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STATUS OF CHIKUNGUNYA
ANTIBODIES IN SUSPECTED
DENGUE PATIENTS

NAME OF THE CANDIDATE

DEPARTMENT OF MICROBIOLOGY
AND IMMUNOLOGY
SUNNYDALE
DHAKA, BANGLADESH
OCTOBER 2018

I

STATUS OF CHIKUNGUNYA
ANTIBODIES IN SUSPECTED
DENGUE PATIENTS

Dissertation is submitted to the Department of Microbiology and
Immunology, SUNNYDALE (SUNNYDALE) in partial fulfilment of the
requirements for the degree of Bachelor of Science in Microbiology and
Immunology
...
:

SUNNYDALE

II

DECLARATION
This is to confirm that the dissertation entitled on “Status of Chikungunya
Antibodies in Suspected Dengue Patients” conducted by NAME OF THE
CANDIDATE (ID: ) under the supervision of Dr
...
This is an
original research work; the results embodied in this dissertation have not been
included in any other thesis or dissertation for the award of any degree
...
He carried out the research work in the
Department of Immunology, SUNNYDALE
...


Supervisor
Dr
...
Mohammad Moniruzzaman MBBS, MPhil (Immunology)
Associate Professor
Department of Immunology
SUNNYDALE

Dated:
IV

Table of Contents
DECLARATION

III

CERTIFICATE

IV

TABLE OF CONTENTS

V

ACKNOWLEDGEMENTS

VIII

LIST OF ABBREVIATION

X

ABSTRACT

XI

CHAPTER 1

CHAPTER 2

Introduction

1

Rationale

7

Research question

8

Objectives

8

Review of Literature

9

Introduction

10

History and origin of the virus

10

Epidemiology of Chikungunya virus

11

Early outbreaks and Periodic Detections

13

Re-Emergence of CHIKV

14

Clinical Manifestation

15

Dengue virus

19

Origin and Natural History

20

General Description of Clinical Dengue Diseases

22

Febrile Phase - commonly lasts for 3-7 days

22

Recovery phase - unusual onset around 6-8 of illness

25

Dengue with warning signs

26

Differential diagnosis

26

Prevention of Dengue

30
V

Association between Chikungunya and Dengue

31

CHAPTER 3

Materials and Methodology

33

CHAPTER 4

Results

38

CHAPTER 5

Discussion and Conclusion

48

CHAPTER 6

Bibliography

50

CHAPTER 7

Appendix

65

Appendix I

66

Appendix II

71

Appendix III

72

Appendix IV

75

Table I

40

Table II

41

Table III

42

Table IV

43

Table V

44

Table VI

45

Table VII

46

Table VIII

46

Table IX

47

Table X

47

LIST OF TABLES

VI

LIST OF FIGURES
Fig: 1
...


4

Fig: 2
...
2

28

Fig: 2
...


The success and final outcome of this work required a lot of guidance of assistance
from many people and I am extremely privileged to have got this all along the
completion of my report
...
I am deeply indebted and
immeasurably gratified to my supervisor Dr
...
I shall always remain grateful for her continuous
support and valuable advices in accomplishing this work
...
Md
...
I am grateful for his endurance and support during the
preparation of the thesis
...
Zahurul Haque Asna, Head of the department, Department of
Microbiology and also the Dean of Basic Sciences because without his kind
instruction and help it was impossible for me to finish this task at all
...
Salima Akter,
Assistant Professor & Head, Dept
...

VIII

A lot of thanks and gratefulness to Md
...


My sincere gratitude towards all the participants of the study; thank you all for
making this dream a reality
...
Abdullah Al-Aff
Discipline: BSc
...
In Microbiology and Immunology
Session: Fall – 13
ID:

SUNNYDALE
IX

LIST OF ABBREVIATION

SUNNYDALE

SUNNYDALE

CHIKV

Chikungunya Virus

DNA

Deoxyribonucleic Acid

ECSA

East – Central South African

ELISA

Enzyme Linked Immune Sorbent Assay

HIV

Human Immunodeficiency Virus

ICT

Immunochromatographic Test

IFA

Immunoflurescence Assay

PCR

Polymerase Chain Reaction

RNA

Ribonucleic Acid

US

United States

WHO

World Health Organization

WNV

West Nile Virus

X

ABSTRACT

Chikungunya Fever is a mosquito-borne illness of humans caused by the
Chikungunya Virus
...
Aedes aegypti and Aedes
albopictus mosquitoes are the main vectors of chikungunya in Asia and the
Indian Ocean Islands
...
Since
then it has been attributed to many outbreaks in a number of countries
...
Sporadic cases
are regularly reported from different countries in the affected regions
...


Aims and objectives: The study was to evaluate presence of Chikungunya
Antibodies (IgM and IgG) in the patients clinically suspected as dengue
...
Subjects with clinical manifestation of dengue fever
attending at BIHS General Hospital were recruited
...

Blood sample (5 ml) was collected, processed and separated serum used to
determine anti-dengue and anti-chikungunya IgM and IgG antibodies using
standard ICT kit
...
05 taken as level of significance
...
5:47
...
Of the total 120
subjects, 32
...
Educational status of the
study subject was heterogeneous
...


Conclusion: It may be concluded that superimposed chikungunya in patients
suspected of dengue fever as suggested by presence of anti-chikungunya IgG and
IgM in single and in combination in those cases positive for anti-dengue
antibodies has been occurred
...
To make
conclusive comment regarding this issue, the need to be substantiated by
recruiting adequate number of patients and antibody determination supported by
standard method comparison would be helpful
...
CHIKV is transmitted to humans
by the bite of Aedes spp
...
e
...

In Western and Central Africa, CHKV is maintained in a sylvatic cycle involving
non-human primates and forest – dewelling Aedes spp
...
In Asia, the
predominant vector is the urban, peri-domestic, anthropophilic, Aedes aegypti
mosquitoes, which is responsible for large-scale outbreaks characterized by long
inter-epidemic periods that may last several decades
...
Human chikungunya virus have been identified
in the following regions:
• Cambodia
• East Timor
• India
• Indonesia
• Laos
• Malaysia
• Maldives
• Myanmar
• Pakistan
• Philippines
• Reunion
• Seychelles
• Singapore
• Taiwan
• Thailand
• Vietnam
• Bangladesh
14

Evolutionary studies have suggested an African origin for CHIKV
...
In the
last decade, CHIKV has re-emerged, causing a series of large outbreaks,
which started in Kenya in 2004 and ravaged the Comoros Islands, the Island
of La Re Union and other Islands in the Southwest Indian Ocean in early

Fig 1
...

Dengue is the most prevalent and dangerous of the emerging Arbovirus
...
A study done in 2103 estimated that 96 million
clinically significant cases occur annually, a dramatic increase from 50 million
in 2009
...
There are four distinct dengue virus
serotypes, with type 2 considered to be the most virulent strain
...
Dengue is not transmitted via sexual contact or
respiratory droplets
...
2: Dengue Virus

16

About 50% of dengue infections are symptomatic
...
Three distinct phases of symptomatic dengue have
been well described:
• Febrile
• Critical and
• Recovery
...
Serum
specimens collected five or more days after the onset of symptoms should be
evaluated for anti CHIKV and anti DENV IgM antibodies by Immunoassay
...

Chikungunya and dengue are both acute febrile illnesses characterized by fever,
myalgia and lethargy
...
Distinguishing features of chikungunya include
potentially debilitating bilateral polyarthralgia and, in some cases, arthritis
...

Such patients should also be evaluated for other serious conditions, such as
malaria, leptospirosis and other bacterial infections
...
Clinical presentation is thought
to vary by age, however, with the very young (neonates) and the elderly being at
greater risk for more severe disease, co-morbidities (underlying diseases) have
also been identified as a risk factor for poor disease outcome
...
There have been rare reports of spontaneous abortions
17

following CHIKV infection in the mother so far
...
So far,
no such studies have been done in Bangladesh to explore the chikungunya status
of the patients affected with dengue infection
...
However, our physicians
at present, are experienced enough to handle this menace
...

Chikungunya virus is most often spread to people by Aedes aegypti and Aedes
albopictus mosquitoes
...
Signs
and symptoms of both dengue and CHIKV disease are almost similar
...
However, it is utmost important to properly
investigate any viral disease in order to intervene in time to protect the
individuals from severity and complications
...


19

Research Question:
Is there any association between Chikungunya Antibodies with Dengue fever?

Objectives
A
...


B
...
To determine antibodies (IgM and IgG) against Chikungunya virus
in patients who are diagnosed clinically as dengue attending at the
BIHS General Hospital
...
To see any association of Dengue and CHKV antibodies in patients
suffering for fever and Arthralgia
...
Caused by the chikungunya virus, an alphavirus belonging to the
togavirus family, the virus has an incubation period of 3 – 7 days (Powers AM,
Louge CH, 2007)
...
However,
severe forms including deaths, often associated with comorbidities have been
reported in the 2005 – 2006 Indian Ocean Islands Outbreaks (Lemant J et al,
2008)
...
The virus since spread to several parts of Central and Latin
America (Morens DM, Fauci AS (2014)
...

The first combined CHIKV fever outbreak was reported in 1952 in Makonde
Plateau in Tanzania, East Africa, where the virus was first isolated (Cavrini F,
Gaibani P, Pierro AM, Rossini G, Landini MP, Sambri V, 2009)
...
Phylogenetic studies placed representative CHIKV strains from all of
these

sub-Saharan

African

locations

into

one

clade

termed

the

East/Central/South American (ECSA) linage (Burt FJ, Rolph MS, Rulli NE,
Mahalingam S, Heise MT, 2012; Schwartz O, Albert ML, 2010)
...
5 was recognized in many
parts of the sub-Saharan Africa and sporadic outbreaks in Africa and Asia
...

In 1963, India reported the first CHKV outbreaks in Calcutta and Madras
[Pialoux G, Gauzere BA, Jaureguiberry S, Strobel M, 2007; Jupp PG, McIntosh
BM, 1988)
...

The Asian CHIKV linage became extinct in India after 1973 but continued to
circulate in Southeast Asia occasionally detected during small-medium sized
epidemics
...
8 kb RNA genome
...

Recently, in additional site of origin has been identified in Asia
...
It is noteworthy that, this diffusion has been mainly sustained by Aedes
albopictus, a new vector to which the virus become adapted due to the mutation
in E1-Ala226Val gene
...
Three years later, the
virus appeared for the first time in the western hemisphere and since then, in less
than 24 months spread to the North and South America
...
1: Epidemiology of Chikungunya Virus
...
These ranged geographically across
central Africa from Senegal to Uganda south to South Africa, across the Indian
Ocean to India and throughout Southeast Asia
...
Large urban
outbreaks were first reported in Thailand in the late 1950s and early 196s
(Halstead SB, 1996) and the scope of these outbreaks was staggering
...

Clear evidence that the virus had been previously established in the area was
seen in age-dependent A
...
Powers 35 v immune rates
...
Similarly high rates were found in rural Thailand [15] and in Vietnam
after a 1964 outbreak (Burke DS, Nisalak A, Nimmannitya S, 1985) further
supporting the idea of long – term epidemic city in the region
...

India also experienced large urban outbreaks of CHKV in both early 1960s t0
1970s
...
The Madras activity involved an estimated 400,000
individuals in this single region in Southern India where the virus had not
previously been documented (Rao TR, 1996)
...
Interestingly,
involvement of the central nervous system in children was identified in the
25

outbreaks that demonstrate early the ability of CHKV to cause atypical disease
(Padbidri VS, Gnaneswar TT, 1979) as had been reported in outbreaks since
2005
...
Even though these outbreaks in India were sufficient, there was no
reported epidemic activity from the country for the next 30 years
...

During the 1950s to 1970s, a number of smaller scale outbreaks were recorded
in Africa as well
...

The epidemiological pattern was quite distinct from that seen in Asia during the
same period, perhaps most significantly due to the association of enzootic
maintenance of CHKV in Africa with a number of alternative vector species
...
During July, an unusual increase in the number of malaria-like
illness was detected in the island community of Lamu
...
In addition, 91% of the
blood smears were negative for parasites; thus, the increase in cases could not be
accommodated for by malaria
...
The scope of this outbreak was quite large for Eastern Africa with an
estimated 13,500 cases
...

26

Approximately eight months later, an outbreak of febrile illness began on the
island of Comoros just off the coast of Tanzania
...
Because of the recent CHIKV activity in Kenya, testing of samples
for CHKV antibody and additional samples were RT-PCR positive
...

Molecular epidemiology further revealed that the virus originated from the
Lamu/Mombasa outbreaks, demonstrating that the activity in Comoros was
simply an extension of the Kenya outbreaks rather than novel outbreaks (Weaver
SC, Lecuit M, 2015)
...
5 years
(Kariuki Njenga et al, 2008)
...
This pattern would characterize CHIKV outbreaks for the next
decade
...
The acute phase is characterized by the following:
1
...
High fever
3
...
Headache
5
...
Rash and
7
...
al, 2008)
...
The
virus replicates in the mosquito can transmit the virus to another person,
throughout its life, mother-to-child transmission can occur during childbirth
when the mother is viremic (Gallian P, de Lamballerie X, Salaz N, Piorowski G,
Richard P, Paturel L
...
al, 2014)
...


Acute stage
Clinical manifestations and symptoms
In contrast to other arboviral diseases, CHIKV infection is symptomatic in more
than 70% of cases and ranges from 72 to 96% (Burt FJ, Rolph MS, Rulli NE,
Mahalingam S, Heise MT, 2012)
...

In the common presentation, chikungunya is a rapid onset febrile disease with
no prodromal phase (Javelle E, Tiong TH, Leparc – Goffarat I, Savini H, Simon
F, 2014)
...
Other laboratory abnormalities include
moderate thrombocytopenia, increased level of aspartate aminotransferase,
alanine aminotransferase and creatinine phosphokinase and mild increase in C
reactive protein levels (about 50-60 mg/L)
...


In the chronic phase, incapacitating arthralgia persists for months
...
Encephalitis
2
...
Myelopathy or myeloneuropathy

Non-neurological systemic syndromes included renal, hepatic, respiratory,
cardiac and hematological manifestations together with atypical manifestations
including
1
...
Oral ulcers and
3
...

CHIKV is most of symptomatic (⁓ 80% of cases); the symptoms last from a few
days to several years depending on the case
...
The mortality rate of CHIKV is comparable to that of
seasonal influenza (⁓ 0
...
1%) and is mainly related to the patient’s age
(increased over 75 years) and/or to chronic co-morbidities (Simon F, Javelle E,
Cabie A, Bouquillard E, Trisgros O, Gentile G et al, 2015)
...
Some of
the clinical features in children include the following:

29

1
...
e
...
Altered levels of consciousness
3
...
Acute flaccid paralysis
...
Acrocyanosis
2
...
Erythematous asymmetrical morbiliform rashes
...
5% of cases, mainly in vulnerable
patients (young children, elderly patients, chronic alcohol abuses, patients
presenting with chronic medical conditions including systemic lupus (Betancur
J-F, Navarro EP, Bravo Bonilla JH, Cortes AD, Velez JD, Echeverry A et al
...
Atypical presentations include –
• Hyperalgesia symptoms
• Gastrointestinal symptoms e
...
diarrhea, vomiting, abdominal pain
• Neurological symptoms e
...
confusion, optic neuritis
• Damage to mucous membranes i
...
oral or genital ulceration,
conjunctivitis
• Malaise i
...
hypotension, dysautonomia

CHIKV can directly include severe atypical presentations (rhabdomyolysis,
bullous darmatosis, fulminant hepatitis, encephalitis or encephalopathy,
Guillain-Barre syndrome, polyneuropathy, myocarditis, multiple organ failure

30

(Ramful D, Carbonnier M, Pasquet M, Bouhmani B, Ghazouani J,
Noormahomed T, Beullier G, Attali T, Sameriz S, Fourmaintraux A, Alessandri
JL, 2007)
...
Vertical
transmission from mother to child has been documented
...
The mortality rate of severe presentations is 50% and
the risk of post encephalopathy psychomotor sequel is important (Lindencach,
B
...
, Theil, H
...
and Rice, C
...
, 2007)
...
J
...
and Markoff L
...
They are:
• DENV – 1
• DENV – 2
• DENV – 3 and
• DENV – 4
All of these can cause mild to severe and fatal disease in humans (Hirsch, A
...
A proposed new serotype (DENV – 5) recently been described from
31

Malaysia (Nikolas Vasilakis, personal communication, October 2013)
...
At this time, the public health
implications of this new serotype are uncertain
...

All four original serotypes however, have similar natural histories including an
enzootic cycle involving non-human primates and canopy dwelling mosquitoes
in Asia and an urban cycle involving humans as the primary vertebrate host and
Aedes mosquitoes of the subgenus Stegomyias, the primary mosquito vector
globally in the topics
...


Origin and Natural History
The origin and evolution of the dengue viruses has been the subject of much
discussion in recent years
...
E
...
,
1956)
...
M
...
J
...
B
...
J
...

The dengue viruses belong to the genus Flavivirus are branched into four
subgroups:
i
...


The vertebrate viruses that have no known arthropod vector
...


The mosquito – borne viruses and

iv
...


32

It is still uncertain which Flavivirus group is the oldest
...
B
...
T
...
R
...
It
seems plausible that the ancestral Flavivirus was a mosquito or tick virus that
diverged by adapting to a variety of vertebrate hosts, including rodents, birds,
bats and non-human primates
...

That the tick-borne and mosquito-borne viruses had a common ancestor is
supported by the fact that several mosquito-borne Flaviviruses (Koutango,
Saboya, West Nile and Yellow Fever) have all been isolated from ticks (Clifford,
C
...
, Yunker, C
...
, Thomas, I
...
, Easton, E
...
and Corwin D
...
Also, it
has been reported that some tick-borne viruses replicate in mosquitoes or
mosquito cell cultures (Kuno, G
...

It is not known whether the divergence of the four Flavivirus subgroups occurred
in Africa, Asia or in both areas
...
The Asian
origin of dengue viruses is supported by both ecological and phylogenetic
evidence (Rudnick A
...
Thus, all four dengue serotypes have been
documented is a sylvatic cycle involving non-human primates and arboreal
mosquitoes in the Malay Peninsula (Cornet, M
...


33

General Description of Clinical Dengue Disease

Timing, evolution and notable features of symptomatic dengue
Following an infectious mosquito bite, there is an incubation period of up to 2
weeks (commonly 5 – 7 days), after which the individual develops symptoms
and the illness typically follows three phases – An initial phase, A critical phase
starting around 4 – 5 days from fever onset when complications may develop,
followed by a spontaneous recovery phase (Phung C
...
, Nhan, N
...
Kneen, R
...
T
...
Thein, C
...
Nega, N
...
T Thut, T
...
Solomon, T
...

Will B
...
In order to ensure a good outcome
for this dynamic and multifaceted disease, it is important that clinicians are aware
of the range of clinical problems that can arise during the different phases of
illness
...
F
...
W
...
P
...
B
...
C
...
P
...
B
...
, Yoon I
...
, Green, S
...
L
...
J
...
G
...
H
...
V
...
J
...
M
...
F
...
and Tomashek, K
...
, 2010; Biswas H
...
, Ortega O, Gordon
A, Standish K, Balmaseda, A
...
, Chanthavanich P
...
Chokejindachai, W
...
Suvannadabba, S
...
Leston, G
...
and Sabchareon, 2012)
...
Other common symptoms
include –
34

• Altered taste sensation
• Colicky abdominal pain
• Constipation or diarrhea
• Occasionally dysuria

Cough, sore throat and rhinorrhea are sometimes present but are seen less
frequently than in usual in other viral illnesses (Gregory, C
...
Santiago, L
...

Arguello, D
...
Hunsperger E
...
M
...
X
...
T
...
Pham, T
...
T
...
V
...
T
...
Thut, T
...
Solomon, T
...
Will B and the Dong Nai Study Group, 2004)
...

On examination during this phase of the illness, facial, flushing, conjunctival
suffusion and generalized truncal erythema may be noted
...

Generalized mild enlargement of the lymph nodes is common
...
Splenomegaly is occasionally observed in
small infants
...
Mucosal bleeding from the
nose, gums, gastrointestinal or genitourinary tracts does sometimes occur but is
not usually clinically significant
...

Laboratory findings include mild to moderate thrombocytopenia and leukopenia,
often with mild evaluation of hepatic transaminases (Dietz, V
...
J
...
Kunno, G
...
Sathar, G
...
Gomez and Vergne E
...
Vaughn, D
...
Nimmannitya, S
...
Suntaykorn, S
...
Viramitachai, W
...
Kiaptolpoj, S
...
L
...
L
...
A, 1997; Wilder Smith, A
...

Paton, N
...
, 2004; Villar – Centeno, L
...
Diaz-Quijano, F
...
and MartinezVega, R
...
, 2008; Trung D
...
, Thao Le, T
...
Hein, T
...
Hung, N
...
Vihn, N
...

Hien, P
...
Chinh, N
...
Simmons, C
...
R
...
T
...
Y
...
C
...
M
...
C
...
S
...
M
...
H
...

The high fever persists for 3 – 7 days and usually terminates abruptly
...
As the disease
progresses, the patient becomes anorexic and may show marked lethargy and
prostration
...
Crucially, however, a small number of individuals go on
to develop complications around the time of effervescence; prompt identification
of these individuals as the transition to the critical phase is essential to facilitate
effective case management
...

The most feared complication is an unexplained ‘vasculopathy’ whereby an
increase in vascular permeability results in a capillary leakage syndrome
...
The altered capillary permeability is often
accompanied by hemorrhagic manifestations and hematological abnormalities
...
X
...
T
...
Thuy, P
...
Van Thien, C
...
T
...
T
...

Stepniewska K
...
, Guezman, M
...
Hammond, S
...
Flores, C
...
Videa, E
...
Perez, L
...

Rodriguez, Y
...
No specific event defiens the timing of onset of this
pahse but vascualr leakage often becomes apparaent from around day 3-4 of illness,
with shock (if it occurs) typically developing within 24 hours of defervescence
...


Recovery phase – unusual onset around day 6 – 8 of illness
The increased vascular pemeability and abnormal hoemostasis are transied and usually
resolve within 48 – 72 hours
...

However, convalescence may be prolonged in adults, who sometimes experience
profound tiredness, asthenia and depression for several weeks after recovery
(Balmaseda A
...
G
...
Robleto, G
...
Tellez, Y
...
Saborio, S
...
Sandoval, E
...
and Harris, 2003)
...
X Gubler, D
...
Liu,

J
...
and Chen, 1993)
...
Rarely,
unusual complications such as hemophagocytic lymphohistocytosis may be identified
in patients with persisting fever during the recovery phase (Srichikul, T
...
Kachanopoom, T
...
Likittanasombat, K
...
, 2008;
Ramachandran, B
...
Abhishek, N
...
G
...
V
...
and Shah, 2012; Tan, L
...
Lum, L
...
Omar,
S
...
and Kan, F
...
, 2012)
...


37

Dengue with warning signs
During the transition from febrile to critical phases, it is crucial for clinicians to be
aware of warning signs that the patient may be developing significant vascular leakage
...
However, it is also important to remember that some patients may progress
to severe dengue whether or not they have warning sings
...


Differential diagnosis
Early and accurate diagnosis of dengue is important for several reasons
...
Secondly, other potentially
serious diagnoses can be identified and treated properly from an early stage
...

The particular disorders that should be considered vary, depending partly on the phase
of illness and the age of the patient as well as the epidemiological pattern of other febrile
illnesses that are common locally
...
Among them measles, rubella, enterovirus infections, adenovirus
infections, influenza, infectious mononucleosis, other arboviral infections and
occassinally, Human Immunodeficiency Virus (HIV) are most common
...

In measles and rubella, for example, the rash is often florid and typically extends from
the head to the trunk and extremitie, while in dengue, the early rash can be quite difficult
to see and usually starts on the trunk, later extending to the face and extremities (Dietz,

38

V
...
Gubler, D
...
Upper respiratory symptoms with
headache and myalgia are common features in many systemic viral infections – by
contrast, patients with dengue tends to experience gastrointestinal cough, sore throat
and occasionally rhinorrhea (Qiu, F
...
J
...
C
...

Among the other arboviral infections, chikungunya in particular should be considered,
especially if there is evidence for current transmission in the locality; the general
symptomatology is very similar to dengue except that a symmetrical polyarthritis,
typically involving the small joints is a common feature in chikungunya infections,
while thrombocytopenia and bleeding are more pronounced with dengue (Kularatne,

S
...
Gihan, M
...
Weerasinghe, S
...
and Gunasena, S
...

Matumoto, K
...
K
...
L
...
and Takasaki T
...


39

Fig 2
...


During the critical phase of the illness, a different range of possible diagnoses needs to
be borne in mind
...

However, the onset of shock 4-6 days into the illness, usually after the fever has
subsided, would be very unusual for bacterial septic shock, which typically followed
by a much more rapid disease course
...

40

Occasionally, surgical conditions such as acute appendicitis or cholecystitis have been
mistaken for dengue, as dengue-associated pain may be very intense during the critical
phase ideally ultrasound studies should be performed to confirm the diagnosis before
surgery is contemplated; life-threatening bleeding with fatal outcome has been reported
in patients with dengue, thrombocytopenia and hemostatic derangements who undergo
operative procedure (Premaratna, R
...
S
...
G
...
J
...
Tenner-Racz, K
...
Emmerich, P
...
Dinges, C
...
Nerlich, A
...
and Gunther, S
...


Fig 2
...


41

Prevention of Dengue
Dengue remain the leading arbovirus cause of morbidity in man
...
6
billion people living in areas of dengue risk, with an estimated 390 million
infections and 96 million symptomatic cases annually (Beatty, M
...
Letson,
G
...
and Morgolis, H
...
, 2009; Bhatt, S
...
W
...
J
...
P
...
W
...
L
...
M
...
S
...
G
...
Meyers, M
...
George, D
...
Jaenisch, T
...
R
...
, Simons, C
...
Scott,
T
...
Farrar, J
...
and Hay, S
...
, 2013)
...
For example, Aedes albopictusis is an excellent
vector of dengue in the laboratory and outbreaks in Hawii (Effler, P
...
Pang, L
...
Vorndarm, V
...
Ayers, T
...
Tom, T
...

Rigau-Perez, J
...
Hayes, J
...
Mills, K
...
Clark, G
...
and Gubler D
...
,
2005) and Taiwan (Lambrechts, L
...
W
...
J
...
Differences in the ability for Aedes
albopictus to develop disseminated infections of dengue viruses may explain its
lower vector competence status relative to Aedes aegypti (Lambrechts, L
...
W
...
J
...
Aedes scutellariscomplex members, Aedes
polynesiensis (Rosen, L
...
E
...
H
...
B
...
J
...
R
...
H
...
A
...
A
...
F
...
Aedes
polynesiensis is suspected of vectoring outbreaks in French Polynesia [82] while
Aedes hensilli (Savage, H
...
Fritz, C
...
Rutstein, D
...
Vorndam, V
...
J
...
M
...
However,
it is another transmission worldwide and is almost exclusively the vector in large,
explosive urban epidemics of dengue (Gubler, D
...
, 1998; Lambrechts, L
...
W
...
J
...


42

What is unique about Aedes aegypti that makes it such an effective vector for
dengue? Aedes aegypti is arguably the most anthropophilic mosquito
(Tabachnick W
...
Most of its behavior from immatures residing within
male-made, water-holding containers to adult females living inside human
domains where they feed almost exclusively on human blood is tightly liked to
man
...


Association between Chikungunya and Dengue
To date, the number of diagnosed cases of DENV-CHIKV coinfections is
surprisingly small and available information is often incomplete, making it
difficult to establish epidemiological trends
...

Indeed, although the first documented cases of DENV-CHIKV coinfections date
back to the 1960s in Vellore, south India, when 14 cases were reported during a
CHIKV epidemic outbreak and in Thailand, where nine cases were documented
...


In 2006, two cases of coinfection corresponding to two female patients were
described in Malaysia, and 20 more were recorded during the CHIKV outbreak
in La Reunion the same year
...
The most recent cases were diagnosed
in South America, India and Nigeria in 2013-2014
...


44

CHAPTER

3

MATERIALS AND
METHODOLOGY

45

MATERIALS AND METHODOLOGY

Type of study
This is a cross sectional study
...


Period of study
From January to September 2018
...


Exclusion Criteria
➢ Dengue patients refused to be part of this study
...


46

Sampling Techniques: Purposive sampling
...
e
...
5 (As true proportion of viral is unknown
in Bangladesh, it was considered as 50%)
q = (1 – p)
= 1 – 0
...
5
d = Acceptable error 5% (here 10% of p)
= 0
...
96 (at 95% confidence interval)

So, N =

(1
...
5 ×0
...
05)2

= 384

However, statistically acceptable size was 384, but due to time and financial
constraint, the study subjects were limited to 120
...
The blood was kept stand still to allow clotting and the serum was
separated by centrifugation at 3000 rpm for 10 minutes
...


Laboratory Methods for Chikungunya Test
Chikungunya IgM or IgG and Dengue IgM or IgG antibodies were qualitatively
analyzed by using ICT kit (SD Biosensor)
...
Both tests
procedures described in details in appendix IV
...

Data relating to clinical information were taken from pre-designed questionnaire
sheets of donors kept in the Blood Bank personnel before donation of blood
...
Data collection sheet
2
...


Data Presentation
Data and result was presented in the form of tables and figures
...

The study subjects were assured about minimum physical and psychological risk
during collection of blood and due consideration was paid to safeguard the
confidentiality of the data
...
A d value of 0
...
Further statistical analysis of the results were done by
using by software with statistical package for social science (SPSS), Version 20
...


49

CHAPTER
RESULTS

50

4

Results
The study was set to detect chikungunya antibodies form the patients that have
dengue fever and to investigate clinical similarities, to gain more insights into
the long-term persistence of CHIKV Ab and its role in driving dengue fever
...
So
presentation and analysis of the collected data was computed using Percentage,
Mean, Mode, Frequency and Cross-table correlation
...
5%

Male

63

52
...
5%) were female and 63 (52
...
Results were expressed as Frequency and Percent in appropriate
...


Education level

Number

Percent

Graduate or above

36

30%

Class 11-12

22

18
...
5%

Class 1-5

20

16
...
5%

Total

120

100%

Considering the education of the studied patients, the most frequent were those
who had Graduate or above degree 36 of 120 (30%), followed by those
completed higher secondary 22/120 (18
...
5%), class 1 – 5 were 20/120 (16
...
5%)
...
5%

Service

11

9
...
17%

Student

4

3
...
33%

Others

45

37
...
5%), service 11/120 (9
...
17%)
...
33%), homemakers 10/120 (8
...


54

Table IV: Distribution of Respondents Based on Socio-Economic Status:

Income group

Frequency

Percent

Income group

Low income group

80

66
...
< 100,000
annually (approx
...
83%

Tk
...
)

High income group

9

7
...
> 500,000
annually (approx
...
67%), followed by middle income
group 31 out of 120 (25
...
5%)
...


55

Table V: Distribution by age

Age Group

Number

Percent

<15

4

3
...
67%

31-45

39

32
...
5%

61-75

19

15
...
17%

Total

120

100%

By the age category, a total of 120 respondents were participated in this study
and their ages were categorized in Six age groups
...
33%)
▪ Group II: 16-30 of 20 (16
...
5%)
▪ Group IV: 46-60 of 33 (27
...
83%)
▪ Group VI: 76-90 of 5 (4
...
33%

6-10 days

46

38
...
5%

16-30 days

5

4
...
67%

Total

120

100%

About knowledge of respondents to the site of Incubation period where
participated in this study and their duration of sign and symptoms like fever, joint
pain etc
...
33%)
▪ Group II: 6-10 days of 46 (38
...
5%)
▪ Group IV: 16-30 days of 5 (4
...
67%)

LVII

Table VII: Anti Dengue antibody status

Anti-Dengue antibody

Number (%)

Both IgM and IgG positive

8 (6
...
33%)

IgM negative but IgG positive

18 (15%)

Both IgM and IgG negative

90 (75%)

Total

120

The table shows that, Out of 120 cases, dengue IgM positive but IgG negative
patients are 18 (15%) and double positive are 8 (6
...


Table VIII: Anti Chikungunya antibody status
Anti-Chikungunya antibody

Number (%)

Both IgM and IgG positive

4 (3
...
83%)

IgM negative but IgG positive

8 (6
...
16%)

Total

120

The table shows that, out of 120 cases, double positive chikungunya patients are
4 (3
...


LVIII

Table IX: Anti Dengue and Anti Chikungunya antibody status

Antibody
Both

IgM

IgM

Both

Total

IgM and

positive

negative

IgM and

IgG

IgG

IgG

IgG

positive

negative

positive

negative

Dengue

8

4

18

90

120

Chikungunya

4

1

8

107

120

This table indicates the diagnostic patients of dengue antibodies and chikungunya
antibodies according to Cross-tabulation
...

▪ The double positive patients both of dengue and of chikungunya were 8
and 4 respectively out of 120
...
Screening tests results can be
obtained within 15 minutes with high sensitivity and specificity
...
5%) were female and 63 (52
...
al, 2016) where 51
...


39 (32
...
33%) out of 120 were in the age ranging
less than 15 years
...
So we can conclude that people who live in slum area and
unhygienic condition, are risk of most of mosquito bite and so they can be affected
by mosquito borne diseases like dengue and chikungunya
...
Because, there is a lack of sufficient
fund and shortage of time, it cannot be shown that patients with dengue antibody
develops chikungunya antibody
...


LXI

CHAPTER

6

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Blood test
is the only reliable wat to identify chikungunya since the symptoms are similar to
much more deadly dengue fever
...

Your locality has been selected for this purpose and your participation in it is
highly appreciated
...

If you feel any discomfort to take part or to participate this study, you are free to
leave
...


Risk
No risk is associated in taking part with this research
...
If
you do not like to participate in this research or want to leave after participation,
you can go
...


Thanks for your helpfulness
...


Name of the participants
...


Signature
...


Date
...

• Permission for the study will be taken from the concerned departments
from where we will collect study samples
...

• Interest of the study subject will be assured to adequate treatment of any
risk development in relation to study purpose
...

• Finally, written consent of all the study subjects will be taken free of duress
and without exploiting any weakness of subjects
...


LXXXI

Appendix III:

Questionnaire
Status of Chikungunya Antibodies in Suspected
Dengue Patients
Interview questionnaire form
Part A: General Information
Consent, Personal and other information
No
Question
Response
A 01
Consent has been read and No ……
...
1
If No, END
A 02
Have you even told by a doctor No …
...
1
If Yes, END
A 03

Age (in completed years)

A 04

Name of the participant

A 05

Father’s name

A 06

Mother’s name

A 07

Spouse name

LXXXII

Code

A 08

Address

A
09
Mobile No
...
Monoclonal anti-human IgM and Monoclonal anti-human IgG are
immobilized at two individual test lines respectively (M, G line) on the
nitrocellulose membrane
...
If human anti-chikungunya IgM or IgG exist in patient serum, the
individual test line appear visible band respectively forming the complex with
anti-human IgM/IgG, human IgM/IgG, inactivated chikungunya virus and antichikungunya EI-gold which means positive test result
...


Intended use of chikungunya test
Standard Q chikungunya IgM/IgG test is an Immunochromatographic assay for
the detection of IgM/IgG antibodies against chikungunya virus in human serum,
plasma or whole blood samples
...
This is intended
for professional use, only for an initial screening test
...
Collect the venous whole blood into the commercially available plain tube,
NOT containing anti-coagulants such as heparin, EDTA or sodium citrate,
by venipuncture and leave to settle for 30 minutes for blood coagulation
and the centrifuge blood to get serum specimen of supernatant
...
If serum in the plain tube is stored in a refrigerator at 2-8c/36-46f, the
specimen can be used for testing in 1 week after collection
...
For prolonged storage, it should be kept at below — 400
C or — 40 F
...
They should be brought to room temperature prior to use
...
Collect the venous whole blood into the commercially available anticoagulants such as heparin, EDTA or sodium citrate and centrifuge blood
to get serum specimen of supernatant
...
If plasma in anti-coagulant tube is stored in a refrigerator at 2 – 80 C or 36
– 46 F, the specimen can be used for testing in 1 week after collection
...
For prolonged storage, it should be at below -400 C
or -40 F
...
They should be brought to room temperature prior to use
...
Capillary whole blood should be collected aseptically by fingertip
...
Clean the area to be lanced with an alcohol swab
...
Squeeze the end of the fingertip and price with a sterile lancet
...
Collect the capillary whole blood to the black line of the SD Ezi tube+ for
the testing
...
The capillary whole blood must be tested immediately after collection
...
Collect the venous whole blood into the commercially available anticoagulant tube such as heparin, EDTA or sodium citrate by venipuncture
...
If venous whole blood in anti-coagulant tube is stored in a refrigerator at 2
– 80 C, the specimen can be used for testing within 1 – 2 days after
collection
...
Do not use hemolysed blood samples
...
Read the instruction
B
...
Check the test device and silica gel pack in the foil pouch
...
Using a SD Ezi tube+, collect the 10 ml of serum/plasma/whole blood to
the black line of the SD Ezi tube+
...
Add the collect serum plasma whole blood to the sample well of the test
device
...
Add 3 drops (90 ml) of assay diluent into the assay well of the test device
...
Read the test result 15 minutes; the test result can be read up to 30 minutes
...
Negative result: Only band (C control line) within the result window
indicates a negative result
...
IgM positive result: Two colored bands (C control line and M test line)
within the result window indicate chikungunya IgM positive
...
IgG positive result: Two colored bands (C control line and G test line)
within the result window indicate chikungunya IgG positive
...
IgM and IgG positive result: Three colored bands (C control line, M test
line and G test line) within the result window indicate chikungunya IgM
and IgG positive
...
Invalid test: If the control band (C control line) is not visible within the
result window, the result is considered invalid
...
The test procedure, precaution and interpretation of results for this test must
be followed strictly when testing
...
This test detects the presence of anti-chikungunya IgM and IgG and should
not be used as the sole for criteria for the diagnosis of chikungunya virus
infection
...
The test result must be considered with other clinical data available to the
physician
...
Failure to follow test procedure and interpretation of test result may
adversely affect test performance and/or products in valid results
...
Monoclonal anti-human IgM and Monoclonal anti-human IgG are
immobilized at two individual test lines respectively (M, G line) on the
nitrocellulose membrane
...
If human anti-dengue IgM or IgG exist in patient serum, the individual
test lien appears visible band respectively forming the complex with anti-human
IgM/IgG, Human IgM/IgG, inactivated dengue virus and anti-dengue EI-Gold that
means positive test result
...


Intended Use of Dengue Test
Standard Q dengue IgM/IgG test is an Immunochromatographic assay for the
detection of IgM/IgG antibodies against dengue virus in human serum, plasma or
whole blood samples
...
This is intended for
professional use, only for an initial screening test
...


Serum
LXXXIX

1
...

2
...
Using the
specimen in the long term keeping more than 1 week can cause specific
reaction
...

3
...


Plasma
1
...

2
...
Using the
specimen in the long-term keeping more than 1 week can cause nonspecific reaction
...

3
...


Whole Blood
Capillary Whole Blood
XC

1
...

2
...

3
...

4
...

5
...

Venous Whole Blood
1
...

2
...

3
...


Test procedures:
Common check-up:
A
...
Check the expiry date
C
...


1
...

2
...

3
...

4
...


Interpretation of the Test Results

XCI

1
...

2
...

3
...

4
...

5
...


Limitation of the test:
1
...

2
...

3
...

4
...


XCII



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