Determination of Etiology of Febrile Illness in Nepal (DEFINe Study)
Phase: NA
Conditions: Febrile Illness, Fever Without Source
Sponsor: Patan Academy of Health Sciences

STUDY TITLE 
Determination of Etiology of Febrile Illness in Nepal  
 
STUDY ID  
 
DEFINe Study 
 
INVESTIGATORS   
Dr. Janak Koirala, MD MPH FACP FIDSA, Professor of Medicine and Infectious diseases (PI) 
Dr. Nora Ranjitkar, MD, Associate Professor of Medicine (Co-PI) 
Dr. Suman Thapa, MD, Assistant Professor of Medicine (Co-PI) 
 
Department of Internal Medicine, Patan Academy of Health Sciences  
Patan Hospital, Lalitpur, Nepal 
 
 
GRANT FUNDING 
University Grant Commission (UGC) Research Grant (NRS 4,05,900.00) 
 
 
 
     
(VERSION DATE: MARCH 2025) 

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STUDY TITLE 
Determination of Etiology of Febrile Illness in Nepal (DEFINe Study) 
 
INVESTIGATORS   
Dr. Janak Koirala, MD MPH FACP FIDSA, Professor of Medicine and Infectious diseases, PAHS (PI) 
Dr. Nora Ranjitkar, MD, Associate Professor of Medicine, PAHS (Co-PI) 
Dr. Suman Thapa, MD, Assistant Professor of Medicine, PAHS (Co-PI) 
GRANT FUNDING 
University Grant Commission (UGC) Research Grant (NRS 4,05,900.00) 
STUDY SITE 
Proposed Study Site: Patan Academy of Health Sciences, Patan Hospital, Lalitpur 
 
1. ABSTRACT 
In the absence of a microbiological lab testing, majority of the cases of febrile illnesses in Nepal are 
diagnosed on a clinical basis alone. These cases of “undifferentiated febrile illness” are treated 
empirically with multiple antibiotics without an etiological diagnosis. Unnecessary use of antibiotics can 
increase the risk of antibiotic resistance and adds unnecessary burden of cost to the patients and their 
families.  
This study aims to determine the etiology of infections using additional microbiological techniques in 
patients admitted to the hospital with acute undifferentiated febrile illness of 3 days to 21 days 
duration, and assess its impact on use of antimicrobial drugs. In addition to the clinical features, the 
etiological diagnosis will be confirmed by using additional tests including culture techniques and ELISA 
based serological tests for 7 additional pathogens which are currently not tested in Nepal. Patients 
meeting definition of undifferentiated fever of 3 days to 21 days duration will be enrolled from a major 
tertiary care center using predefined inclusion and exclusion criteria.  
This study will help to identify additional infectious diseases prevalent in Nepal, decrease unnecessary 
empirical use of multiple antibiotics, and improve patient outcome. Additionally, identification of new 
causes of febrile illness will help in prevention and control of these infectious diseases at public health 
level.   
 
2. BACKGROUND AND RESEARCH GAP 
Nepal is located between latitudes 26° and 31°N while its altitude ranges from 60m to 8848m from sea 
level allowing for a large variation in climate. Southern part of Nepal has tropical climate suitable for 
vector-borne tropical diseases whereas northern parts have more temperate and sub-arctic climate 
where these diseases are less common. Additionally, water-borne diseases have persisted because of a 

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lack of sanitation and safe drinking water. Most hospitals in Nepal lack support of well equipped 
microbiology laboratories for diagnosis of infectious diseases, and hence, most clinicians treat their 
patients empirically based on their decisions on clinical features alone [1,2]. A study done in the region 
has shown poor predictability of clinical features alone to distinguish enteric fever from other causes of 
febrile illness [3].  
Either from the lack of or limited access to confirmatory microbiological tests, majority of the cases of 
febrile illnesses in Nepal are diagnosed on clinical basis only. These cases of “undifferentiated fever” or 
“undifferentiated febrile illness” are treated empirically without etiological diagnosis. In the absence of 
correct diagnosis, patients receive multiple empirical antibiotics which increase the risk of antibiotic 
resistance and adds unnecessary burden of cost to the patients and their families. A hospital-based 
study of febrile patients published in 2004 revealed identifiable causes in only 37% of the patients [4]. 
The most common causes of febrile illness in Nepal at the time were enteric fever, murine typhus, scrub 
typhus, Leptospirosis, and bacterial infections including Streptococcus pneumoniae, Staphylococcus 
aureus, and Escherichia coli. Studies show a trend in increasing cases of rickettsial infections including 
Rickettsia typhi and Orientia tsutsugamushi [4-6]. 
 
Preliminary work done at Patan Hospital 
Patan Hospital is a tertiary care medical center with 450 beds that admits between 10 to 30 febrile 
patients per week depending on the season. Over 30% of these patients do not have localizing signs and 
symptoms for clinical diagnosis nor do they have a laboratory-based diagnosis. These cases are labelled 
as undifferentiated fever and receive empirical treatment with two or more antimicrobial agents. 
Previous studies done at Patan Hospital have demonstrated that Salmonella typhi and Salmonella 
paratyphi A were causes of undifferentiated fever of >3 days duration in 34% patients and Rickettsia 
typhi in 17% cases, whereas another study in 2008 found 7% cases of febrile illness has Rickettsia typhi, 
which is the cause of Murine typhus [5,6]. These studies show an increase in cases of rickettsial 
infections including Riackettsia typhi and Orientia tsutsugamushi. Studies at Patan Hospital have also 
identified Streptococcus pneumoniae, Leptospira spp., Orientia tsutsugamushi, Hantavirus, and Coxiella 
burnetii as casues of febrile illnesses [4-6].  
 
Research Gap 
The epidemiology of infectious disease has changed with massive within country urban migrations, 
increased migrant workers to India and overseas countries, change in climate, and several other 
reasons. Previous common causes of febrile illnesses such as malaria and enteric fever have declined 
over past decade whereas dengue and scrub typhus are on the rise [7-9]. Meanwhile, new diseases such 
as COVID-19, dengue, and Scrub typhus have become commonplace [5,10,11]. Other causes of febrile 
illnesses, such as zoonoses (e.g. bartonellosis and Q fever), rickettsial (e.g. Q fever), and viral infections 
(e.g. arboviruses) remain mostly undiagnosed. This study aims to fill the gap in research by identifying 
additional causes and estimating burden of these infectious diseases. This will also help to reduce the 
unnecessary use of antibiotics. 
 

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3. SIGNIFICANCE AND INNOVATION 
A. Significance 
This study is expected to identify additional infectious diseases prevalent in Nepal with the laboratory 
confirmation of the etiologic agents and improve the appropriate use of antimicrobial agents with 
targeted therapy. The laboratory tests used in this study will help to determine the presence of 
additional infectious diseases whose prevalence is currently unknown. This information will be helpful to 
the clinicians to design initial empiric antibiotic therapy based on epidemiological and clinical features. A 
more appropriate choice of antibiotics will decrease the unnecessary use of multiple broad-spectrum 
antibiotics, which in turn will help to reduce antimicrobial resistance, and improve patient outcome. 
New information discovered from the study will be useful for making policies to prevent and control 
various infectious diseases such as vector-borne infections and other communicable diseases.  
B. Innovation and discovery 
The investigators aim to introduce and use selected lab technology with a higher sensitivity and 
specificity for laboratory confirmation of the diagnosis. We expect to identify new organisms causing 
febrile illness which are not detected with the currently used routine laboratory tests.  Besides 
confirmation of the diagnosis, this study also plans to compare the clinical diagnosis alone as a guiding 
tool for the use of empirical antimicrobial therapy versus use of microbiological diagnosis. Hence, we 
will estimate the overuse and inappropriate use of antibiotics that may lead to antimicrobial resistance. 
The results and data from this study will be published and shared to educate the clinicians, reduce 
inappropriate antimicrobial therapy, and improve patient outcome.  
 
4. OBJECTIVES AND SPECIFIC AIMS 
A. Overall Objectives and Hypothesis 
This study aims to determine the etiology of infections using laboratory methods in patients admitted to 
the hospitals with acute undifferentiated febrile illness and assess its impact on the use of 
antimicrobials. In addition to the clinical features of the patients, rapid serological tests and culture 
techniques will be used to confirm the etiological diagnoses.  
We hypothesize that improved determination of etiological diagnoses in patients presenting with 
undifferentiated febrile illness can decrease the use of broad-spectrum antimicrobials. 
B. Specific Aims  
Primary:  
1. To estimate the proportion of additional infectious etiologies of febrile illness among patients 
presenting with an undifferentiated fever of 3 to 21 days duration.  
2. To compare the use of antimicrobials in patients with undifferentiated fever in whom additional 
microbiological tests have been performed versus patients with clinical diagnosis alone. 
 
 
 

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Secondary: 
3. To evaluate the appropriateness of the empiric antibiotic therapy based on clinical diagnosis 
alone and after laboratory confirmation.    
4. To estimate the correlation between clinical diagnosis with laboratory diagnosis in patients with 
undifferentiated fever. 
5. To compare the outcome among patients whose treatment is based on microbiological 
diagnosis compared to the patients whose treatment is based on clinical diagnosis alone.  
  
5. APPROACH: STUDY DESIGN AND METHODOLOGY 
This will be a randomized, open-label, prospective study which will include patients admitted to the 
hospital with an undifferentiated febrile illness of 3-days or longer duration. In addition to the 
history, physical exam and routine laboratory tests and X-ray, when clinically indicated, a battery of 
laboratory investigations will be performed to identify the etiology of fever.  
a. Definition of Acute Undifferentiated Fever (AUF) 
Acute undifferentiated fever (AUF) will be defined as a febrile illness with a temperature of 38°C 
(100.4°F) or higher for at least a duration of 3 days to 21 days, which presents without an 
obvious cause on the basis of clinical, radiological, and rapidly available laboratory 
investigations, and not associated with nosocomial infection, neutropenia, or 
immunosuppressing conditions [12].  
b. Study population 
This study will be conducted in the inpatient ward of Patan Hospital.  
Patients admitted with undifferentiated fever of at least for 3 days to 21 days duration will be 
enrolled in this study.  
 
c. Inclusion criteria 
1. Age 16 years or older 
2. Documented fever (T ≥100.4F or ≥38C) of 3 days to 21 days duration 
3. No obvious diagnosis found on the basis of clinical, radiological, or initial routine laboratory 
tests*  
4. Admitted to the medical unit of hospital 
5. Signed ICF (informed consent form) 
 
(* Initial routine laboratory tests for an etiologic diagnosis of febrile illness work up include- 
blood and urine cultures, malaria RDT or thick and thin smears, sputum culture and 
GeneXpert for MTB (if suspected on chest X-ray), and serologies for scrub typhus, dengue, 
Dengue, Scrub typhus, Leptospirosis, Brucellosis, Kala azar, and HIV, as indicated based on 
clinical picture)    
 
d. Exclusion criteria 
1. Fever of less than 3 days or more than 21 days duration  
2. Outpatients 

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3. Patients being discharged from Emergency Room  
4. Children (younger than 16 years in age)** 
5. Immunocompromised patients 
6. Neutropenia (ANC <500 per cumm) 
7. Known case of HIV infection 
8. Admission to a hospital for 48-hours or longer within past 30 days  
(**Children under 16 years will be excluded since they have different causes of febrile 
illnesses requiring a different set of lab tests which is beyond the scope of this study.) 
 
e. Informed Consent 
 
Patients admitted to the Medical Ward with febrile illness will be screened for the study 
inclusion and exclusion criteria. Patients who meet all inclusion criteria and none of the 
exclusion criteria will be enrolled in the study after taking written voluntary consent on an 
Informed Consent Form (ICF) approved by the institutional research committee (IRC). If patient 
is unable to give consent, his or her legal guardian will be contacted for written consent on 
behalf of the patient.   
 
f. Clinical and Laboratory Procedures 
The main purpose of the current study is to find additional causes of febrile illnesses in Nepal 
which are not tested or detected with the routine laboratory tests, and are generally labelled as 
an undifferentiated fever. In a patient who meets above inclusion and exclusion criteria and has 
undergone standard of care evaluation as listed below, the following baseline data including 
results of the standard of care evaluations will be collected. Then, additional lab tests as noted 
below will be performed to find an etiology of the febrile illness. These new tests are neither 
readily available nor routinely performed as a part of the fever work up in Nepal.    
Standard of Care Evaluation 
Standard of care evaluation of a febrile patient is done by the treating medical team which 
usually consists of the following set of clinical, basic laboratory, radiological and microbiological 
evaluations:   
1. History and Physical Examination 
2. Basic Laboratory investigations: As a part of standard of care, following routine laboratory 
tests are done on patients with a febrile illness:  
Complete blood count (CBC), Renal function test (RFT), Liver function test (LFT), CRP, 
ESR, Urine routine exam  
3. Radiological investigations: Chest X-ray, USG abdomen, and other Radiological investigations 
(as indicated) 
4. Microbiological investigations:  
a. Blood cultures x 2  
b. Urine culture/sensitivity in symptomatic patients or in patients with >10 pus cells in 
urinalysis 
c. Culture of sputum, pus, and other samples as indicated based on clinical findings 

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d. Routine serologies- currently the following serological tests are routinely done for 
evaluation of fever:  
HIV, Dengue, Scrub typhus, Leptospirosis, Brucellosis, Kala azar, malaria 
(RDT/thick and thin smears) 
 
Additional Evaluations for the Study 
For patients without an obvious cause of fever and an inconclusive work up after standard of 
care evaluations, additional serological tests as a part of undifferentiated febrile illness work up 
will be performed, including: 
a. Clinical evaluation: includes thorough history and physical examinations based on a 
standard questionnaire and data collection tool 
b. Additional Microbiological Evaluations: If above listed laboratory and 
microbiological tests do not indicate a conclusive diagnosis, the patient’s blood will 
be processed for additional Serological tests as a part of undifferentiated febrile 
illness work up. These tests will help to establish diagnosis of the following 
infections: 
- 
Murine typhus  
- 
Bartonellosis  
- 
Q-fever  
- 
Chikungunya  
- 
Zika  
- 
Japanese B encephalitis 
- 
West Nile virus 
 
5. Use of antimicrobials in the patients with and without an etiological diagnosis and their 
outcome at the time of discharge from the hospital will be recorded.  
 
g. Laboratory Methods 
For the purpose of the study, following samples will be collected from 50% of randomly assigned 
patients for additional microbiological testing.  
1. Blood cultures, 2 sets from 2 different sites from all study patients (20 mL total) 
2. Serum samples for serological testing (4 mL) 
After obtaining appropriate consent, total 25 mL blood will be collected from each patient 
assigned to the additional testing arm. Blood samples will be cultured in liquid media and held in 
the incubator for 7-days.  
Urine will be taken to the lab for routine examination and culture will be performed only if there 
are >10 pus cells or patient reports urinary symptoms.  
Gram stain, AFB stain and Genexpert will be performed on sputum samples.  
As a part of routine febrile illness work up, the serum samples will be tested for HIV, scrub 
typhus, dengue, leptospirosis, brucella, malaria, and kala azar.  

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Additional serum sample will be processed for further serological testing for Chikungunya, Zika, 
Coxiella burnetii (Q-fever), Bartonella henselae (Bartonellosis), Rickettsia typhi (murine typhus), 
Japanese B encephalitis, and West Nile virus using ELISA and/or Rapid Diagnostic Test (RDT), as 
applicable. Although molecular tests have a higher sensitivity and specificity, molecular testing 
has not been planned for this study because of the budget limits.    
 
h. Data collection  
After taking written informed consent from patient or his/her legal guardian, data will be 
collected from patients who meet all of the inclusion criteria and none of the exclusion criteria. 
Standard questionnaires will be used and recorded on a designated laptop device protected and 
secured with password. Only the study data collection and management team will have access 
to the collected data. Physical exams and initial clinical diagnosis will be transferred from 
patient’s chart, or by interviewing patients, if needed, using a standardized questionnaire. All 
available lab data and treatment (antimicrobials and other relevant therapy) will be recorded. 
Chest X-ray, USG, CT scan, and echocardiogram reports will be collected, if applicable to the 
diagnosis. 
 
i. 
Follow up and Expected Outcomes 
During the hospital stay, patients will be followed up for new laboratory results, changes in 
treatment plan and outcomes. The patient outcomes will be recorded as: 
a. time to defervescence;  
b. de-escalation or readjustment in antimicrobials; 
c. complete recovery and discharge vs. partial recovery and discharge vs. death.  
 
j. 
Data Collection, Data Analysis and Sample Size  
Demographic, clinical, radiological, and laboratory data will be collected and entered into a 
password protected database. To minimize the errors, data will be directly entered into the 
database using REDCap Database questionnaires.  
 
Data analysis will include the followings: 
1. Demographics and baseline characteristics of patients will be analyzed and compared 
between the two groups.  
2. Clinical diagnosis and laboratory confirmation of diagnosis will be used to estimate the 
proportions of various infectious etiologies of febrile illness in patients presenting with a 
febrile illness of 3-21 days duration. (Specific aim-1) 
3. Number, type, dose and duration of antimicrobials used to treat the patients will be 
collected and compared between the two groups: clinical diagnosis alone versus clinical plus 
microbiological diagnosis. (Specific aim 2) 
4. Appropriateness of the empiric antibiotic therapy will be determined by an independent 
physician expert after the laboratory confirmation of the diagnosis. Comparison between 
the two groups (clinical diagnosis vs. microbiological diagnosis) will be done by calculating 
the proportion of cases who received appropriate antibiotics divided by the total number of 
cases for whom empiric antibiotics were administered. (Specific aim-3)  

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5. Pretest probability for clinical diagnosis and posttest probability after laboratory-based 
diagnosis will be calculated and correlated for bacterial, rickettsial, viral infections versus 
other causes of fever. (Specific aim-4)  
6. Clinical outcomes will be compared for patients whose treatment is based on clinical 
diagnosis alone (Group 1) versus the patients whose treatment is based on clinical plus 
microbiological diagnosis (Group 2). Composite Positive outcome (complete or partial 
recovery) and Negative outcome (in-hospital death) will be compared between the two 
groups. (Specific aim-5) 
 
Demographics and baseline characteristics of patients will be expressed in percentages, 
means±SD, 95% confidence intervals, and/or median±IQR, as appropriate. Proportions will be 
compared between the two groups with Chi-square tests. Numerical variables will be expressed 
as Means/SD/95% confidence intervals and Median/IQRs, whereas Student T test or Mann-
Whitney test, respectively, will be used for statistical comparisons. Alpha will be set at 95% 
(level of significance, p <0.05). 
 
For Specific aims 2 and 3, total daily defined dose (DDD) for each antimicrobial agent received 
by patients will be calculated following WHO guidelines. The total DDD, number of antibiotics, 
and Duration of antibiotics used will be compared between the groups by calculating 
Means/SD/95% confidence intervals. Similarly, appropriateness of antibiotic uses will be tested 
with cross-tabulation using Chi-square analysis.  
 
For specific aim 4, pre-test probability and post-test probability will be determined by both using 
standard calculations and using Fagan’s Nomogram. Correlations between pre- and post-test 
probabilities for bacterial, rickettsial, viral vs. other causes will be evaluated.    
  
For specific aim 5, the hazard ratio using the Cox proportional hazard model will be calculated to 
compare the outcomes between the groups. Since multiple factors, such as comorbidities, 
severity of illness at baseline, type of infection and host immune status, can influence the 
outcomes, adjustment for these factors will be considered during the univariate and 
multivariate outcome analyses. Updated Charlson comorbidity index (Quant et al.) will be 
calculated and used for comparison between the groups [15]. Similarly, qSOFA will be used to 
stratify the patients by severity of infection [16].  
 
REDCap (Research Electronic Data Capture) software will be used for questionnaire and 
database entry. SPSS version 25 (IBM, USA) will be used for statistical analysis.  
 
 
Sample Size: 
For this study, a total of 234 randomly selected patients with acute undifferentiated febrile 
illness will be enrolled with a fever of 3 days duration. Of these 234 patients, 117 will be 
randomly assigned to undergo additional microbiological testing.  

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Based on previous studies and number of patients needed to include less common seasonal 
infections, this study will enroll patients throughout a period of 12 months. Cluster sample 
technique will be used to select 20 patients per month including 10 patients who will be 
randomly selected to undergo additional microbiological testing.   
 
Sample size determination and assumptions:  
For specific aim 1, a reduction of antimicrobial use in about 15% patients is expected after 
additional laboratory confirmation of the diagnosis including viral infections. Assuming this 
decrease in antibiotics from 90% to 75%, 95% confidence interval, and 80% power, required 
sample size would be 97 in each arm.  With similar assumptions for specific aim 3, an 
improvement in antimicrobial therapy from 70% to 90% would require 59 patients. 
For specific aim 2, assuming an improvement in identification of causative agents from 30% to 
50%, 95% confidence interval, and 80% power, required sample size would be 91.  
For specific aim 5, an improvement in clinical outcome for patients whose treatment is based on 
microbiological diagnosis by 20%, from 60% to 80%, 95% confidence interval, and 80% power, 
the required sample size would be 79 in each arm. 
Based on above calculations, minimum sample size needed would be 97 in each arm. Including a 
20% loss for failure to consent and loss to follow up, total sample size needed will be 117 in each 
arm.  
 
 
k. Limitations and Alternative Strategies 
This study has been proposed for only one center but depending on the findings from this study, 
it will be expanded to other centers in the future. Additional microbiological methods will be 
limited to serological testing alone, mainly due to budgets limits. Although molecular tests are 
more sensitive and specific, molecular testing has not been planned for this study because of 
the budget limits.  
 
This one center study will serve as a pilot study with limited number of patients has been 
planned because of the budget limit. However, the results of this study will be useful for 
treatment of febrile patients. Additionally, the results of this study will be useful for designing 
larger, multi-centered studies in the future.     
 
 
 
6. PROTECTION OF HUMAN SUBJECTS (ETHICAL CONSIDERATIONS) 
This clinical research project will be submitted for a full board approval by the PHAS Institutional 
Research Committee. Patients will be enrolled from Patan Hospital’s inpatient ward after taking 
informed consent on an informed consent form (ICF) as approved by the PAHS IRC. All patients will be 
provided written information about the study in Nepali language before enrollment. For patients who 

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are unable to understand Nepali, either a family member or a hospital staff will be used as an 
interpreter to overcome the language barrier and to help the patient understand the study process and 
its potential harms and benefits.  
This project will have equal opportunities for inclusion of women and minorities. Children will be 
excluded because of a different spectrum of infectious agents outside the scope of this study.  
All personal data collected for the study will be stored in a confidential manner in a secure computer 
with password protection. Only the research team and assigned data analyst will have access to the 
database. Any new information received during the study will be revealed to the patient. Patients 
enrolled in the study will be able to withdraw their consent at anytime they wish. Any serious adverse 
events will be reported to the IRC. 
Registration: After IRC approval, this study will be registered at Clinical Trial.gov. 
 
7. DATA SAFETY AND MONITORING PLAN 
All data entered into the database will be secured and password protected. The study principal 
investigator and co-investigators will review data from time to time for consistency in data collection. An 
interim data analysis will be done after enrollment of 25% and 50% of the patients.  
Data safety and monitoring will be done by an independent committee known as DSM board consisting 
of two senior level medical doctors including one internal medicine physician and one microbiologist, 
and a representative of ethics committee or IRC.   
The DSMB will review the interim data and events reports to conclude with their recommendations to 
whether the study should continue without change, be modified, or be terminated. 
 
8. EXPECTED OUTPUTS (STUDENT TRAINING AND PUBLICATIONS) 
This study will help the internal medicine residents and infectious disease fellows at PAHS to learn to 
design and conduct clinical studies. They will also participate in patient enrollment, data collection, 
analysis and presentation/publication phases of the study.  
The results of this study will be shared within and outside the institution in the form of scientific 
presentations and publication in peer-reviewed journals.  
 
9. ORGANIZATION OF THE STUDY 
Study staff will consist of the followings: 
 
 
Staff category 
Number  
Role in study 
Principal Investigator 
1 
Design, Supervision, 
Conduction, Data monitoring, 
Analysis, Write up for 

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publication, and Scientific 
presentations  
Co-Investigators 
2 
Design, Enrollment, Data 
collection supervision, Patient 
monitoring, Scientific 
presentation and publication  
Researchers (Infectious 
Diseases fellows and Internal 
medicine residents) 
2-3 
 
Patient enrollment, consent, 
clinical follow up and data 
collection, data entry 
Statistician 
1  
Data analysis 
 
 
10. STUDY TIMELINE 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
11. DETAILED BUDGET 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Events 
Details 
Timeline 
Preparatory Phase 
IRC approval 
Jan 2025 
Team preparation 
Oct-Dec 2024 
Lab setup/supplies 
Oct-Dec 2024 
Interventional Phase 
Enrollment (12 months) 
1 Feb 2025- 31 Jan 2026 
Laboratory tests 
1 Feb 2025- 31 Jan 2026 
Follow up 
1 Feb 2025- 31 Jan 2026 
Data Collection 
1 Feb 2025- 31 Jan 2026 
Data Analysis and 
Reporting Phase 
Statistical analysis 
1 Feb 2026- 31 Mar 2026 
Report and Manuscript 
preparations  
Apr-Jun 2026 
Budget Item 
Unit Cost (Rs.) 
Number 
Total Cost (Rs.) 
A. Personnel 
Dr. Janak Koirala (10% effort) 
Dr. Nora Ranjitkar (20% effort) 
Dr. Suman Thapa (10% effort) 
 
129011. 
82074. 
76867. 
 
0.10 
0.20 
0.10 
 
12,900 
16,414. 
7686. 
B. Equipment, Consumables, Services 
Serological test by ELISA and/or RDT 
Other lab supplies for sample 
collection, processing and 
transportation of samples 
 
500 
 
200 
 
7x97 
 
97 
 
339,500 
 
19,400 
C. Travel/Transportation of sample 
NA 
NA 
NA 
D. Facilities and Administrative  
Presentation and publication 
 
 
10,000 
 
 
1 
 
10,000 
TOTAL =   
 
Rs. 405,900.  

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12. ASSOCIATION TO NATIONAL PRIORITY 
Under the Epidemic and Outbreak Surveillance Programme of Government of Nepal, Ministry of Health 
and Population (MOHP), the Early Warning and Reporting System (EWARS) has been set up in 81 sites of 
the country. However, the laboratory testing for various new infectious diseases prevalent in the 
Southeast region are not available at these sites or in major hospitals of Nepal. This study is expected to 
identify additional infectious diseases prevalent in Nepal with the laboratory confirmation of the 
etiologic agents. 
Increasing antimicrobial resistance has been identified one of the major causes of morbidity and 
mortality in hospitals. Therefore, control of antimicrobial resistance has been another national priority. 
and improve the appropriate use of antimicrobial agents with targeted therapy. The laboratory tests 
used in this study will help to identify additional infectious diseases. This information will be helpful to 
the clinicians to design initial empiric antibiotic therapy based on epidemiological and clinical features. A 
more appropriate choice of antibiotics will decrease the unnecessary use of multiple broad-spectrum 
antibiotics, which in turn will help to reduce antimicrobial resistance, and improve patient outcome. 
New information discovered from the study expected to be useful for making policies to prevent and 
control various infectious diseases such as vector-borne infections and other communicable diseases.  
 
 
13. BIBLIOGRAPHY AND REFERENCES 
 
1. Pokharel S, Karki M, Acharya B, Marasini B, Arjyal A. Outbreak of acute undifferentiated febrile 
illness in Kathmandu, Nepal: clinical and epidemiological investigation. BMC Infect Dis. 2020 Jan 
30;20(1):89. PMID: 32000695. 
2. Koirala KD, Chappuis F, Verdonck K, Rijal S, Boelaert M. Persistent febrile illnesses in Nepal: A 
systematic review. Indian J Med Res. 2018 Oct;148(4):385-395. PMID: 30666001. 
3. Aiemjoy K, Tamrakar D, Saha S, Naga SR, Yu AT, Longley A, Date K, Hemlock C, Qamar FN, Saha SK, 
Luby SP, Garrett DO, Andrews JR, Bogoch II. Diagnostic Value of Clinical Features to Distinguish 
Enteric Fever From Other Febrile Illnesses in Bangladesh, Nepal, and Pakistan. Clin Infect Dis. 2020 
Dec 1;71(Suppl 3):S257-S265. PMID: 33258936. 
4. Murdoch DR, et al. The etiology of febrile illness in adults presenting to Patan Hospital in 
Kathmandu, Nepal. Am. J. Trop. Med. Hyg., 70(6), 2004. 
5. Thompson CN, Blacksell SD, Paris DH, Arjyal A, Karkey A, Dongol S, Giri A, Dolecek C, Day N, Baker S, 
Thwaites G, Farrar J, Basnyat B. Undifferentiated febrile illness in Kathmandu, Nepal. Am J Trop Med 
Hyg. 2015 Apr;92(4):875-878. 
6. Zimmerman MD, Murdoch DR, Rozmajzl PJ, Basnyat B, Woods CW, Richards AL, Belbase RH, 
Hammer DA, Anderson TP, Reller LB. Murine typhus and febrile illness, Nepal. Emerg Infect Dis. 2008 
Oct;14(10):1656-9. PMID: 18826840. 
7. Rijal KR, Adhikari B, Adhikari N, Dumre SP, Banjara MS, Shrestha UT, Banjara MR, Singh N, Ortegea L, 
Lal BK, Thakur GD, Ghimire P. Micro-stratification of malaria risk in Nepal: implications for malaria 
control and elimination. Trop Med Health. 2019 Mar 27;47:21. PMID: 30976192. 

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8. Poudyal P, Sharma K, Dumre SP, Bastola A, Chalise BS, Shrestha B, Poudel A, Giri A, Bhandari P, Shah 
Y, Poudel RC, Khadka D, Maharjan J, Ngwe Tun MM, Morita K, Pandey BD, Pandey K. Molecular 
study of 2019 dengue fever outbreaks in Nepal. Trans R Soc Trop Med Hyg. 2021 Jun 2;115(6):619-
626. PMID: 32987406. 
9. Gupta BP, Haselbeck A, Kim JH, Marks F, Saluja T. The Dengue virus in Nepal: gaps in diagnosis and 
surveillance. Ann Clin Microbiol Antimicrob. 2018 Jul 16;17(1):32. PMID: 30008269. 
10. Basnet S, Koirala S, Pandey B, Koirala J. COVID-19 Containment Efforts of a Low-Resource Nation: 
The First Four Months in Nepal. Cureus. 2020 Jul 1;12(7):e8946. PMID: 32765991. 
11. Lamichhane P, Pokhrel KM, Alghalyini B, Zaidi ARZ, Alshehery MZ, Khanal K, Bhattarai M, Yadav A. 
Epidemiology, clinical characteristics, diagnosis, and complications of scrub typhus infection in 
Nepal: a systematic review. Ann Med Surg (Lond). 2023 Sep 5;85(10):5022-5030. PMID: 37811079. 
12. Susilawati TN, McBride WJ. Undiagnosed undifferentiated fever in Far North Queensland, Australia: 
a retrospective study. Int J Infect Dis. 2014 Oct;27:59-64. Epub 2014 Aug 27. PMID: 25173425. 
13. Epidemic and Outbreak Surveillance Programme. Government of Nepal, Ministry of Health and 
Population, Epidemiology and Disease Control Division. Available at: https://mohp.gov.np/program/ 
epidemic-and-outbreak-surveillance-programme/en (Accessed on 8 Dec 2023) 
14. National Action Plan For Antimicrobial Resistance Nepal. Government of Nepal, Ministry of Health 
and Population, 2021-2026. 
15. Quan, H. et al. Updating and validating the Charlson comorbidity index and score for risk adjustment 
in hospital discharge abstracts using data from 6 countries. Am. J. Epidemiol. 173, 676–682. 
16. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The third 
international consensus definitions for sepsis and septic shock (Sepsis-3) JAMA. 2016;315(8):801–
810. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX – 1. 
 
DEFINe Study Questionnaire for Database 
 
 
Screening Questions 
A. Definition of Acute Undifferentiated Fever (AUF) 
Acute undifferentiated fever (AUF) will be defined as a febrile illness with a temperature of 38°C 
(100.4°F) or higher for at least a duration of 3 days to 21 days, which presents without an 
obvious cause on the basis of clinical, radiological, and rapidly available laboratory 
investigations, and not associated with nosocomial infection, neutropenia, or 
immunosuppressing conditions [10].  
No. 
Question 
Yes 
No 
a1 
Temperature 38C (100.4F) or higher? 
 
 
a2 
Duration of fever > 3 days and <21 days? 
 
 
a3 
Cause of fever unknown after routine work up including 
clinical, radiology and lab evaluations? 
 
 
a4 
Is it a Nosocomial infection? 
 
 
a5 
Neutropenia ruled out? 
 
 
a6 
Immunosuppression present? 
 
 
a7 
Does the patient meet all criteria for undifferentiated fever? 
 (Needs to answer “Yes” to 1,2&3, and “No” to 4,5,&6) 
 
 
 
 
 
 
 
If answer to a7 is “Yes”, proceed with questions in “b. Inclusion/Exclusion criteria” 
 
B. Inclusion/Exclusion criteria 
Patients needs to meet al of the following Inclusion Criteria before enrollment: 
No. 
Question 
Yes 
No 
b1 
Age 16 years or older 
 
 
b2 
Documented fever (T ≥100.4F or ≥38C) of 3 days to 21 days 
duration 
 
 
b3 
No obvious diagnosis found on the basis of clinical, 
radiological, or initial routine laboratory tests* 
 
 
b4 
Admitted to the medical unit of Patan Hospital 
 
 
b5 
Informed consent form (ICF) signed 
 
 
B6 
Patient meets all of the above inclusion criteria 
 
 
 
 
 
 
 

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(* Initial routine laboratory tests for an etiologic diagnosis of febrile illness work up include- 
blood and urine cultures, malaria RDT or thick and thin smears, sputum culture and 
GeneXpert for MTB (if suspected on chest X-ray), and serologies for scrub typhus, dengue, 
Dengue, Scrub typhus, Leptospirosis, Brucellosis, Kala azar, and HIV, as indicated based on 
clinical picture)    
 
 
Patient should meet none of the Exclusion criteria below: 
 
No. 
Question 
Yes 
No 
b7 
Children (younger than 16 years in age)** 
 
 
b8 
Fever of less than 3 days or more than 21 days duration 
 
 
b9 
Outpatients 
 
 
b10 
Patients being discharged from Emergency Room 
 
 
b11 
Immunocompromised patients 
 
 
b12 
Neutropenia (ANC <500 per cumm) 
 
 
b13 
Known case of HIV infection 
 
 
b14 
Hospital admission for 48-hours or longer within past 30 days  
 
 
b15 
Patient does not meet any of the above exclusion criteria 
 
 
 
(**Children under 16 years will be excluded since they have different causes of febrile 
illnesses requiring a different set of lab tests which is beyond the scope of this study.) 
 
If patient meets all of Inclusion criteria and none of the exclusions, proceed with 
patient consent followed by randomization and enrollment. 
 
Randomization Result 
 
 
Information 
Remarks 
Patient’s study ID no. 
D001, D002, D003,…….. 
 
Date of Birth (in AD) 
DD/MM/YYYY 
 
Address (District where patient lives) 
District name 
 
Admission date (in AD) 
DD/MM/YYYY 
 
Unit location 
Ward, HDU, ICU 
 
Randomization Arm 
 
 
Group A, Group B 
 
 
 
 
 
 
 
 

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C. Clinical and Laboratory Questionnaire (Standard of Care Evaluation for all participants) 
No. 
Questions/ Variables 
Data / value 
Unit  
 
BASELINE CLINICAL DATA 
 
 
C1 
Duration of fever 
No. of days 
 
C2  
Clinical Diagnosis 
 
 
C3 
System involvement 
RTI, GI, UTI, CVS, CNS, 
SSTI, Blood, Sepsis, 
Lymphatic, Other 
 
C4 
Comorbidities  
None, DM, HTN, 
COPD/chronic 
pulmonary dis., CHF, 
MI, PVD, CLD/Liver 
failure, PUD, CVA, 
hemiplegia, dementia, 
CTD, AIDS, solid 
tumor, lymphoma, 
leukemia, other  
 
C5 
Temperature in Celsius (baseline at 
presentation) 
 
Celcius 
C6 
BP (baseline at presentation) 
 
mmHg 
C7 
Pulse (baseline at presentation) 
 
per min. 
C8 
Resp rate (baseline at presentation) 
 
per min. 
C9 
SaO2 (baseline at presentation) 
 
% 
C10 
WBC (baseline at presentation) 
 
per cumm 
C11 
ANC (baseline at presentation) 
 
per cumm 
C12 
ALC (baseline at presentation) 
 
per cumm 
C13 
AEC (baseline at presentation) 
 
per cumm 
C14 
Hgb (baseline at presentation) 
 
g/dL 
C15 
Plt 
 
per cumm 
C16 
CRP 
 
 
C17 
ESR 
 
per hour 
C18 
Cr 
 
 
C19 
ALT 
 
 
C20 
AST 
 
 
C21 
UA: pus cells  
 
 
 
 
 
 
 
MICROBIOLOGICAL DATA 
 
 
M1 
Blood culture result 
Organism name 
 
M2 
Urine culture result 
Organism name 
 
M3 
Sputum culture result 
Organism name 
 
M4 
Pus culture  
Organism name 
 
M5 
GeneXpert 
Positive/negative 
 
M6 
Dengue serology 
NS1, IgM, IgG 
 
M7 
Scrub typhus 
Positive/negative 
 

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M8 
Malaria RDT 
P falciparum, P vivax, 
other 
 
M9 
Kala Azar 
Positive/negative 
 
M10 Brucella 
Positive/negative 
 
M11 MTB 
Positive/negative 
 
M12 Leptospirosis  
Positive/negative 
 
M13 Hepatitis A 
IgM 
 
M14 Hepatitis E 
IgM 
 
M15 Hepatitis B 
HBsAg 
 
M16 Hepatitis C 
HBC Ab 
 
M17 HIV 
 
 
M18 Other test results (…………………………) 
 
 
M19 Other test results (…………………………) 
 
 
M20 Other test results (…………………………) 
 
 
 
 
 
 
 
 
 
 
 
RADIOLOGICAL DATA 
 
 
R1 
X-ray finding 
Consolidation, 
Infiltrates, Pleural 
effusion, CHF, ARDS, 
other 
 
R2 
CT Chest 
Consolidation, 
Infiltrates, Pleural 
effusion, CHF, ARDS, 
other 
 
R3 
CT Abdomen 
Hepatomegaly, 
pancreatitis, 
Cholecystitis, Gall 
stone, Ascites, 
appendicitis, typhlitis,  
intrabdominal 
abscess, 
lymphadenopathy, 
other  
 
R4 
USG Abdomen 
Hepatomegaly, 
pancreatitis, 
Cholecystitis, Gall 
stone, Ascites, 
appendicitis, typhlitis,  
intrabdominal 
abscess, 
lymphadenopathy, 
other 
 
R5 
CT/MRI skull 
Abscess, Meningitis, 
Encephalitis, 
parameningeal focus, 
other 
 

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R6 
Echocardiogram 
Valvular vegetation, 
Pericardial effusion, 
other 
 
 
 
 
 
 
ADDITIONAL SEROLOGICAL EVALUATIONS  
(for Group B patients only) 
Results 
 
D1 
Murine typhus  
Positive, negative, NA 
 
D2 
Bartonella 
Positive, negative, NA 
 
D3 
Q-fever  
Positive, negative, NA 
 
D4 
Chikungunya  
Positive, negative, NA 
 
D5 
Zika  
Positive, negative, NA 
 
D6 
Japanese B encephalitis 
Positive, negative, NA 
 
D7 
West Nile virus 
Positive, negative, NA 
 
 
 
 
 
 
 
D. Severity and Comorbidity indices 
i. qSOFA score 
 
Altered mental status GCS <15 
Yes  
No 
Respiratory rate ≥22/min 
Yes  
No 
Systolic BP ≤100 mmHg 
Yes  
No 
Total Score 
 
 
 
 
ii. Updated Charlson Comorbidity index score  
 
Index 
Score 
CHF 
2 
Chronic pulmonary disease 
1 
Connective tissue disease 
1 
Dementia  
1 
DM with end organ damage 
1 
Liver disease-  Mild 
 
 
Moderate to severe 
2 
4 
Renal disease- moderate to severe 
2 
Hemiplegia 
2 

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Page 19 
 
Tumor without metastasis 
Tumor with metastasis 
2 
6 
Leukemia 
2 
Lymphoma 
2 
AIDS 
2 
 
Total Score 
 
 
 
 
E. Antimicrobials Dose and Durations 
   
 
Antimicrobial agent used 
Route  
Total Dose per 
day (Grams) 
Total No. of 
days used 
AM1  
PO, IV 
grams 
No. of days 
AM2  
 
 
 
AM3  
 
 
 
AM4  
 
 
 
AM5  
 
 
 
 
 
 
 
 
AM6 Appropriateness of 
Antimicrobials used  
(to be determined by an 
independent physician) 
Yes, No, 
Partially  
 
 
 
 
 
 
 
 
 
F. Outcomes 
 
 
 
 
 
 
 
 
 
Outcome variable 
Results 
 
Outcome at the end of hospitalization 
Discharge- complete recovery,  
Discharge- partial recovery, 
Death 
 
Composite outcome at the end of 
hospitalization 
Good Outcome (complete plus 
partial recovery) 
Poor outcome (Death)