Dengue Virus Infection Amongst Patients With Acute Febrile Illness: Clinical Characteristics, Molecular and Serological Profiles for Vaccine Trial and Roll-out
Conditions: Dengue
Sponsor: Tangerang District Hospital

Protocol V2.0: TNG205 dated 08 January 2024 
 
 
 
 
 
Dengue virus infection amongst patients with acute febrile illness: 
clinical characteristics, molecular and serological profiles for 
vaccine trial and roll-out 
 
 
 
 
dr. Dewi Lokida SpPK (K) 
dr. Achmad Muchlis, MARS 
dr. Susilowati, SpA 
dr. Nathania Nadia, SpPD 
dr. Salmawati, MKM 
dr. Herman Kosasih, PhD 
 
 
 
 
 
 
 
 
 
 
Tangerang District Hospital 
Tangerang District Health Office 
2023 

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Protocol V2.0: TNG205 dated 08 January 2024 
Table of Content 
 
Table of content….…………………………………………………………………………… 
List of abbreviations………………….…..………………………………………………….. 
I. 
Background……………………………………………………………………………… 
II. 
Objectives 
2.1 Primary objectives………………………………………………………………….. 
2.2 Secondary objectives..…………………………………………………………….. 
III. Clinical hypotesis…. …………………………………………………………………… 
IV. Ethical approval………………………………………………………………………… 
V. Method 
5.1 Study design………………………………………………………………………... 
5.2 Inclusion and exclusion criteria…………………………………………………… 
5.3 Study duration.……….…………………………………………………………….. 
5.4 Procedure………………….……………………………………………………….. 
5.5 Clinical data and specimen collection time………………… …………………… 
5.6 Laboratory test……………………………………………………………………... 
VI. Statistical analysis and sample size justification …………………………………… 
6.1 Variables...………………………………………………………………………….. 
6.2 Statistical analysis…..…………………………………………………………….. 
6.3 Sample size……….………………………………………………………………... 
VII. References……..……………………………………………………………………….. 
 
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LIST OF ABBREVIATIONS 
 
 
 
 
AFIRE                                  The Etiology of Acute Febrille Illness Requiring Hospitalization 
CRF                                     Case Report Form 
DENV  
                   Dengue virus  
DBD                                     Demam Berdarah Dengue 
ELISA                                   Enzyme-Linked Immunosorbent Assay 
ICF 
INA-RESPOND 
Informed Consent Form 
Indonesia Research Partnership on Infectious DIseases 
IRB 
Institutional Review Board 
IgM 
IgG 
Immunoglobulin M 
Immunoglobulin G 
NS-1 
PCR 
PRNT 
Dengue virus non structural-1 protein 
Polymerase Chain Reaction 
Plaque Reduction Neutralization Test 
RDT 
RSUD 
Rapid Diagnostic Test 
Rumah Sakit Umum Daerah 
RT 
Research Team 
SAE 
Serious Adverse Event 
SID 
Subject Identification Number 
SoC 
Standard of Care 
SOP 
Standard Operating Procedure 
SRB 
Site Regulatory Binder 
SAE 
Kejadian Tidak Diinginkan yang Serius (KTDS) 

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Protocol V2.0: TNG205 dated 08 January 2024 
I. 
BACKGROUND 
 
Dengue is a significant public health problem in Indonesia, with 25.000-79.000 
cases reported each year from 2013 to 2022. The disease is caused by four serotypes of 
Dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4), transmitted primarily by the 
Aedes aegypti. 
 Comprehensive knowledge of the prevalence, immune response, and clinical 
characteristics associated with each serotype is essential for the development and 
implementation of effective dengue prevention and control strategies, especially in the 
context of vaccine development. 
Previous studies have consistently shown that DENV-4 infections are relatively 
rare compared to other serotypes. A study in Vietnam found a low number of DENV-4 
cases, illustrating the lower incidence of DENV-4 than other serotypes (Ta et al., 2019). 
A similar study in the Philippines, assessing the efficacy of a tetravalent dengue vaccine 
candidate (TAK-003) for DENV-4 in eight dengue endemic countries in Asia and Latin 
America was difficult due to the small number of DENV-4 infection cases, only 7 cases 
were virologically confirmed (Biswal et al., 2019). 
A study on the prevalence and clinical aspects of DENV-1, DENV-2, DENV-3, and 
DENV-4 in Indonesia showed that DENV-4 was the least common circulating serotype 
(Harapan et al., 2019). A study conducted in many hospitals examining acute febrile 
illness in hospitalized patients (AFIRE), found that DENV-4 infection was less common 
among the cases studied (Utama et al., 2019). Interestingly, DENV-4 is often found in 
outpatients or during dengue outbreaks. A study in outpatients in West Java found that 
DENV-4 infection occurred as frequently as other serotypes, but most cases were not 
clinically typical (Kosasih et al., 2016). In the 2004 Jakarta outbreak, DENV-4 was the 
second most common serotype after DENV-3 (Suwandono et al., 2006). A study 
conducted during a dengue outbreak in Jember in 2019, has identified genetic factors 
associated with DENV-4, suggesting that a combination of selection pressure and 
changes in genetic mutations may contribute to the dominance of DENV-4 in East Java, 
Indonesia (Wardhani et al., 2023; Aryati et al., 2020). 
One possible explanation for the low prevalence of DENV-4 is that most studies 
are hospital-based, which may not include untreated mild DENV-4 infections. Besides, 
NS-1 screening, which is used for detection and identification of DENV-4 cases, has low 
sensitivity, which further complicates accurate diagnosis (Mata et al., 2020; da Costa et 
al., 2014). In addition, the sensitivity of NS1 detection varies among secondary infections, 
with the highest sensitivity of DENV-3 (47.1%), followed by DENV-1 (40.9%), DENV-2 
(30%), and DENV-4 (27%) (Kosasih et al., 2013). This is important as secondary 

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infections of DENV-2, DENV-3, and DENV-4 in Southeast Asia are commonly associated 
with severe clinical manifestations, indicating the potential for a significant health burden 
(Soo et al., 2016). 
Considering the consequences of undiagnosed dengue infection, the inability to 
detect DENV-4 using NS1 rapid test may lead to misclassification of cases as non-
dengue, resulting in different therapy/ management. This may adversely affect patient 
therapy, leading to delayed or inappropriate management potentially leading to prolonged 
illness or worsening of symptoms. 
Overcoming the limitations of current diagnostic methods is critical to ensure quick 
and precise treatment for individuals infected with DENV-4. This is particularly relevant in 
the context of vaccine development and clinical trials, where accurate serotype 
identification is essential to evaluate vaccine efficacy and effectiveness. Improving 
diagnostic capabilities and awareness of the presence and potential impact of DENV-4 
infection will contribute to more effective management and control strategies for dengue, 
ultimately reducing the burden of disease in affected populations. By addressing these 
issues, healthcare providers and researchers can improve patient prognosis and reduce 
the challenges posed by DENV-4 infection. 
Tangerang, located in Banten province, Indonesia, has a high risk of dengue virus 
transmission due to its dense population and favorable environmental conditions for 
mosquito breeding. Despite vector control efforts, dengue remains a significant health 
challenge in the region. In 2022, as many as 50-100 dengue cases were reported in each 
public health center and more than 200 patients were hospitalized due to dengue 
infection. 
Focused study in Tangerang, which includes hospitals and public health centers, 
is essential to obtain local data that can be used as a source of information for 
interventions that will improve a broader understanding of dengue epidemiology in 
Indonesia. Tangerang District Hospital is a referral hospital for all public health centers 
that has several national and multinational clinical studies. Including intervention studies, 
Tangerang District Hospital has a well-equipped infectious disease laboratory to conduct 
molecular and serological tests. Collaborating with INA-RESPOND lab, we are also able 
to conduct Plaque Reduction Neutralization Test (PRNT). 
This prospective observational study in Tangerang aims to examine the 
prevalence, immunologic, virologic, and clinical aspects of dengue virus infection, 
especially DENV-4. The results of this study will improve our understanding of dengue 
epidemiology in Tangerang, Indonesia, the relationship between host factors, viral factors, 
and severity. This study will provide evidence-based information for dengue control, and 

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contribute to global efforts in developing an effective vaccine for all dengue virus 
serotypes. 
The comprehensive screening efforts of this study will provide important data on 
other serotypes, enabling a holistic understanding of dengue infection and facilitating 
inter-serotype characterization. This study will have a significant impact on dengue 
research, public health policies, and global initiatives to combat this challenging disease. 
 
II. OBJECTIVES 
2.1. Primary objective 
To determine the clinical characteristics, specific immune responses, and viral 
serotypes in patients with confirmed dengue virus infection in Tangerang District, 
Indonesia. 
 
2.2. Secondary objectives 
• 
To assess the prevalence of dengue virus infection among patients with acute 
fever 
• 
To assess the prevalence of infection with DENV-4 and other serotypes as well as 
clinical characteristics, immunologic and virologic profiles important for vaccine 
trials and implementation 
• 
To assess human and viral factors associated with severity of febrile illness due 
to dengue virus infection 
• 
To increase the capacity of Tangerang District Hospital’s Infectious Disease 
Laboratory/ INA RESPOND Reference Laboratory to perform plaque reduction 
neutralization test (PRNT). 
• 
To identify other etiologies of fever in Tagerang district 
 
III. CLINICAL HYPOTESIS 
Dengue fever is a hyperendemic disease in Tangerang district, Indonesia.  
DENV-4 infection is more frequently found at the primary health centers than at 
the hospital because the clinical symptoms are milder. 
Several human and viral factors, such as age, nutrition status, immune status, 
serotypes, types of infections (primary/secondary) are associated with severity of 
symptoms. 
 
 
 

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IV. ETHICAL APPROVAL 
This study will be conducted in accordance with ethical guidelines and obtained 
permission from the relevant institutions and the ethics committee of Tangerang District 
Hospital. 
 
V. METHOD 
5.1. Study design 
This study is a prospective observational study that will be conducted in 
Tangerang Regency, Indonesia. In this study, data will be collected from patients who 
seek treatment at the Tangerang District Hospital, Kelapa Dua Public Health Center and 
Bojong Nangka Public Health Center in a one-year period from January 2024 to 
December 2024. Both public health centers have densely populated areas and have 
experience in conducting clinical research on COVID-19 vaccination.  
Patients suspected of dengue infection will be screened using inclusion and 
exclusion criteria. Selected participants will be seen at the first visit as clinical data will be 
collected, routine hematology test and NS-1 RDT will be conducted. Blood specimens will 
be collected for further testing at Tangerang District Hospital’s Infectious Disease 
Laboratory. 
 
5.2. Inclusion and exclusion criteria 
Inclusion criteria: 
1. Patients age ≥ 4 years old with fever (≥ 37.5°C) or history of fever with antipyretic 
use, lasting less than or equal to 7 days 
2. Presence at least one of the following dengue-like symptoms: 
• Headache 
• Retro-orbital pain 
• Nausea/ vomiting 
• Muscle, bone, or joint pain 
• Rash or bleeding manifestations 
3. Patient or patient's guardian giving consent to participate in the study 
 
Exclusion criteria: 
1. Have a condition that may interfere with study procedures and compliance (based 
on doctor's assessment) 
2. Suspected of focal infection 
 

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5.3. Study duration 
The study will be conducted over a period of one year, from January 2024 to 
December 2024. Data will be collected from eligible participants visiting Tangerang 
District Hospital, Kelapa Dua Public Health Center and Bojong Nangka Public Health 
Center. Study period will be extended until it reaches the specified number. 
 
5.4. Study procedure 
• Recruitment of participants 
Patients who seek treatment at Tangerang District Hospital, Kelapa Dua Public 
Health Center and Bojong Nangka Public Health Center with dengue-like symptoms 
will be selected. All those who meet the enrollment criteria will be offered to 
participate in this study. 
• Informed consent 
Potential participants who meet the criteria, or their guardians, will be provided with 
detailed information about the study. Informed consent will be obtained from 
potential participants or their guardians prior to participating in the study. 
• Data collection 
Data will be collected through structured interviews and medical record review. 
Information on demographic characteristics, clinical symptoms, medical history, 
laboratory results, therapy and previous dengue infection will be recorded in the 
case report form (CRF). 
Data on the number of patients presenting with fever to Tangerang District Hospital, 
Kelapa Dua Public Health Center and Bojong Nangka Public Health Center will be 
collected for use in identifying the proportion of dengue fever. 
• Data management 
Participants will be anonymized to ensure participant confidentiality and the 
collected data will be stored in a locked cabinet with limited access. 
5.5. Clinical data and specimen collection time 
• Hospitalized participants 
The data and specimens to be collected are data at the time of the first visit to the 
hospital and at the second visit. For participants with a positive NS1 RDT result at the 
first visit, second visit data will be collected when the participant is scheduled to be 
discharged from hospital inpatient care. For participants with a negative NS1 RDT 
result at the first visit, data for the second visit will be collected within 7-14 days of the 
first visit. 
  

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• Participants from Public Health Centers 
Data and specimens to be collected are data and specimens at the time of the visit to 
the public health centers, while the second data and specimens are only taken as much 
as 25% of the number of participants of the first visit, with a window period of 7-14 days 
after the first visit. The determination of participants for the second visit is determined 
by study’s team based on clinical judgment. 
• Clinical evaluation 
Participants will undergo a clinical assessment to evaluate the severity of the disease, 
including the presence of signs of severe clinical manifestations. 
• Type and amount of specimens to be taken 
- 
Serum 
Serum obtained with a 4 mL yellow cap gel separator tube, all serum specimens 
obtained will be sent to Tangerang District Hospital’s Infectious Disease 
Laboratory/INA-RESPOND lab for viral culture and PRNT.  
- 
Whole blood dan plasma 
Whole blood specimens in 3 mL purple cap EDTA tubes (adult) or 2 mL purple cap 
EDTA tubes (pediatric) will be used for hematology test, RDT IgM/IgG, and RDT-NS1 
testing, the remaining plasma will be sent to Tangerang District Hospital’s Infectious 
Disease Laboratory for ELISA and PCR testing. 
 
5.6. Laboratory test 
• Hematology 
Hematology test is conducted at PHCs and Tangerang District Hospital using an 
automatic hematology analyzer, and the results will be given to the doctor in charge 
as a basis for diagnosis and therapy. 
• Antigen 
NS1 antigen will be tested at PHCs and Tangerang District Hospital using RDT, and 
the results will be provided to the doctor in charge as a basis for diagnosis and 
therapy. The results will be confirmed by NS1 antigen by ELISA. 
• Molecular  
Blood from participants will be subjected to molecular testing. Polymerase Chain 
Reaction (PCR) to detect and confirm the presence of DENV,and identify dengue 
serotypes. 
 
 
 

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Protocol V2.0: TNG205 dated 08 January 2024 
• Serology 
Enzyme-Linked Immunosorbent Assay (ELISA) will be used to detect dengue 
specific antibodies (IgM and IgG), Rapid Diagnostic Test (RDT) to detect IgM for 
acute dengue infection, and IgG to determine primary or secondary infection.  
• Plaque Reduction Neutralization Test (PRNT)  
PRNT will also be performed to assess serotype-specific neutralizing antibodies in 
a subset of specimens (specimens from all severe cases and randomized mild 
cases as controls). 
 
VI. STATISTICAL ANALYSIS AND SAMPLE SIZE JUSTIFICATION 
6.1. 
Variable 
Variables collected include age, gender, demographic characteristics, clinical 
symptoms, medical history, and history of previous dengue infection. The prevalence, 
immunologic and virologic characteristics, clinical features, and severity of DENV1-4 
infection will also be recorded. 
 
6.2. 
Statistical analysis 
Statistical analysis will include descriptive statistics to summarize the demographic and 
clinical characteristics of participants. Prevalence estimates of DENV1-4 will be 
calculated with their respective confidence intervals. Prevalence, demographic and 
clinical comparisons between DENV-4 and other serotypes will be made using 
appropriate statistical tests, such as chi-square or Fisher's exact test.  
 
6.3. 
Sample size 
This study aims to collect data for one year. In accordance to the data reported in 2022, 
we will collect  100 dengue infection cases from each public health center and 200 
cases from the Tangerang District Hospital.  
It is expected that the Tangerang District Hospital, Kelapa Dua Public Health Center 
and Bojong Nangka Public Health Center can represent the population in Tangerang 
District. 
 
 
 
 
 
 
 

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