission:
The virus is transmitted to humans through the bites of infected female
mosquitoes, primarily the Aedes aegypti mosquito. Other species within the
Aedes genus can also act as vectors, but their contribution is secondary to
Aedes aegypti.
After feeding on an DENV-infected person, the virus replicates in the mosquito
midgut, before it disseminates to secondary tissues, including the salivary
glands. The time it takes from ingesting the virus to actual transmission to a
new host is termed the extrinsic incubation period (EIP). The EIP takes about
8-12 days when the ambient temperature is between 25-28°C. Variations in
the extrinsic incubation period are not only influenced by ambient
temperature; a number of factors such as the magnitude of daily temperature
fluctuations, virus genotype, and initial viral concentration can also alter the
time it takes for a mosquito to transmit virus. Once infectious, the mosquito is
capable of transmitting virus for the rest of its life.
Human-to-mosquito transmission:
Mosquitoes can become infected from people who are viremic with DENV.
This can be someone who has a symptomatic dengue infection, someone who
is yet to have a symptomatic infection (they are pre-symptomatic), but also
people who show no signs of illness as well (they are asymptomatic).
Human-to-mosquito transmission can occur up to 2 days before someone
shows symptoms of the illness, up to 2 days after the fever has resolved. Risk
of mosquito infection is positively associated with high viremia and high fever
in the patient; conversely, high levels of DENV-specific antibodies are
associated with a decreased risk of mosquito infection (Nguyen et al 2013
PNAS). Most people are viremic for about 4-5 days, but viremia can last as
long as 12 days.

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Fig 4.2: Transmission of Dengue fever

Other modes of transmission:


The primary mode of transmission of DENV between humans involves
mosquito vectors. There is evidence however, of the possibility of maternal
transmission (from a pregnant mother to her baby). While vertical
transmission rates appear low, with the risk of vertical transmission
seemingly linked to the timing of the dengue infection during the pregnancy.
When a mother does have a DENV infection when she is pregnant, babies may
suffer from pre-term birth, low birthweight, and fetal distress.

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4.4 PATHOGENESIS

Cell and tissue tropism of DENV may have a major impact on the
outcome of DENV infections. The absence of an appropriate animal disease
model largely hampers our understanding of the role played by DENV
tropism. In vitro data and autopsy studies suggest that three organ systems
play an important role in the pathogenesis of DHF/DSS: the immune system,
the liver, and endothelial cell (EC) linings of blood vessels. The tropism of
DENV for cells of the respective systems, the corresponding pathological
effects of DENV infection of these systems, and the relevance of these events
for the overall pathogenesis of DENV infection will be described.

Fig 4.3: Pathogenesis of Dengue

Cells of the immune system - During the feeding of mosquitoes on humans,


DENV is presumably injected into the bloodstream, with spillover in the
epidermis and dermis, resulting in infection of immature Langerhans cells
(epidermal dendritic cells [DC]), and keratinocytes. Infected cells then migrate
from site of infection to lymph nodes, where monocytes and macrophages are
recruited, which become targets of infection. Consequently, infection is
amplified and virus is disseminated through the lymphatic system. As a result
of this primary viremia, several cells of the mononuclear lineage, including
blood-derived monocytes, myeloid DC, and splenic and liver macrophages are

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infected. DENV has also been shown to have tropism for circulating
mononuclear cells in blood and for cells residing in the spleen, lymph nodes,
and bone marrow of infected AG129 mice. Leukocytes also have been shown
to be infected with DENV in experimentally infected nonhumans primates. It
should be noted that during secondary infections with heterologous DENV,
high concentrations of DENV-specific immunoglobulin G (IgG) will complex
newly produced virus that adheres to and is taken up by mononuclear cells.
Organ pathology - Although thousands of patients with confirmed dengue
have been recognized in Southeast Asia and the Americas in the past 60 years,
autopsies have been performed on only a small number of these patients, and
whether those cases are representative in reflecting the viral tropism in the
acute phase of infection is unclear. Histopathological research is difficult to
perform because fatal cases of DHF/DSS are rare and occur mainly in remote
parts of the world where appropriate laboratory technology is largely lacking
and thus fresh or frozen patient materials are rare.
EC - EC play an important role in the coagulation response upon severe
systemic inflammation. The integrity of the EC bed is physiologically regulated
by many factors. The tropism of DENV for EC in vivo remains controversial.
Early studies of skin biopsy specimens indicated that the microvasculature
located in the dermal papillae is the main site affected, although DENV antigen
was not detected in EC but was detected in cells surrounding the
microvasculature.

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4.5 SIGN & SYMPTOMS

Dengue is a severe, flu-like illness that affects infants, young children


and adults, but seldom causes death. Symptoms usually last for 2–7 days, after
an incubation period of 4–10 days after the bite from an infected mosquito.
The World Health Organization classifies dengue into 2 major categories:
dengue (with / without warning signs) and severe dengue. The sub-
classification of dengue with or without warning signs is designed to help
health practitioners triage patients for hospital admission, ensuring close
observation, and to minimise the risk of developing the more severe dengue.

Fig 4.4: Schematic depiction of the symptoms of Dengue fever

Dengue
Dengue should be suspected when a high fever (40°C/104°F) is accompanied
by 2 of the following symptoms during the febrile phase:
• severe headache
• pain behind the eyes
• muscle and joint pains

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• nausea
• vomiting
• swollen glands
• rash
Severe dengue
A patient enters what is called the critical phase normally about 3-7 days after
illness onset. It is at this time, when the fever is dropping (below 38°C/100°F)
in the patient, that warning signs associated with severe dengue can manifest.
Severe dengue is a potentially fatal complication, due to plasma leaking, fluid
accumulation, respiratory distress, severe bleeding, or organ impairment.
Warning signs that doctors should look for include:
• severe abdominal pain
• persistent vomiting
• rapid breathing
• bleeding gums
• fatigue
• restlessness
• blood in vomit
If patients manifest these symptoms during the critical phase, close
observation for the next 24–48 hours is essential so that proper medical care
can be provided, to avoid complications and risk of death.

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4.6 EPIDEMIOLOGY

Dengue is common in more than 120 countries. In 2013 it caused about 60


million symptomatic infections worldwide, with 18% admitted to hospital and
about 13,600 deaths. The worldwide cost of dengue case is estimated US$9
billion. For the decade of the 2000s, 12 countries in Southeast Asia were
estimated to have about 3 million infections and 6,000 deaths annually. In
2019 the Philippines declared a national dengue epidemic due to the deaths
reaching 622 people that year. It is reported in at least 22 countries in Africa;
but is likely present in all of them with 20% of the population at risk. This
makes it one of the most common vector-borne diseases worldwide.
Infections are most commonly acquired in the urban environment. In recent
decades, the expansion of villages, towns and cities in the areas in which it is
common, and the increased mobility of people has increased the number of
epidemics and circulating viruses. Dengue fever, which was once confined to
Southeast Asia, has now spread to Southern China, countries in the Pacific
Ocean and America, and might pose a threat to Europe.
Rates of dengue increased 30 fold between 1960 and 2010. This increase is
believed to be due to a combination of urbanization, population growth,
increased international travel, and global warming. The geographical
distribution is around the equator. Of the 2.5 billion people living in areas
where it is common 70% are from Asia and the Pacific. An infection with
dengue is second only to malaria as a diagnosed cause of fever among
travelers returning from the developing world. It is the most common viral
disease transmitted by arthropods, and has a disease burden estimated at
1,600 disability adjusted life years per million population. The World Health
Organization counts dengue as one of seventeen neglected tropical diseases.

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Fig 4.5: Distribution of Dengue, worldwide, 2016

Like most arboviruses, dengue virus is maintained in nature in cycles that


involve preferred blood-sucking vectors and vertebrate hosts. The viruses are
maintained in the forests of Southeast Asia and Africa by transmission from
female Aedes mosquitos—of species other than Ae. aegypti—to their offspring
and to lower primates. In towns and cities, the virus is primarily transmitted
by the highly domesticated Ae. aegypti. In rural settings the virus is
transmitted to humans by Ae. Aegypti and other species of Aedes such as Ae.
Albopictus. Both these species had expanding ranges in the second half of the
20th century. In all settings the infected lower primates or humans greatly
increase the number of circulating dengue viruses, in a process called
amplification. One projection estimates that climate change, urbanization, and
other factors could result in more than 6 billion people at risk of dengue
infection by 2080.

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Dengue Fever outbreak in India

In the 2006 dengue outbreak in India, cases of dengue fever were reported
first from New Delhi in early September and by the end of September other
states also started to report deaths. At least 3613 confirmed cases of dengue
fever were reported and over 50 people died in the outbreak.
• New Delhi: By early October more than 590 cases of dengue fever were
reported from Delhi and over 367 from neighbouring states who had
come to New Delhi for treatment.
• Rajasthan: By 12 September, more than 35 patients were treated for
dengue fever.
• Chandigarh: 159 cases of dengue fever were reported. These were
reported from the Post-graduate Institute of Medical Education and
Research, the government run multi-specialty hospital. However out of
159 only 29 were from Chandigarh and the remaining were from
Punjab, Harayana and Uttar Pradesh who had come to chandigarh for
treatment.
• Uttar Pradesh: Over 214 suspected cases of the diseases were reported.
• Andhra Pradesh: One person succumbed to the disease and at least five
were treated.
• West Bengal: Over 30 people were treated for dengue fever in Kolkata.
By 9 October 2006 more than fifty deaths were reported to dengue fever and
more than 3613 patients were treated for this disease.

The Government of India's Health Department released the statistical data


related to dengue fever in a press statement on 8 October 2006. Nationwide
data on the dengue outbreak, released by the Ministry of Health:

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State Cases reported
New Delhi 886
Kerela 713
Gujarat 424
Rajasthan 326
West Bengal 314
Tamil Nadu 306
Maharashtra 226
Uttar Pradesh 79
Harayana 65
Karnataka 59
Andhra Pradesh 9

Table : 2 Cases reported in 2006 outbreak of Dengue in India

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4.7 DIAGNOSIS
The diagnosis of dengue is typically made clinically, on the basis of reported
symptoms and physical examination; this applies especially in endemic areas.
However, early disease can be difficult to differentiate from other viral
infections. A probable diagnosis is based on the findings of fever plus two of
the following: nausea and vomiting, rash, generalized pains, low white blood
cell count, positive tourniquet test, or any warning sign in someone who lives
in an endemic area. Warning signs typically occur before the onset of severe
dengue. The tourniquet test, which is particularly useful in settings where no
laboratory investigations are readily available, involves the application of a
blood pressure cuff at between the diastolic and systolic pressure for five
minutes, followed by the counting of any petechial hemorrhages; a higher
number makes a diagnosis of dengue more likely with the cut off being more
than 10 to 20 per 1 inch2 (6.25 cm2).The diagnosis should be considered in
anyone who develops a fever within two weeks of being in the tropics or
subtropics. It can be difficult to distinguish dengue fever and chikungunya, a
similar viral infection that shares many symptoms and occurs in similar parts
of the world to dengue. Often, investigations are performed to exclude other
conditions that cause similar symptoms, such as malaria, leptospirosis, viral
hemorrhagic fever, typhoid fever, meningococcal disease, measles, and
influenza. Zika fever also has similar symptoms as dengue.
The earliest change detectable on laboratory investigations is a low white
blood cell count, which may then be followed by low platelets and metabolic
acidosis. A moderately elevated level of aminotransferase (AST and ALT) from
the liver is commonly associated with low platelets and white blood cells. In
severe disease, plasma leakage results in hemoconcentration (as indicated by
a rising hematocrit) and hypoalbuminemia. Pleural effusions or ascites can be
detected by physical examination when large, but the demonstration of fluid
on ultrasound may assist in the early identification of dengue shock
syndrome. The use of ultrasound is limited by lack of availability in many
settings. Dengue shock syndrome is present if pulse pressure drops to ≤ 20
mm Hg along with peripheral vascular collapse. Peripheral vascular collapse is
determined in children via delayed capillary refill, rapid heart rate, or cold
extremities. While warning signs are an important aspect for early detection
of potential serious disease, the evidence for any specific clinical or laboratory
marker is weak.

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Laboratory Tests
The diagnosis of dengue fever may be confirmed by microbiological
laboratory testing. This can be done by virus isolation in cell cultures, nucleic
acid detection by PCR, viral antigen detection (such as for NS1) or specific
antibodies (serology). Detection of NS1 during the febrile phase of a primary
infection may be greater than 90% sensitive however is only 60–80% in
subsequent infections. All tests may be negative in the early stages of the
disease. PCR and viral antigen detection are more accurate in the first seven
days. In 2012 a PCR test was introduced that can run on equipment used to
diagnose influenza; this is likely to improve access to PCR-based diagnosis.
These laboratory tests are only of diagnostic value during the acute phase of
the illness with the exception of serology. Tests for dengue virus-specific
antibodies, types IgG and IgM, can be useful in confirming a diagnosis in the
later stages of the infection. Both IgG and IgM are produced after 5–7 days.
The highest levels (titres) of IgM are detected following a primary infection,
but IgM is also produced in reinfection. IgM becomes undetectable 30–90 days
after a primary infection, but earlier following re-infections. IgG, by contrast,
remains detectable for over 60 years and, in the absence of symptoms, is a
useful indicator of past infection. After a primary infection, IgG reaches peak
levels in the blood after 14–21 days. In subsequent re-infections, levels peak
earlier and the titres are usually higher. Both IgG and IgM provide protective
immunity to the infecting serotype of the virus. In testing for IgG and IgM
antibodies there may be cross-reactivity with other flaviviruses which may
result in a false positive after recent infections or vaccinations with yellow
fever virus or Japanese encephalitis. The detection of IgG alone is not
considered diagnostic unless blood samples are collected 14 days apart and a
greater than fourfold increase in levels of specific IgG is detected. In a person
with symptoms, the detection of IgM is considered diagnostic.

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Elisa Test for Dengue Fever
Although the hemagglutination-inhibition (HI) test has been the standard test
used by the World Health Organization (WHO) for the classification of
serologic response in dengue infections, it is slow, requiring paired specimens.
Furthermore, not all investigators have accepted the classification. Thus, there
is an urgent need to develop a rapid test which employs a single specimen. We
developed an enzyme-linked immunosorbent assay (ELISA) for rapid
classification of serologic responses in dengue infections based on the ratio of
IgM and IgG in a single specimen. Using the criteria established by the WHO
(1986) for comparison, concordant results were obtained in 81% and 95% of
primary and secondary infections, respectively, when serum specimens were
tested as pairs. When tested as single specimens, the diagnoses by ELISA and
HI agreed in 41% and 52% of acute specimens of primary and secondary
infections, respectively. The lower rate of concordance in acute-phase samples
was due to the absence of detectable IgM in acute specimens collected at
outpatient clinics. On the other hand, diagnoses by ELISA and HI agreed in
79% and 95% of primary and secondary infections when single convalescent
specimens were used. Analysis of the discordant results between the two tests
revealed that the interpretation by the IgM-IgG ratio agreed better with HI
classifications practised by some investigators than it did with the WHO.

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Principle of the assay
The qualitative immunoenzymatic determination of specific antibodies is
based on the ELISA (Enzyme-linked Immunosorbent Assay) technique.
Microplates are coated with specific antigens to bind corresponding
antibodies of the sample. After washing the wells to remove all unbound
sample material a horseradish peroxidase (HRP) labelled conjugate is added.
This conjugate binds to the captured antibodies. In a second washing step
unbound conjugate is removed. The immune complex formed by the bound
conjugate is visualized by adding Tetramethylbenzidine (TMB) substrate
which gives a blue reaction product.
The intensity of this product is proportional to the amount of specific
antibodies in the sample. Sulphuric acid is added to stop the reaction. This
produces a yellow endpoint colour. Absorbance at 450/620 nm is read using
an ELISA microwell plate reader.
Test Precautions
The test procedure, the information, the precautions and warnings in the
instructions for use have to be strictly followed. The use of the test kits with
analyzers and similar equipment has to be validated. Any change in design,
composition and test procedure as well as for any use in combination with
other products not approved by the manufacturer is the responsibility of the
user.
• Only for in-vitro diagnostic use.
• All materials of human or animal origin should be regarded and handled
as potentially infectious.
• All components of human origin used for the production of these
reagents have been tested for anti-HIV antibodies, anti-HCV antibodies
and HBsAg and have been found to be non-reactive.
• Do not interchange reagents or strips of different production lots.
• No reagents of other manufacturers should be used along with reagents
of this test kit.
• Do not use reagents after expiry date stated on the label.
• Use only clean pipette tips, dispensers, and lab ware.

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• Do not interchange screw caps of reagent vials, to avoid cross-
contamination.
• Close reagent vials tightly immediately after use to avoid evaporation
and microbial contamination.
• After first opening and subsequent storage check conjugate and
standard/control vials for microbial contamination prior to further use.
• To avoid cross-contamination and falsely elevated results pipette
samples and dispense reagents without splashing, accurately into the
wells.
• The ELISA is only designed for qualified personnel who are familiar
with good laboratory practice.
• Bacterial contamination or repeated freeze-thaw cycles of the sample
may affect the absorbance values.

Reagents and Materials

Reagent Component Volume / Qty


MTP Antigen coated 8-well snap-off strips 12 x 8 well strip
AG LYO Dengue Virus Antigen, lyophilized 6 x 2 ml
CONJ Enzyme Conjugate 1 x 15 mL
CON- Negative Control 1 x 2 mL
CON+ Positive Control 1 x 2 mL
CUT OFF Cut-off Control 1 x 3 mL
DIL Sample Diluent 1 x 80 mL
SOLN STOP Stop Solution 1 x 15 mL
SUB TMB TMB Substrate solution 1 x 15 mL
Table - 3

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Storage and Stability

Store kit components at 2-8oC and do not use after the expiry date on the box
outer label. Before use, all components should be allowed to warm up to
ambient temperature (20-25°C) and mixed thoroughly. After use, the plate
should be resealed, the bottle caps replaced and tightened and the kit stored
at 2-8°C. The opened reagents are stable up to the expiry date stated on the
label when stored at 2-8°C.
Dengue Virus Coated Microplate (IgM): 12 break-apart 8-well snap-off strips
coated with anti-human IgM-class antibodies; in resealable aluminium foil.
Sample Diluent: 1 bottle containing 80 ml of phosphate buffer (10 mM) for
sample dilution; pH 7.2 ± 0.2; coloured yellow; ready to use; white cap.
Stop Solution: 1 bottle containing 15 ml sulphuric acid, 0.2 mol/l; ready to use;
red cap.
Washing Buffer (20x conc.): 1 bottle containing 50 ml of a 20-fold concentrated
phosphate buffer (0.2 M), pH 7.2 ± 0.2, for washing the wells; white cap.
Dengue Virus Antigen, lyophilized: 6 bottles containing lyophilized Dengue
virus antigen; red cap.
Dengue Virus Conjugate: 1 bottle containing 15 ml of peroxidase labelled
antibody to Dengue virus; coloured red; black cap.
TMB Substrate Solution: 1 bottle containing 15 ml 3,3’,5,5’-
tetramethylbenzidine (TMB), < 0.1 %; ready to use; yellow cap; < 5 % NMP
(N-Methyl-2-pyrrolidone)
Note: NMP may damage an unborn child. Wear protective gloves and eye
protection when handling TMB Substrate Solution. Get medical advice in case of
exposure.
Dengue Virus IgM Positive Control: 1 vial containing 2 ml control (human
serum or plasma); coloured yellow; ready to use; red cap.
Dengue Virus IgM Cut-off Control: 1 vial containing 3 ml control (human serum
or plasma); coloured yellow; ready to use; green cap.
Dengue Virus IgM Negative Control: 1 vial containing 2 ml control (human
serum or plasma); coloured yellow; ready to use; blue cap.
For potential hazardous substances please check the safety data sheet.

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Materials required but not provided

• ELISA microwell plate reader, equipped for the measurement of


absorbance at 450/620 nm
• Incubator 37°C
• Manual or automatic equipment for rinsing wells
• Pipettes to deliver volumes between 10 and 1000 µl
• Vortex tube mixer
• Distilled water
• Disposable tubes

Incubator 37°C Vortex Shaker

Disposal Pipettes Wash Buffer 1X

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Samples Testing
Use human serum or plasma (citrate, heparin) samples with this assay. For
CSF please use the instruction for use ABVL0001. If the assay is performed
within 5 days after sample collection, the samples should be kept at 2-8°C;
otherwise they should be aliquoted and stored at -20°C or -70°C. If samples
are stored frozen, mix thawed samples well before testing. Avoid repeated
freezing and thawing. Heat inactivation of samples is not recommended.
Sample DilutionBefore assaying, all samples should be diluted 1+100 with IgM
Sample Diluent. Dispense 10 µl sample and 1 ml IgM Sample Diluent into
tubes to obtain a 1+100 dilution and mix thoroughly with a vortex.
Assay Steps
Please read the instructions for use carefully before performing the assay.
Reliable results depend on strict adherence to the instructions for use, as
described. The following test procedure is only validated for manual
procedures. If performing the test on automatic ELISA systems we
recommend increasing the washing steps from three to five and the volume of
Washing Buffer from 300 µl to 350 µl, to avoid washing effects. Prior to
commencing the assay, the distribution and identification plan for all samples
and standards/controls (duplicates recommended) should be carefully
established on the plate layout supplied in the kit. Select the required number
of microtiter strips or wells and insert them into the holder.

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Assay Procedure for NS1 Antigen:

Preparation of Reagents for NS1 Antigen:


It is very important to bring all reagents and samples to room temperature
(20-25 °C) and mix them thoroughly before starting.
Coated Microplate: The break-apart snap-off strips are coated with Dengue
Virus antigens. Immediately after removal of the strips, the remaining strips
should be resealed in the aluminium foil along with the desiccant supplied and
stored at 2-8 °C.
Washing Buffer (1x conc.): Dilute 10X wash buffer to 1X using biological or
high grade water .To prepare a 1X wash buffer solution ,mix 120 mL 10X wash
buffer with 1080mL distilled water mix to ensure that any precipitate is
dissolved and the solution is uniform.
TMB Substrate Solution: The reagent is ready to use and has to be stored at 2-8
°C, away from the light. The solution should be colourless or could have a
slight blue tinge. If the substrate turns into blue, it may have become
contaminated and should be thrown away.
Dengue Virus Antigen: The bottles contain lyophilized Dengue virus antigen.
The content of each vial has to be resolved in 2 ml Conjugate Solution by
turning it slowly (do not vortex) and 15 min incubation at room temperature
(20-25°C). This Antigen Conjugate Solution is stable for 3 hours at room
temperature (20-25°C).

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Procedure for NS1 antigen test:
• Perform all assay steps in the order given and without any delays.
• A clean, disposable tip should be used for dispensing each
standard/control and sample.
• Adjust the incubator to 37 ± 1 °C.
Now,
1. Positive , negative and cut off controls should be assayed in duplicate.
Unknown serum samples may be tested in singlet.
2. Using a pipettor , aliquot 50 µl of sample dilution buffer into each of the
required wells.
3. Add 50 µl of each undiluted sera (test samples and control samples)
directly to the center of the wells containing the sample dilution buffers.
Use a clean , disposable pipette tip for each control and test samples.
Gently rock the plate by hand from side to side 5 times to ensure the
samples are mixed well.
4. Cover wells with the foil supplied in the kit.
5. Incubate for 1 hour ± 5 min at 37 ± 1 °C.
6. When incubation has been completed, remove the foil, aspirate the
content of the wells and wash each well three times with 300 µl of 1X
Washing Buffer. Avoid overflows from the reaction wells. The interval
between washing and aspiration should be >5 sec. At the end carefully
remove remaining fluid by tapping strips on tissue paper prior to the
next step. Note: Washing is important! Insufficient washing results in
poor precision and false results.
7. Prepare the conjugate solution (120 µl of 100X conjugate:12mL of
conjugate diluent ) and dispense 100 µl per well of this Antigen
Conjugate Solution into all wells except for the Substrate Blank well A1.
8. Incubate for 30 min at 37 ± 1 °C. Do not expose to direct sunlight.
9. Wash the plate 6 times with 1X wash buffer.
10. Dispense 100 µl TMB Substrate Solution into all wells.
11. Incubate for exactly 15 min at room temperature (20-25 °C) in the dark.
A blue colour occurs due to an enzymatic reaction.
12. Dispense 50 µl Stop Solution into all wells in the same order and at the
same rate as for the TMB Substrate Solution, thereby a colour change
from blue to yellow occurs.

30
13. Measure the absorbance at 450/620 nm within 30 min after addition of
the Stop Solution.
14. Adjust the ELISA microwell plate reader to zero using the Substrate
Blank. If for technical reasons the ELISA microwell plate reader cannot
be adjusted to zero using the Substrate Blank, subtract its absorbance
value from all other absorbance values measured in order to obtain
reliable results.
15. Measure the absorbance of all wells at 450 nm and record the
absorbance value for each standard/control and sample in the plate
layout.
16. Bichromatic measurement using a reference wavelength of 620 nm is
recommended.
17. Where applicable calculate the mean absorbance values of all
duplicates.

31
Assay Procedure for Igm Antibody:

• Perform all assay steps in the order given and without any delays.
• A clean, disposable tip should be used for dispensing each
standard/control and sample.
• Adjust the incubator to 37 ± 1 °C.
NOW
1. Remove required number of strip and fox with strip holder, wash the
strip 3 time with wash buffer.
2. Add 50 µl negative control in A1, add 50 µl positive control in B1
3. Transfer 50 µl of diluted sample (sample + dilution ) into the deep
wells C1 D1 E1……..
4. Cover the plate with aluminium foil , Keep the plate in humidified
box and incubate at 37 °C for 1 hour.
5. At the time end of l hour wash the plate 5 times with wash buffer and
tap the plate after last wash on a tissue paper.
6. Add 50 µl of dengue antibody each well of the plate
7. Cover the plate with aluminium foil , Keep the plate in humidified
box and incubate at 37°C for 1 hour
8. At the time end of 1 hour wash the plate 5 times with wash buffer
and tap the plate after last wash on a tissue paper.
9. Add 50 µl of Avidin-HRP To all each well of the plate.
10.Cover the plate with aluminium foil. Keep the plate in humidified box
and incubate at 37 °C for 30 Minutes
11.At the time end 30 minutes wash the plate 5 times with wash buffer
and tap the plate after last wash on a tissue paper
12. Add 100 µl of Liquid TMB Substrate to cach well
13. Incubate at room Temperature in dark for 10 minutes
14. Add 100 pl of Stop solution exactly after 10 minutes
15.Measure the absorbance at 450nm within 10 minutes after
termination of reaction

32
Assay Procedure for Igg Antibody:

• Perform all assay steps in the order given and without any delays.
• A clean, disposable tip should be used for dispensing each
standard/control and sample.
• Adjust the incubator to 37 ± 1 °C.

Now,
1.Controls and diluted samples are incubated in microtiter wells
coated with monoclonal antibody bound to DENRA

2.After incubation and washing wells are treated with IgG antibody labeled
with HRP.

3. After a second incubation and washing, wells are incubated with TMB
substrate.

4. Acid stop is added and absorbance at 450 nm is measured.

5. Ratio of the absorbencies of the DENRA and the control wells determines
whether a result is positive or negative.

33
Calculation For Dengue

Kit validation
Negative Control OD < 0.18
Positive Control OD > 5x NC
Result
NC OD x 2-COV1 cut off value
NC OD x 5-COV2
Patient OD>COV2 = Positive
Patient OD <COV1 = Negative
COV1 <= patients OD >COV2 = Equivocal

Yellow colour indicates that dengue specific IgM is present

No colour change indicates that dengueNS1 antigen is not present

34
Interpretation of Results
Cut-Off 10 U Results
Positive >11U Antibodies against the pathogen are present.
There has been contact with the antigen.
Equivocal 9–11U Antibodies against the pathogen could not be
detected clearly. It is recommended to repeat the
test with a fresh sample in 2 to 4 weeks. If the
result is equivocal again the sample is judged as
negative.
Negative <9U The sample contains no antibodies against the
pathogen. Exposure to the antigen is unlikely.
Table – 4 Interpretation of the test results
Assay Characteristics
Intra-assay n Mean (E) CV (%)
#1 24 0.530 3.23
#2 24 1.019 2.44
#3 24 0.986 2.75
Inter-assay n Mean (U) CV (%)
#1 12 18.77 6.60
#2 12 8.96 6.16
#3 12 5.32 5.79
Table – 5
Diagnostic Specificity: The diagnostic specificity is defined as the probability of
the assay of scoring negative in the absence of the specific analyte. It is 98.5%
(95% confidence interval: 91.72 – 99.96%).
Diagnostic Sensitivity: The diagnostic sensitivity is defined as the probability
of the assay of scoring positive in the presence of the specific analyte. It is
100% (95% confidence interval: 89.42% - 100.0%).
Interferences: Interferences with hemolytic, lipemic or icteric samples are not
observed up to a concentration of 10 mg/ml hemoglobin, 5 mg/ml
triglycerides and 0.5 mg/ml bilirubin.
Cross Reactivity: Cross reactions with antibodies against West Nile Virus and
with rheumatoid factors cannot be excluded. However, cross reactivity with
other flaviviruses should be considered for result interpretation.

35
4.8 TREATMENT

There is no specific treatment for dengue fever.


Fever reducers and pain killers can be taken to control the symptoms of
muscle aches and pains, and fever.
• The best options to treat these symptoms are acetaminophen or
paracetamol.
• NSAIDs (non-steroidal anti-inflammatory drugs), such as ibuprofen and
aspirin should be avoided. These anti-inflammatory drugs act by
thinning the blood, and in a disease with risk of hemorrhage, blood
thinners may exacerbate the prognosis.
For severe dengue, medical care by physicians and nurses experienced with
the effects and progression of the disease can save lives – decreasing mortality
rates from more than 20% to less than 1%. Maintenance of the patient's body
fluid volume is critical to severe dengue care. Patients with dengue should
seek medical advice upon the appearance of warning signs.
Apart from attempts to control the spread of the Aedes mosquito there are
ongoing efforts to develop antiviral drugs that would be used to treat attacks
of dengue fever and prevent severe complications. Discovery of the structure
of the viral proteins may aid the development of effective drugs. There are
several plausible targets. The first approach is inhibition of the viral RNA-
dependent RNA polymerase (coded by NS5), which copies the viral genetic
material, with nucleoside analogs. Secondly, it may be possible to develop
specific inhibitors of the viral protease (coded by NS3), which splices viral
proteins. Finally, it may be possible to develop entry inhibitors, which stop the
virus entering cells, or inhibitors of the 5′ capping process, which is required
for viral replication.

36
4.9 PREVENTION & CONTROL

If you know you have dengue, avoid getting further mosquito bites during the
first week of illness. Virus may be circulating in the blood during this time, and
therefore you may transmit the virus to new uninfected mosquitoes, who may
in turn infect other people.
The proximity of mosquito vector breeding sites to human habitation is a
significant risk factor for dengue as well as for other diseases that Aedes
mosquito transmit. At present, the main method to control or prevent the
transmission of dengue virus is to combat the mosquito vectors. This is
achieved through:
Prevention of mosquito breeding:
• Preventing mosquitoes from accessing egg-laying habitats by
environmental management and modification;
• Disposing of solid waste properly and removing artificial man-made
habitats that can hold water;
• Covering, emptying and cleaning of domestic water storage containers
on a weekly basis;
• Applying appropriate insecticides to water storage outdoor containers;
Personal protection from mosquito bites:
• Using of personal household protection measures, such as window
screens, repellents, insecticide treated materials, coils and vaporizers.
These measures must be observed during the day both inside and
outside of the home (e.g.: at work/school) because the primary
mosquito vectors bites throughout the day;
• Wearing clothing that minimises skin exposure to mosquitoes is
advised;
Community engagement:
• Educating the community on the risks of mosquito-borne diseases;
• Engaging with the community to improve participation and
mobilization for sustained vector control;
Reactive vector control:
• Emergency vector control measures such as applying insecticides as
space spraying during outbreaks may be used by health authorities;
Active mosquito and virus surveillance:

37
• Active monitoring and surveillance of vector abundance and species
composition should be carried out to determine effectiveness of control
interventions;
• Prospectively monitor prevalence of virus in the mosquito population,
with active screening of sentinel mosquito collections;

Fig 4.8: Prevention & Control measures for dengue

In addition, there is ongoing research amongst many groups of international


collaborators in search of novel tools and innovative strategies that will
contribute in global efforts to interrupt transmission of dengue, as well as
other mosquito-borne diseases. The integration of vector management
approaches is encouraged by WHO to achieve sustainable, effective locally
adapted vector control interventions.

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5. CONCLUSION

Dengue infection is a common mosquito-borne infectious disease that can be


seen in many countries around the world. Since it is still an important global
public health problem, the issue of management of cases remains an
important topic in medicine. This viral infection seems to be difficult to
diagnose, since it can be mimicked by other common infections that share the
same endemic geography. Although there are many new advanced laboratory
investigation tools to help diagnose dengue infection, problems can still be
observed. The issue of error in the laboratory diagnosis of dengue has to be
mentioned, and the general practitioner has to keep in mind the important
points in the diagnosis of dengue.
Dengue has evolved as a global life-threatening public health concern,
affecting around 2.5 billion individuals in more than 100 countries. The
physician should be aware about the varied clinical manifestations of this
condition and ensure an early and adequate treatment plan. Future directions
to combat this dreadful disease aim at methods of mosquito control,
development of vaccine, and antiviral drug regimen. There is no antiviral
therapy or vaccination available for dengue at this time, leaving only early
detection and symptomatic treatment with fluid resuscitation essential for
management of severe cases. The known social, economic, and disease burden
of dengue internationally is alarming and it is evident that the wider impact of
this disease is grossly underestimated.

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