RRC APPLICATION FORM
RESEARCH PROTOCOL
NUMBER: 2008-025
FOR OFFICE USE ONLY
RRC Approval:
ERC Approval:
AEEC Approval:
Yes /
Yes /
Yes /
No
No
No
Date: 19 August 2008
Date:24 Sep 2008
Date:
Protocol Title: Hospital based selected febrile illness study in Bangladesh
Short title (in 50 characters including space): Hospital based selected febrile illness study
Theme: (Check all that apply)
Nutrition
Emerging and Re-emerging Infectious Diseases
Population Dynamics
Reproductive Health
Vaccine Evaluation
HIV/AIDS
Environmental Health
Health Services
Child Health
Clinical Case Management
Social and Behavioural Sciences
Key words: selected, febrile, illness, study, Bangladesh --------------------------------------------------------------------Relevance of the Protocol:
In Bangladesh, there are a lot of infectious diseases that cause substantial morbidity and mortality, but their
contributions to febrile illnesses is unknown due to lack of systematic observation. Malaria, dengue,
chikungunya, rickettsia, leptospira and bartonella are circulating in the South Asian region but we know little
about their geographical distribution in Bangladesh. Bangladesh is geographically surrounded by India where 151
districts of 10 states had experienced outbreaks of chikungunya fever since 2005. All the extrinsic and intrinsic
risk factors associated with leptospira are also evident in Bangladesh. Population based surveillance for febrile
illness in Kamalapur community in urban Dhaka has revealed that Leptospira species are circulating within that
surveillance area. Recently in a district in Bangladesh scrub typhus and Indian tick typhus have been detected in a
small scale survey. Bartonella was recently reported from mammals of Bangladesh and human disease has been
reported from Indonesia and Thailand. As a first step in better understanding the contribution of these pathogens
to the infectious disease burden in Bangladesh, we propose a 12 month national study to investigate their role in
causing human febrile illness in Bangladesh. This will be a useful first step towards appropriate treatment and
control of these potentially important pathogens.
Centre’s Priority (as per Strategic Plan, to be imported from the attached Excel Sheet):
4.1: Define the epidemiology and burden of selected infectious diseases and identify effective strategies for
prevention and control.
Programmes:
Child Health Programme
Nutrition Programme
Programme on Infectious Diseases & Vaccine Science
Poverty and Health Programme
Principal Investigator (Should be a Centre’s staff)
Stephen P Luby, MD., Head, PIDVS, HSID, ICDDR,B
Health and Family Planning Systems Programme
Population Programme
Reproductive Health Programme
HIV/AIDS Programme
DIVISION:
CSD
HSID
LSD
PHSD
Address (including e-mail address):
ICDDR,B, Mohakhali, Dhaka 1212, Bangladesh
Email: [email protected]
1
Co-Principal Investigator(s): Internal
Labib Imran Faruque
Co-Principal Investigator(s): External:
Mahmudur Rahman
(Please provide full official address including e-mail address and Gender)
Male
Director, Institute of Epidemiology, Disease Control and Research (IEDCR)
Mohakhali, Dhaka 1212, Bangladesh
Phone: +880-2-8821237
Email: [email protected]
Co-Investigator(s): Internal:
Emily Gurley
Rashidul Haque
Rashid Zaman
Co-Investigator(s): External
(Please provide full official address including e-mail address and Gender)
Renee Galloway, CDC/CCID/NCZVED,USA and [email protected]
Michael Kosoy, CDC/CCID/NCZVED,USA and [email protected]
Ying Bai, CDC/CCID/NCZVED,USA and [email protected]
Ann Powers, CDC/CCID/NCZVED,USA and [email protected]
Robert Massung, CDC/CCID/NCZVED,USA and [email protected]
William L. Nicholson, CDC/CCID/NCZVED,USA and [email protected]
A. S. M. Alamgir, IEDCR, Bangladesh and [email protected] (Male)
Student Investigator(s): Internal (Centre’s staff):
Student Investigator(s): External:
(Please provide full address of educational institution and Gender)
Collaborating Institute(s): Please Provide full address
Institution # 1
Country
Contact person
USA
Renee Galloway
Michael Kosoy
Ying Bai
Ann Powers
Robert Massung
William L. Nicholson
Department
(including Division, Centre, Unit)
(CDC/CCID/NCZVED)
Institution
(with official address)
Center for Disease Control and Prevention (CDC)
Fort Collins, Atlanta
Directorate
(in case of GoB i.e. DGHS)
Ministry (in case of GoB)
2
Institution # 2
Country
Contact person
Department
(including Division, Centre, Unit)
Bangladesh
Prof. Mahmudur Rahman
Director
Institution
(with official address)
Institute of Epidemiology, Disease Control and Research
(IEDCR)
Mohakhali, Dhaka 1212, Bangladesh
Directorate
(in case of GoB i.e. DGHS)
Directorate General of Health Service (DGHS)
Ministry (in case of GoB)
Ministry of Health and Family Welfare (MoHFW)
Institution # 3
Country
Contact person
Department
(including Division, Centre, Unit)
Institution
(with official address)
Directorate
(in case of GoB i.e. DGHS)
Ministry (in case of GoB)
Note: If more than 3 collaborating institutions are involved in the research protocol, additional block(s) can be
inserted to mention its/there particular(s).
Population: Inclusion of special groups (Check all that apply):
Gender
Male
Female
Age
0 – 4 years
5 – 9 years
10 – 19 years
20 – 64 years
65 +
NOTE
Pregnant Women
Fetuses
Prisoners
Destitutes
Service Providers
Cognitively Impaired
CSW
Others (specify
)
Animal
It is the policy of the Centre to include men, women, and children in all research projects involving human
subjects unless a clear and compelling rationale and justification (e.g. gender specific or inappropriate with
respect to the purpose of the research) is there. Justification should be provided in the `Sample Size’ section of
the protocol in case inclusiveness of study participants is not proposed in the study.
3
Project/study Site (Check all the apply):
Dhaka Hospital
Matlab Hospital
Matlab DSS Area
Matlab non-DSS Area
Mirzapur
Dhaka Community
Chakaria
Abhoynagar
Mirsarai
Patyia
Other areas in Bangladesh
Outside Bangladesh
Name of Country:
Multi Centre Trial
(Name other countries involved):
Type of Study (Check all that apply):
Case Control Study
Community-based Trial/Intervention
Program Project (Umbrella)
Secondary Data Analysis
Clinical Trial (Hospital/Clinic)
Family Follow-up Study
NOTE: Does the study meet the definition of clinical studies/trials
Journal Editors (ICMJE)? Yes
No
Cross Sectional Survey
Longitudinal Study (cohort or follow-up)
Record Review
Prophylactic Trial
Surveillance/Monitoring
Others:
given by the International Committee of Medical
Please note that the ICMJE defined clinical trial as “Any research project that prospectively assigns human
subjects to intervention and comparison groups to study the cause-and-effect relationship between a medical
intervention and a health outcome”.
If YES, after approval of the ERC, the PI should complete and send the relevant form to provide required
information about the research protocol to the Committee Coordination Secretariat for registration of the study
into websites, preferably at the www.clinicaltrials.gov. It may please be noted that the PI would require to provide
subsequent updates of the research protocol for updating protocol information in the website.
Targeted Population (Check all that apply):
Expatriates
Immigrants
Refugee
No ethnic selection (Bangladeshi)
Bangalee
Tribal group
Consent Process (Check all that apply):
Written
Oral
None
Bengali Language
English Language
Proposed Sample Size:
720 samples in a year
Sub-group (Name of subgroup (e.g. Men, Women) and Number
Name
(1)
(2)
Number
Name
Number
(3)
(4)
Total sample size: 0
Determination of Risk: Does the Research Involve (Check all that apply):
Human exposure to radioactive agents?
Fetal tissue or abortus?
Investigational new device?
(specify:
)
Existing data available from Co-investigator
Human exposure to infectious agents?
Investigational new drug
Existing data available via public archives/sources
Pathological or diagnostic clinical specimen only
Observation of public behaviour
New treatment regime
4
Yes
No
Is the information recorded in such a manner that study participants can be identified from information
provided directly or through identifiers linked to the study participants?
Yes
No
Does the research deal with sensitive aspects of the study participants’ behaviour; sexual behaviour,
alcohol use or illegal conduct such as drug use?
Could the information recorded about the individual if it became known outside of the research:
Yes
No
Place the study participants at risk of criminal or civil liability?
Yes
No
Damage the study participants’ financial standing, reputation or employability, social rejection, lead to
stigma, divorce etc.?
Do you consider this research (Check one):
Greater than minimal risk
Only part of the diagnostic test
No more than minimal risk
Minimal Risk is "a risk where the probability and magnitude of harm or discomfort anticipated in the proposed research are
not greater in and of themselves than those ordinarily encountered in daily life or during the performance of routine physical,
psychological examinations or tests. For example, risk of drawing a small amount of blood from a healthy individual for
research purposes is no greater than the risk of doing so as a part of routine physical examination".
Yes/ No
Is the proposal funded?
If yes, sponsor Name: (1)
Center for Disease Control and Prevention (CDC) -----------------------------------
(2)
Yes/No
Is the proposal being submitted for funding?
If yes, name of funding agency:
(1)
(2)
Do any of the participating investigators and/or member(s) of their immediate families have an equity relationship (e.g.
stockholder) with the sponsor of the project or manufacturer and/or owner of the test product or device to be studied or serve
as a consultant to any of the above?
IF YES, a written statement of disclosure to be submitted to the Centre’s Executive Director .
Dates of Proposed Period of Support
(Day, Month, Year - DD/MM/YY)
Beginning Date : 01/10/2008
End Date
: 31/09/2009
Cost Required for the Budget Period ($)
Years
Year-1
Year-2
Year-3
Year-4
Year-5
Total
Direct Cost
41000
Indirect
Cost
0
41000
0
Total
Cost
41000
0
0
0
0
41000
5
Certification by the Principal Investigator
I certify that the statements herein are true, complete and accurate to the best of my knowledge. I am aware that any false,
fictitious, or fraudulent statements or claims may subject me to criminal, civil, or administrative penalties. I agree to accept
the responsibility for the scientific conduct of the project and to provide the required progress reports including updating
protocol information in the SUCHONA (Form # 2) if a grant is awarded as a result of this application.
___________
____________
Signature of PI
Date
Approval of the Project by the Division Director of the Applicant
The above-mentioned project has been discussed and reviewed at the Division level as well by the external reviewers. The
protocol has been revised according to the reviewers’ comments and is approved.
Name of the Division Director
Signature
Date of Approval
6
Table of Contents
RRC APPLICATION FORM .................................................................................................................. 1
Project Summary...................................................................................................................................... 8
Description of the Research Project ......................................................................................................... 9
Hypothesis to be Tested: ...................................................................................................................... 9
Specific Aims: ...................................................................................................................................... 9
Background of the Project including Preliminary Observations ....................................................... 10
Research Design and Methods ........................................................................................................... 12
Sample Size Calculation and Outcome Variable(s) ........................................................................... 24
Facilities Available ............................................................................................................................ 24
Data Safety Monitoring Plan (DSMP) ............................................................................................... 25
Data Analysis ..................................................................................................................................... 25
Ethical Assurance for Protection of Human Rights ........................................................................... 26
Use of Animals .................................................................................................................................. 26
Literature Cited .................................................................................................................................. 27
Dissemination and Use of Findings ................................................................................................... 31
Collaborative Arrangements .............................................................................................................. 31
Biography of the Investigators ............................................................................................................... 32
Detailed Budget ..................................................................................................................................... 38
Budget Justifications .............................................................................................................................. 39
Other Support
Appendix A: Voluntary consent form (with Bengali translation) ..................................................... 41
Appendix B: Voluntary assent form (with Bengali translation) ........................................................ 45
Appendix C: Standerdized assessment form ..................................................................................... 49
Appendix D: Laboratory report form ................................................................................................ 50
Appendix E: Follow-up questionnaire .............................................................................................. 51
Appendix F: Specimen collection procedure ..................................................................................... 52
Appendix G: External reviewer’s comments [1] ............................................................................... 53
Appendix H: Responses to external reviewer’s comments................................................................ 55
Appendix I: External reviewer’s comments [2] ................................................................................. 63
Appendix J: Responses to external reviewer’s comments ................................................................. 65
Appendix K: External reviewer’s comments [3] ............................................................................... 72
Appendix L: Responses to external reviewer’s comments ................................................................ 74
Check-List
Check here if appendix is included
7
Project Summary
Describe in concise terms, the hypothesis, objectives, and the relevant background of the project. Also describe concisely
the experimental design and research methods for achieving the objectives. This description will serve as a succinct and
precise and accurate description of the proposed research is required. This summary must be understandable and
interpretable when removed from the main application.
Principal Investigator(s): Stephen P Luby
Research Protocol Title: Hospital based selected febrile illness study in Bangladesh
Total Budget US$: 41,000
Beginning Date : 1st October, 2008
Ending Date: 31st September, 2009
In Bangladesh there are numerous infectious diseases that cause substantial morbidity and mortality. Though
fever is the common complaint of most of those, in many cases the microbial etiologies of illness are unknown.
It is assumed that there are several agents that may contribute importantly to morbidity and mortality in
Bangladesh, but their contribution to febrile illnesses in Bangladesh remains unknown due to lack of systematic
observation.
The study will investigate the relative importance of dengue, malaria, leptospira, chikungunya, ricketsia and
bartonella among patients who present with fever across Bangladesh. Although malaria and dengue are known
common causes of fever in Bangladesh, their distribution in a wider geographical area and other differentials of
febrile illness like chikungunya, leptospira, rickettsia and bartonella are yet to be determined. The re-emergence
of chikungunya virus in the Indian sub-continent since December 2005 are due to a variety of social,
environmental, behavioural and biological changes which have also occurred in Bangladesh. Leptospira is
already identified in urban Dhaka. Ecological niches that would support leptospirosis are common throughout
Bangladesh. Rickettsial disease was identified in a small scale study in Mymensingh in Bangladesh, but its
contribution to febrile illness in other settings remains unknown. Bartonella is identified in the South East Asia
Region in both rodents and humans. ICDDRB collaborators are interested in a variety of pathogens. It is
efficient to look for all these pathogens together rather than single pathogen in multiple single pathogen studies.
We hypothesize that these organisms are present and cause febrile illness in Bangladesh. The objective of this
study is to identify individuals across Bangladesh and test their biological specimens for these pathogens.
This study will be implemented under direct collaboration of ICDDR,B and IEDCR. Currently, hospital based
surveillance for influenza is ongoing in twelve hospitals, six government and six non-government hospitals
across the country. Regular active surveillance for febrile patient will be carried out at both adult medicine and
paediatric inpatient and outpatient departments of six of these hospitals to characterize the presence and
distribution of different unknown causes of fever in Bangladesh. Up to 10 blood specimens will be collected
maintaining standard procedure each month from each facility and will be analyzed in ICDDR,B and US CDC.
ELISA or PCR for dengue and chikungunya, immuno chromatography test and microscopic diagnosis for
malaria, Indirect fluorescent antibody (IFA) assay and PCR for Rickettsia and culture for leptospira and
bartonella will be performed.
8
KEY PERSONNEL (List names of all investigators including PI and their respective specialties)
Name
1. Stephen P Luby
Professional Discipline/ Specialty
Medical Epidemiologist
Role in the Project
Principal Investigator
2. Mahmudur Rahman
Medical Epidemiologist
Co-Principal Investigator
3. Labib Imran Faruque
Research Fellow
Co-Principal Investigator
4. Renee Galloway
Microbiologist
Co-Investigator
5. Michael Kosoy
Research Biologist
Co-Investigator
6. Ying Bai
Researcher
Co-Investigator
7. Ann Powers
Molecular Virologist
Co-Investigator
8. Robert Massung
Microbiologist
Co-Investigator
9. William L. Nicholson
Microbiologist
Co-Investigator
10. Emily Gurley
Public Health Specialist
Co-Investigator
11. Rashidul Haque
Parasitologist
Co-Investigator
12. A. S. M. Alamgir
Virologist
Co-Investigator
13. Rashid Zaman
Medical Researcher
Co-Investigator
Description of the Research Project
Hypothesis to be Tested:
Concisely list in order, the hypothesis to be tested and the Specific Aims of the proposed study. Provide the scientific basis of the
hypothesis, critically examining the observations leading to the formulation of the hypothesis.
Leptospira, chikungunya, rickettsia and bartonella cause febrile illness in Bangladesh.
Specific Aims:
Describe the specific aims of the proposed study. State the specific parameters, biological functions/ rates/ processes that will be
assessed by specific methods.
Aim:
To determine the existence and distribution of hospital patients with dengue, malaria, chikungunya, rickettsia,
leptospira and bartonella in Bangladesh.
9
Background of the Project including Preliminary Observations
Describe the relevant background of the proposed study. Discuss the previous related works on the subject by citing specific
references. Describe logically how the present hypothesis is supported by the relevant background observations including any
preliminary results that may be available. Critically analyze available knowledge in the field of the proposed study and discuss the
questions and gaps in the knowledge that need to be fulfilled to achieve the proposed goals. Provide scientific validity of the
hypothesis on the basis of background information. If there is no sufficient information on the subject, indicate the need to
develop new knowledge. Also include the significance and rationale of the proposed work by specifically discussing how these
accomplishments will bring benefit to human health in relation to biomedical, social, and environmental perspectives.
For health care providers and public health officials, exploration of unrecognized agents which cause febrile
illness is crucial for well-informed treatment and the guidance to prioritize the usage of scarce health resources [1] and
for the formulation of new policy on the prevention of infectious diseases [2]. Measuring the incidence and prevalence
of febrile illness caused by various pathogens poses a major public health challenge because of scanty or unreliable
data. Cost-effectiveness and feasibility of community surveillance or catchments area survey also contribute to this
challenge. Notably, unreliable clinical diagnosis, scarcity of technology, paucity of investment in training and
infrastructure for the diagnostic tools are also well observed in many disease endemic developing nations [3].
Consequently, the incidence and relative importance of the etiologies which cause febrile illness remain poorly
characterized in developing parts of the world. Therefore, presumptive treatment, even for a relatively easily diagnosed
cause of fever such as malaria, remains the standard of care in developing countries [4].
As a result, effective interventions against vector borne diseases like dengue, malaria, chikungunya and
rickettsia can not be assessed and evaluated. Besides, awareness of people about occupational risk factors of leptospira
and immunocompromized patients of bartonella is poorly defined. So sufferings in young people especially the
children can not be reduced.
In Sub-Saharan Africa and Southeast Asian countries with limited resources for long-term routine diagnostic
microbiology facility, sentinel hospital-based studies have been performed to provide clinical and public health
information to the policy makers. This approach helps to establish the relative importance and epidemiological
patterns of common pathogens and to provide clinical predictors in well-defined patient populations [3, 5-9].
Additionally, application of these methods has resulted in the identification of emerging or previously unrecognized
pathogens among the same populations [10, 11].
For the past few years, thousands of people in Bangladesh have been attacked by dengue fever on an annual
basis [12]. Bangladesh experienced dengue outbreaks in 2000, 2001 and 2002 [13]. Cases were reported from the
metropolitan cities (Dhaka, Chittagong, Khulna and Rajshahi) [13]. The weakness of this reporting is that it is
conducted almost entirely in urban areas and during ‘outbreak’ periods. We know little about endemic illness and very
little about disease in rural areas.
Since 2002, the WHO Roll Back Malaria Department has compiled information on malaria burden in a global
database for which WHO regional offices request information from country officials and the National Malaria Control
Programme [14]. That country profile shows in 1990 in Bangladesh annual malaria cases were reported as 53,875
while in 2002 it is as static as the past reported 55,646, mostly from Chittagong 54,939 (97%) and Dhaka 873 (only
2%) [15]. In 2003 Probable or clinically diagnosed malaria cases were reported as 434, 723 and Malaria deaths as
1250 where as Laboratory confirmed Malaria cases were 56,879, severe (inpatient or hospitalized) cases were 10,332
and Malaria deaths were 574 [15]. These small proportions of confirmed cases relative to probable cases raise the
serious public health concern- what are the other closely related etiologies besides malaria causing these large number
of illness? This also calls into question the reliability of diagnostics. Moreover, the weakness of this reporting is that
many people are treated at home or in private facilities that do not report to the national Health Information System.
The malaria control programme in Bangladesh is characterized by a weak surveillance system, limitation in
supervision and monitoring at various levels of programme implementation and shortage of staffs [15]. Therefore a
systematic study across a broad geography would be helpful to understand the discrepancy of the above reporting with
close assessment of the ongoing surveillance system.
10
Although a few studies of dengue or malaria have been conducted in one or two hospitals in Bangladesh, but
we have no data from these hospitals for classifying the patients of these diseases by age group [16-28]. Disease
registers are not strictly maintained with confirmed laboratory tests. People have to pay for their own medical care,
and since most people recover without spending money on testing, there is little interest in diagnostic testing.
Leptospirosis is a zoonotic infectious disease with worldwide distribution caused by pathogenic bacteria
spirochetes of the genus Leptospira that are transmitted directly or indirectly from animals to humans through contact
of skin abrasions with water or soil contaminated with the urine of an infected animal [29, 30]. An illness caused by
leptospira can be characterized by fever, headache, chills, myalgia, conjunctival suffusion, and less frequently by
meningitis, rash, jaundice, or renal insufficiency [31].
Leptospira is a potentially important pathogen in Bangladesh. Known risks for leptospirosis are very common
in Bangladesh through more frequent occupational exposure to environments contaminated with urine from rodents or
other animals [32]. In rural Bangladesh farming [33-35], rice field working [36-38], livestock farming and dairy
farming and in sea-shore districts fish farming [39, 40] and fish workers are common occupations. Recreational
exposures in riverine areas for example swimming, boating [41, 42] and avocational exposures such as barefooted
walking or gardening with bare hands [43] are very common practice in rural communities. We have some evidence of
leptospira infection in Bangladesh. Sixty-three leptospirosis patients were detected in a study at Dhaka Medical
College Hospital and Holy Family Red Crescent Hospital in 2001 [32]. The case-fatality rate among hospitalized
leptospirosis patients in Dhaka was 5%. This study emphasized the need for increased awareness of leptospirosis,
further information on wide geographical variation and optimal management regimens that can be applied in
Bangladesh [32].
Chikungunya is a viral disease, belongs to the genus Alpha virus under the Togaviridae family, transmitted by
Aedes mosquitoes [44, 45]. The disease typically consists of an acute illness with fever, skin rash, and incapacitating
arthralgia [45]. The latter distinguishes chikungunya virus from dengue [46, 47]. In early 2006, WHO reported
chikungunya outbreak in islands of Indian Ocean i.e. Maldives, Mauritius, Madagascar, Mayotte, Seychelles and La
Reunion Islands as well as the coastal region of India [44]. One fifty one districts located in ten states/provinces of
India have experienced chikungunya fever outbreaks [48].
Public health officials, epidemiologists and laboratory researchers suspected that chikungunya virus could be
circulating in Bangladesh due to 2006 outbreak notified in the subcontinent [49]. However a study was conducted by
IEDCR and ICDDR,B in urban Dhaka of 175 blood samples [49]. The study site was only limited to Dhaka. The
sample size was small. Non-specific sampling design of the study could not eliminate the risk of emergence of
chikungunya virus in Bangladesh. Finally the study did not establish any evidence of chikungunya virus in
Bangladesh [49]. Most importantly both the dengue and chikungunya viruses share the same vectors, similar
symptoms and geographical distribution [46, 47]. Still we do not know the related epidemiology of both of these
pathogens in Bangladesh [49].
Bartonellaceae are fastidious, aerobic, short, pleomorphic gram-negative coccobacillary or bacillary rods that
cause animal disease that is communicable to man through vectors (i.e. sand flies for B. bacilliformis or human body
lice for B. Quintana) [50, 51]. Clinical manifestations of bartonella infection in general include fever, malaise, fatigue,
anorexia, emesis, headache, lymphadenopathy and skin manifestation as well. But the severity and presentation of
disease is related to immune status of the patient such as persistent bacteremia, endocarditis and bacillary
angiomatosis have been reported [52]. In the 1990s, however, several major discoveries expanded knowledge of the
genus Bartonella. It is defined as a causative agent for AIDS-related bacillary angiomatosis, cat-scratch disease [5355] and culture-negative endocarditis [56, 57].
Several factors have lead to the emergence of Bartonellae as a recognized agent of zoonotic infections.
Emergence of immunocompromising diseases, organ transplant and cancer therapy [50], co-infection [58, 59],
increased outdoor activity [60-63] and close contact with their domestic animals [50] are found to be the influencing
factors. These factors are also visible in Bangladesh.
Many new Bartonella species have been identified in South East Asia [64, 65] in Indonesia, Philippines,
Singapore, Thailand and some of them could be a source of human infection [50]. In addition, a zoonotic study in
11
Bangladesh reported that prevalence of Bartonella was high in three mammalian species: lesser bandicoots, black rats,
and house shrews [66]. Bartonella is transmitted to humans from animals. These animals are common in Bangladesh.
At the same time, this study has revealed that these animals do have bartonella infections as well. Therefore people in
Bangladesh are probably susceptible to acquire bartonella infection. So bartonella is included as an emerging disease
in our febrile illness study.
Rickettsial pathogens are highly specialized gram-negative, obligate intracellular bacterium causing rickettsial
fever, and rash usually transmitted to man by the contamination of the bite site or skin abrasions with Rickettsiacontaining flea feces or by direct bite of ticks.
Rickettsial diseases are widely distributed throughout the world and recent studies in (Rangoon) Myanmar and
Nepal reveal their continued presence in several parts of the Indian subcontinent, particularly that of scrub typhus [67,
68]. High infectivity, low index of suspicion and the lack of diagnostic facility are usually associated with significant
morbidity and mortality from rickettsial diseases [69, 70]. Suburban expansion and environmental modifications
through destruction and reduction of natural habitats and commensal rats such as R. rattus and R. norvegicus, and their
fleas, in particular the oriental rat flea X. cheopis.are present in Bangladesh [71, 72]. Recently 40 ricketttsial cases
have been detected in a study in Bangladesh in Mymensingh Medical College Hospital, but this was a small study
with small population size. The study area was limited to only one district. Only suspected cases were selected. No
routine surveillance system or standard case definition was followed for clinical diagnosis. The sampling design was
not representative. Weil–Felix agglutination reaction was performed for laboratory diagnosis. The test was not reliable
and confirmatory due to poor sensitivity and specificity. The test was also performed from various local laboratories
outside the hospital which were not the reference laboratories [73].
Other potential causes of febrile illness in Bangladesh include typhoid fever and brucellosis. Typhoid fever is
a common cause of fever in Bangladesh [74-80]. Brucella is another potential pathogen that can be transmitted either
through contact with infected animals or from incompletely pastereurized dairy products. Because the confirmed
diagnosis of these two pathogens requires a substantial volume of blood in a separate blood culture, we have chosen
not to include them in the present study. Indeed, we are not attempting to conduct a comprehensive study of all causes
of febrile illness in Bangladesh. Instead, we are focusing on a handful of pathogens that there is very little data on to
assess if they are important or occasional causes of fever. The list of pathogens that we are assessing is limited by
cost and logistics
Research Design and Methods
Describe in detail the methods and procedures that will be used to accomplish the objectives and specific aims of the project.
Discuss the alternative methods that are available and justify the use of the method proposed in the study. Justify the scientific
validity of the methodological approach (biomedical, social, or environmental) as an investigation tool to achieve the specific
aims. Discuss the limitations and difficulties of the proposed procedures and sufficiently justify the use of them. Discuss the
ethical issues related to biomedical and social research for employing special procedures, such as invasive procedures in sick
children, use of isotopes or any other hazardous materials, or social questionnaires relating to individual privacy. Point out safety
procedures to be observed for protection of individuals during any situations or materials that may be injurious to human health.
The methodology section should be sufficiently descriptive to allow the reviewers to make valid and unambiguous assessment of
the project.
Surveillance with collaborators:
International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) and Institute of Epidemiology,
Disease Control and Research (IEDCR) are conducting Hospital Based Influenza Surveillance since April 2007 in
twelve hospitals across Bangladesh. Out of these twelve, six are government and six are non-government hospitals.
These sites are distributed throughout all six divisions of Bangladesh, while still representing the distribution of the
population within those divisions. In addition to that, geographical area, enthusiasm, capacity and strength of the
hospitals to conduct large scale surveillance were also considered. The Hospital Based Selected Febrile Illness Study
will be launched on top of the existing Human Influenza Surveillance network in the following 6 hospitals.
12
Settings
The government hospitals selected for the surveillance are:
 Rajshahi Medical College Hospital, Rajshahi
 Khulna Medical College Hospital, Khulna
 Sher-e-Bangla Medical College Hospital, Barisal
The remaining three hospitals are non-governmental. These are:
 Jahurul Islam Medical College Hospital, Kishorganj
 Bangabandhu Memorial Hospital, USTC, Chittagong
 Jalalabad Ragib-Rabeya Medical College Hospital, Sylhet
These hospitals are tertiary medical college hospitals, mostly represent as referral hospitals for each division.
Kishorganj is located in rural setting and Khulna, Barisal & Chittagong are located near the Bay of Bengal. Therefore
occupational exposures responsible for leptospirosis are commonly observed in these regions. Khulna, Rajshahi and
Sylhet are quite close to the Indian border where chikungunya infection may be more likely. Sylhet, Barisal &
Chittagong are metropolitan cities so dengue cases may be higher there due to mosquito breeding places and
congested areas. On the other hand, hilly areas like Chittagong and Sylhet can present malaria cases more than any
other region.
The following map of Bangladesh shows the distribution of these hospitals across the country.
Fig 1: Map of Bangladesh showing the hospitals selected for Hospital Based Selected Febrile Illness Study.
13
Objective
Objective: To assess the contribution of dengue, malaria, leptospira, chikungunya, rickettsia and bartonella to febrile
illness from the hospital patients of Bangladesh.
Case definition:
Patients who present with a complaint of fever more than >38º C with onset within the preceding 10 days.
Flow chart showing enrollment of patients
Inclusion criteria:
Clinicians will be trained to identify cases for possible inclusion and to complete a standardized clinical and
laboratory evaluation of all patients who meet the following case criteria:
1. Patients being evaluated at the participating institution.
14
2. Patients both from the inpatient and outpatient medicine and pediatric departments of the hospitals.
We will at first collect samples from the patients of the inpatient department. We will be able to specify the
documented fever from inpatients and all types including seriously ill patients will be included. Besides most of the
times when people get more complicated illness they got admitted in the inpatient department. As in malaria, dengue
fever and leptospirosis people get serious illness so we include inpatients.
On the other hand, increasing awareness about dengue and malaria in the community make people more
conscious about fever and rash. Verbal consultation with family physicians followed by self medication with
paracetamol is a more common practice for mild fever. But for high grade fever with prostration and weakness,
headache or myalgia, very often people visit outpatients to get effective management and to take admission if advised
by attending physician. So we will also include outpatients.
3. We will collect up to ten samples from each hospital every month.
Out of these 10 specimens, we will collect 5 specimens from the adult medicine clinics or ward and 5 from the
pediatric clinics or wards through purposive sampling technique. The objective of the study is not particularly focused
on any age group; rather circulating etiologies will be identified for any age group. Therefore no age group will be
excluded for collecting samples from the inpatient or outpatient department. But the commonality of fever in young
children risks under-sampling adult disease. So using purposive sampling we will collect 5 samples from adult and
same from pediatrics. The medical college hospitals included in the study usually define 14 years as the higher limit of
pediatric age groups.
4. History of fever with onset within the preceding 10 days.
We will enroll febrile patients based on the history. But if we have the opportunity to select patient of
documented fever we always appreciate to get that case.
5. Temperature at admission, consultation or noted at temperature chart > 38°C (oral or rectal).
Exclusion criteria:
1. We will exclude the nosocomial cases, defined as:
Any patient who develops a new onset of fever >38º C after 72 hours of hospitalization will be defined as a
case of hospital infection or nosocomial infection.
2. The Surveillance physicians will be instructed to exclude the following symptoms:




Fever for more than 10 days.
Cough with productive sputum.
Urgency, frequency, hesitancy during micrurition.
Cellulites/abscess/boil/local skin infection in the body.
3. Confirmed laboratory diagnosis of other diseases rather than the study organisms.
Field operation:
Two consecutive days in each month will be designated for specimen collection from a hospital. We will vary
the days of specimen collection for each hospital each month to avoid any confounding from factors that affect when
patients come to hospital. This will be incorporated with the specimen collection days of Hospital Based Influenza
Surveillance. On those days, field assistant and laboratory technician from ICDDR,B will visit the selected hospitals in
order to assist the surveillance physicians in sample collection and to transport the samples to the ICDDR,B
laboratory. This team will also collect data from the surveillance physician. Attending physicians will maintain a
register book to organize the data to gain the ability to comment on disease burden. This could be done if the
15
physicians collect data on the total number of patients admitted, total patients meeting the case definition and finally
total evaluated patients for the whole month. They will provide these data to the study team.
On the day designated for specimen collection, the surveillance physician will detect the clinical cases from
the inpatients. The surveillance physician will select those patients who presents with a complaint of fever with onset
within the preceding 10 days. He will explain to the participant about the study procedure. If a participant has any
queries, physician will try to resolve his concern and will discuss with the study participant about the issues. Then if
participant agrees to join the study, the surveillance physician will take inform consent and assent from him
(Appendix A and B). Then he will examine the participant and collect blood sample from up to 10 of the participants.
But if he did not get 10 samples from inpatients then he will visit the outpatient department and he will collect the rest
of the samples from the outpatients.
Most of the patients may have a cell phone number. If it is not possible then they will name someone in their
village or neighborhood, with whom we can contact. If that is also impossible then they may provide the contact
number of those who rents cell phone services in their locality. Thus, if we find number of patients with a disease of
interest, we will have the option to reconnect with them and collect global positioning system (GPS) reading and
potentially other information from them. We will do a follow-up of all patients after two months. We will have a way
to contact them through their cell phone numbers. We will administer a short questionnaire together (Appendix E).
We can collect some information from the people with a disease of interest about the residual affect of illnesses. We
will also be able to know about some other socio-economic variables whether they are related to our disease of interest
or not.
Surveillance personnel
A senior level physician will act as focal point voluntarily at each hospital and they will be assigned for the
coming study from the collaborators for ongoing influenza surveillance programme. A surveillance physician in each
hospital identifies the potential influenza affected patients according to the ideal case definition, informs them about
the surveillance activities, takes informed consent from them, completes the questionnaire form and collects the nasal
and throat swabs from the patients. For this febrile illness study we will provide extensive training to the study
physicians in study protocol and procedures. They will then be responsible for collecting data and specimens and
reporting this information to ICDDR,B. The surveillance personals from the existing staffs of each hospital have been
selected based upon their interest in participation, clinical skill and likelihood of staying in the hospital for more than
one year. They are already provided a small honorarium (3000 Taka i.e. 45 $US per month) for the extra efforts. In
case of transfer of the surveillance physician from the hospitals, replacements will be made within 30 days and the
focal point will supervise the surveillance activities during the window period.
The laboratory technicians appointed by ICDDR,B will collect specimen from the patients. They will prepare
blood specimens for the laboratory analysis.
The field assistants allotted by ICDDRB for influenza surveillance will deliver continuous support in
collection, transportation and storage of samples in febrile illness study. They will carry the lab materials including
reagents and centrifuge machines to the sample collection sites and also assist in blood collection, centrifugation and
aliquoting procedure. They will label the blood specimen collected by the laboratory technicians. They will keep the
specimens in an organized way for transportation and storage of the specimens in the laboratory for further analysis.
Surveillance physician appointed at each surveillance site will be taught accordingly. However, initially
experienced ICDDR,B study personnel will play a forward role in training and supervising specimen collection.
Responsibility for data and specimen collection at each site will shift from ICDDR,B personnel to surveillance
physicians in phases. Surveillance physicians will first observe activities done by ICDDR,B personnel, then will
perform under close supervision and guidance and will ultimately be entirely conducted by the surveillance physicians
alone.
We have added additional duties for surveillance physician but in the same time we have also reduced their
workload for influenza surveillance. In influenza surveillance they are collecting data and sample both from the
16
patient. But when we will launch our study they will have to collect the data only. They don’t need to collect samples
from the patients which will be performed by our newly recruited laboratory technicians for the study.
Laboratory tests in relation to specimen collection period:
Blood will be collected from febrile patients within 10 days of symptom onset. For leptospira we are
interested to culture the organism so we need to collect blood within 10 days of symptoms onset otherwise beyond that
time we will not find any leptospira pathogen in the blood. For dengue usually we will find antibodies 5-7 days and
Chikungunya 4-7 days of symptom onset. So for early days of fever, virus isolation through PCR may be better
approach for the specimen collected during that period as we have little chance to get any antibody in the blood at that
period. But with the specimen collected after that period ELISA can be done. However, we will perform ELISA test
for all the specimens. We will mark only those cases for PCR whose samples are collected in first 5 days of fever and
whose ELISA results are negative. For chikungunya we will take only those samples with history of joint pain. We
will have the option for performing PCR to identify ricketsial pathogens for samples collected at earlier period of
illness. But we will perform indirect fluorescent antibody (IFA) assays to detect rickettsial antibody for all the
samples. Furthermore for malaria we want to detect specific antigen (proteins) for malaria parasites which will release
immediately in blood through breakdown of RBC with the onset of fever by doing rapid kit test. We will also perform
microscopic examination to identify the malaria species. Actually we are selectively analyzing the samples depending
on their course of illness. We can provide that information through assigning a tag or label to the specimen mentioning
the period of illness and so that the laboratory can perform the analysis of the samples accordingly.
1-2 days
3-4 days
5-6 days
7-8 days
9-10 days
>10 days of fever
chikungunya
PCR
ELISA
PCR
ELISA
PCR
IFA
dengue
rickettsia
leptospira
Culture
bartonella
Culture
malaria
Rapid kit test and
Microscopic malaria parasite diagnosis
Figure: laboratory diagnostic procedure consistent with specimen collection time.
17
Description of Laboratory methods with sensitivity and specifity of the tests
The sensitivity and specificity of the Dengue IgM Capture ELISA are reported to be 94.7 % (with CI 85.4 98.9 %) and 100% (with CI 95.7 - 100%), respectively. The dengue IgM Capture ELISA determines the level of IgM
antibodies to dengue in a patient’s serum. A positive result (> 11 Panbio units) is indicative of either an active primary
or secondary dengue infection. < 9 Panbio units will be counted as negative results [81].
We will have the option to perform PCR for dengue by attaching labels to the samples. Multiplex Reverse
Transcriptase-PCR (MRT-PCR) for dengue is highly sensitive & specific compare to ELISA and confirms the
diagnosis with serotype identification. Gel based PCR will be used. The reaction mixture will contain 50 mM KCl, 10
mM Tris (pH 8.5), 0.1% TritonX-100, 0.01% gelatin. Dengue RNA can be detected by using serotype specific
primers, reverse transcriptase and thermostable polymerase [82]. Multiplex Reverse Transcriptase-PCR (MRT-PCR)
is highly sensitive, specific along with cost-effective and less time consuming. Without proper precaution,
contamination can lead to false-positive results [82, 83].
Malaria will be diagnosed by rapid diagnostic test (RDT) based on the detection of P.falciparum-specific
antigen and plasmodium vivax-specific antigen. The trade name of this Rapid Diagnostic Test is “FalciVax” and it is
being produced by Zephyr Biomedicals, India and “BinaxNow” produced by Inverness medical, USA. Falcivax is
rapid self-performing, qualitative, two-site sandwith immunoassay utilizing whole blood for the detection of
P.falciparum specific histidine rich protein-2 (pf, HRP-2) and P.vivax specific pLDH. The test can be used for specific
detection and differentiation of P.falciparum and P.vivax malaria. The standardization of this test has already been
done by Zephyr Biomedicals. Sensitivity of the RDT is similar to that commonly achieved by good field microscopy.
Sensitivity and specificity of the Rapid Diagnostic Test used for the detection of P.falciparum and P.vivax will be
more than 95 % and now been recommended for use in the malaria control program by the WHO [84-86].
Both thick and thin film will be done for diagnosis of malaria by microscopy. The blood films will be stained
with Giemsa stain in phosphate buffer saline and examined under the microscope at a magnification of x 1000 for the
presence of malaria parasites. Blood films were defined as negative if no parasite were observed in 300 oil immersion
fields (magnification, x 1000) on thin film by an experienced microscopist [87].
Microscopy diagnosis for malaria is fairly sensitive and highly specific. False negative results may be seen in
conditions of very low parasitemia, maturation of sequestered parasites in the bloods, partially treated with
antimalarials or on chemoprophylaxis, and may be due to technical factors; (poorly prepared slides, poorly stained
slides, poor quality microscope, examination of only thin films, inexperienced technician etc). False positive results
are seen due to stained particles, which may be confused for malarial parasite by an inexperienced microscopist. In a
study in Bangladesh, it has been shown that for both types of malaria the sensitivity was 94% (95% CI 71.3–99.9), and
the specificity was 93% (95% CI 90.0–98.5) [26].
In ICDDR,B laboratory we will perform microscopic diagnosis for malaria for all the samples to confirm that
malaria and also able to identify the specific species. If there is any discrepancy between these two malaria test results,
we will inform the results to the attending physician. Therefore, we will get more reliable results with specific spices
identification by performing both of the tests. We will classify plasmodium vivax and plasmodium falciparum. The
degree of parasitaemia in each case will be reported as scanty (1-10 parasites per 100 high-power fields), moderate
(10-100 parasites per 100 high-power fields), or heavy (>100 parasites per 100 high-power fields) [88].
Leptospira culture is the optimum way to confidently identify infection, but a negative culture does not
exclude Leptospira infection. Serology should be sought for identification of the infecting serovar or serogroup prior
to typing of the isolate [89].
Indirect immunofluorescence antibody (IFA) assay for rickettsia pathogen is the “gold standard” technique
and is used as a reference technique in most laboratories. There is no other serologic test is better than the sensitivity
and specificity of these assays. Serum antibodies bind to fixed antigens on a slide and are detected by a fluoresceinlabeled conjugate. The sensitivity of the IFA assay is substantially dependent on the period of sample collection. As
the illness progresses to 7–10 days, the sensitivity of IFA serology increases. The IFA is expected to be 94%–100%
sensitive after 14 days and sensitivity is increased if paired samples are tested [90, 91] . For the detection of R.
18
rickettsii responsible for Rocky Mountain spotted fever (RMSF), sensitivity, as tested with 60 paired serum
specimens, including specimens with stationary titers (5%) and fourfold rising titers (95%), was 100% [92]. In a study
with patients with no rickettsial diseases, a titer of ≥ 1:64 had a specificity of 100% and a sensitivity of 84.6%, and a
titer of ≥ 1:32 had a specificity of 99.8% and a sensitivity of 97.4% [93]. For scrub typhus, the sensitivity of IFA is
low if high specificity is required that can be explained as follows: for a titer of ≥ 1:100, sensitivity is 84% and
specificity is 78%, for a titer of ≥ 1:200, sensitivity is 70% and specificity is 92%, and for a titer of ≥ 1:400, sensitivity
is 48% and specificity is 96% [94]. A fourfold increase to a titer of ≥ 1:200 is 98% specific and 54% sensitive [95].
Usually the indirect microimmunofluorescence assay is not positive when the patient is acutely unwell, but it has been
the most sensitive and specific of the traditional serological tests [96]. Culture techniques are very sensitive but can
take up to 2 months to get a positive result that may limit their clinical usefulness [97]. Real-time PCR is both highly
specific and extremely sensitive for the diagnosis of rickettsioses and may offer the advantages of speed,
reproducibility, quantitative capability, and low risk for contamination, compared with conventional PCR [98, 99] .
Bartonella culture is considered confirmatory diagnosis for bartonella species. If Bartonella-like colonies are
seen on an agar plate, material from one colony will be streaked onto a new agar plate. Presumptive Bartonella
cultures will be used for the extraction of Bartonella DNA and PCR.
We performed a pilot study in Rajshahi Medical College Hospital and consulted with our collaborators for
Hospital Based Influenza Surveillance in other hospitals about this coming protocol. They have informed us that in
most cases febrile illness of 10 days will present in the outpatient department. So in most cases we will get samples
from outpatients who usually take consultation for once and will be difficult to track them within 7 days unless their
illnesses become complicated. Therefore, it will not be feasible to collect convalescent sample for serological tests.
Specimen collection, handling and aliquoting
The locally recruited surveillance physicians will be responsible for informing the patients about the study and
obtain written informed consent and assent (Appendix A & B) for collection of 7-10 ml of venous blood sample from
the patients. For all patients from whom specimens are collected, the surveillance physicians will also obtain studyrelated demographic and clinical information and will record that in the standardized assessment form (Appendix C).
They will not collect specimens or data from any patients who meet the criteria but are unwilling to give written
consent. While taking consent, the surveillance physicians will inform the patient to wait for 15 minutes to get the test
results of the rapid kit test for malaria.
The laboratory technician will wear a long sleeve gown that does not open at the front and disposable gloves.
He will be aware of contamination of pathogens and safety of the study participants. In every case, he will use
disposable syringe to collect the blood specimen. He will assure the study participants, if they feel very anxious and
worried about the procedure. He will mention what he will be going to do and how he will collect blood from them. If
the participants refuse to give blood then he will accept it easily. If the participant agrees, then he will collect 7-10 ml
antecubital venous blood aseptically. All needles syringes will be kept in puncture resistant sharp container and
disposable gloves and any other used materials will be kept in a biohazard bag.
He will do the rapid kit test “ICT for malaria” by using 1 drop of blood in the field. He will also complete a
laboratory report form (Appendix D) for that specimen and keep it with the kit. He will prepare the slides for
microscopic malaria diagnosis.
The laboratory technician will inoculate the Ellinghausen, McCullough, Johnson, and Harris (EMJH) medium
for leptospira culture with 3 drops of fresh whole human blood from the collected sample. He will do these
inoculations for each sample for laboratory analysis in US CDC. The field assistants will be responsible for labeling
the specimen.
The laboratory technician will separate one third of total blood in an EDTA tube for real time PCR for
ricketsial antibody detection. The assay will be performed in US CDC. The field assistant will label the specimen.
19
20
The rest of the blood sample will be initially collected in sterile vacutainer tube and field assistants will label
the samples. The vacutainer tube will be centrifuged in the field to get the blood clot and serum separated necessary
for bartonella culture and ELISA or PCR tests respectively. After spinning of the rest of the sample, the centrifuge will
be allowed to sit for 5 minutes before opening. During opening of the centrifuge, all protective precaution will be
taken. Then blood clot will be transferred into sterile 2 ml cryovials with sterile forceps for bartonella culture. One
aliquot of serum will be preserved in screw cap labelled cryovials for dengue and chikungunya serological tests.
Another cryovial will contain serum for Indirect immunofluorescence antibody (IFA) assay which will be performed
at US CDC for rickettsial antibody detection. ELISA for dengue will be performed in the ICDDR, B. But cryovials of
samples of patients with history of joint pain will be sent to US CDC for chikungunya serological tests and testing for
bartonella will also be performed there. Rest of the serum will be preserved for long term storage. All these specimens
will be labeled and kept in 8 degree centigrade (cool box/refrigerator) in a rack in upright position.
Specimen transportation to ICDDR,B laboratory
Blood for leptospira culture will be kept in room temperature into a sterile container of EMJH tubes.
Cryovials of clot for bartonella culture and serum for serological tests will be stored in cold boxes at 2-8°C
temperature. Soon after specimen collection for both febrile illness study and influenza surveillance activities, the field
assistants will transport the specimens to the ICDDR,B laboratory. In order to maintain the required temperature every
specimen will reach the ICDDR,B laboratory within 72 hours after specimen collection.
After receiving samples in the ICDDR.B laboratory, the samples will be handled with special precaution
wearing gloves, lab coats, face mask, safety glasses. The rack containing the blood tubes will be checked for leaking.
At ICDDR,B parasitology laboratory the laboratory technician will bring all the samples, culture medias and
already tested ICT kits and microscopic slides for malaria along with the field team and make arrangements for the
storage.
At ICDDR,B the laboratory technician will perform malaria slide test. We have single laboratory personnel for
both rapid test and microscopic test. Therefore, we have an alternative approach to make the technician blinded about
the rapid test results. The approach is stated below.
When laboratory technician will prepare the slides in the field he will assign a unique ID number to the
respective slide. Then he will cover that ID number with a translucent scotch tap like the electric tap. At ICDDR,B he
will bring the slides and observe under the microscopy. Thereafter, he will point out the microscopic results and
finally remove the scotch tap. But he has to write with the permanent ink. He will confirm the already done rapid kit
test “ICT for malaria” by inspection of the 2 redlines in T and C windows of rapid kit test. He will re-check the
laboratory report form for that specimen completed in the field and keep that for record.
At ICDDR,B virology laboratory dengue virus will be detected by ELISA method, MAC ELISA for IgM
antibody. For primary and secondary dengue infections MAC ELISA can measure a rise in dengue specific IgM even
in sera samples collected at 1 or 2 day of interval of acute phase. In cases where a single specimen is available,
detection of anti dengue IgM permits the diagnosis of recent dengue virus infection even in primary cases where the
level of heamagglutination-inhibition antibody will not be diagnostic. We will perform ELISA for all the collected
specimens. But we will have the option for performing PCR for that samples collected from a febrile patient before 45 days of symptom onset and among them whose ELISA comes negative. Specimens from patients with joint pain will
be preserved for serological test for chikungunya virus in US CDC.
21
Storage and shipment of specimen to CDC, Atlanta
Storage of specimen will be depending on laboratory capacity. Because of the shipment procedure of these
specimens, these will be stored for long periods until shipment to US CDC. For leptospira culture temperature will be
30 degree set by incubator and for bartonella culture the blood clots be frozen at −70°C before shipment on dry ice to
US CDC laboratory and for serological tests samples will be kept in a freezer at -70°C temperature until time of
analysis.
We can send the leptospira cultures in every three months which do not require ice storage. On the other hand,
the shipments for rickettsia, bartonella and chikungunya that require ice preservation are expensive. Therefore we will
send them every six months. Shipments will be scheduled so that they will reach at US CDC during working hours on
Monday through Friday.
We will separate the serum in cryovials for the specific laboratory tests mentioned in our protocol. We will
also preserve the rest of the serum for the long-term storage. A new pathogen can be discovered with a new diagnostic
at any time. Therefore the left over aliquots of specimens will give us an opportunity to test the sample quickly and
efficiently. In future we may look for additional infections that can cause fever.
Laboratory analysis in CDC
All forwarded specimens will be processed in corresponding laboratories of US CDC.
After decontamination, samples will be analyzed using the following procedure:
For chikungunya antibody detection by ELISA or PCR.
With maintaining the case definition, if blood is collected after 4-7 days of illness then ELISA will be done with that
blood sample to detect the antibody. Again if blood is collected from a febrile patient before that period PCR will be
done with that sample. The field assistant will label the specimen mentioning the period of febrile illness and the
history of joint pain.
For rickettsia identification
Indirect fluorescent antibody (IFA) assays will be done at CDC for all samples for detection of rickettsia antibody
from the serum. Besides there will have the option for real-time PCR assay based on the rickettsial citrate synthase
gene and can also be used to quantify rickettsial DNA copy numbers [99].
For leptosiprosis and bartonella blood culture will be done.
For leptospira culture:
Culture media and method: blood inoculated 6 mL of Ellinghausen, McCullough, Johnson, and Harris (EMJH)
medium will be supplemented with 3% rabbit serum and 0.1% agarose, then incubated aerobically at room
temperature (25°C–30°C) for 6 months and examined every week for 2 months, every 2 weeks during months 3 and 4,
and once a month during months 5 and 6. Leptospiral growth will appear as either a Dinger's ring in the EMJH
semisolid or as turbidity in the EMJH liquid medium. Examination will be done by placing 1 drop of culture onto a
microscopic glass slide and viewing by dark-field microscopy at 300 × magnifications. In addition to isolate
leptospires, serovar level will be identified by using PFGE (pulsed field gel electrophoresis) method.
For bartonella culture:
Culture media and method: In laboratory in CDC they will apply several different approaches for isolation in their
laboratory:
1) Blood clots (100 µl) diluted 1:4 in brain heart infusion media are plated on heart infusion agar plates containing 5%
rabbit blood;
2) Blood clots (100 µl) are co-cultivated with Vero E6 cells at 35oC with 5% carbon dioxide for 7 days and then subcultured onto rabbit blood-enriched agar;
3) Blood clots (100 µl) are inoculated into a pre-enrichment liquid, Bartonella/alpha-Proteobacteria growth medium
(BAPGM) developed by Maggi et al. (2005) and after 7 days of incubation at 35oC with 5% carbon dioxide plated
onto rabbit blood agar.
22
All of the agar plates will be incubated at 35oC in an aerobic atmosphere of 5% carbon dioxide for up to 30 days.
When Bartonella-like colonies are seen on an agar plate, material from one colony will be streaked onto a new agar
plate. Pure culture will be collected in a 10% glycerol solution for PCR confirmation and further characterization.
Presumptive Bartonella cultures will be used for the extraction of Bartonella DNA and PCR.
Time line for the study:
The study will be conducted for 1 year so we perform data and sample collection at least 1 year long completely.
Monitoring and evaluation
Each of the sample collection form will be reviewed by the investigators and any missing or questionable
information reviewed with the physician who completed the form. Besides investigators will check all the positive
malaria test reports as whether patients or attendants are informed the results or not.
Throughout the study period, monitoring and evaluation will be important both for individual sites and for the
entire system as a whole. A small team composed of IEDCR and ICDDR,B personnel will be responsible for this. For
the first three months of study, monthly visits to each site will include a strong monitoring component. After this
initial period, assuming satisfactory performance, such visits will occur every third month. During these visits,
activities at each site will be monitored to ensure that study protocol is followed and data collection is accurate and
efficient.
At the end of the year of study, a full evaluation will take place to examine the progress of the year. The
evaluation will consider observations from regular monitoring activities as well as annual study reports. The principal,
23
co-principal and other co-investigators will be responsible for addressing any issues that may arise during monitoring
and evaluation. The final monitoring system and tools will be developed in collaboration with IEDCR before
commencement of the study.
Sample Size Calculation and Outcome Variable(s)
Sample size
For the objective (to characterize the patient with febrile illness of diversified aetiologies), we attempt to take
a geographically diverse sampling as an estimate of national patterns. The chosen 6 facilities will represent the diverse
catchments populations across the country. We conduct this study within this area and not attempt to extrapolate cases
for facilities that are not under surveillance. But it may be a matter of representativeness rather than sample size
calculation.
If we assume that 200,000 people seek care for febrile illness at these hospitals per year, and the real
prevalence of infection with one of these organisms is 0.75%, then a sample of 675 will provide a 95% probablity of
identifying a point prevalence between 0.1% and 1.4%. Or stated another way with a sample of 675 study subjects we
have a 95% chance of not missing a pathogen that is causes 0.75% of the febrile illnesses in this population.
We will collect 10 specimens from each hospital in each month. This will provide 720 specimens in a year.
The additional specimens provide some additional power.
The primary outcomes to be measured in the study are:
Percentage of patients presenting with fever of unknown origin at our facilities with considering the facts of
the following specific etiologies of interest.
1.
2.
3.
4.
5.
6.
Number of patients with Malaria.
Number of patients with Dengue fever.
Number of patients with leptospirosis.
Number of patients with chikungunya fever.
Number of rickettsial illness.
Number of patients with Bartonelllosis.
Facilities Available
Describe the availability of physical facilities at the place where the study will be carried out. For clinical and laboratory-based
studies, indicate the provision of hospital and other types of patient’s care facilities and adequate laboratory support. Point out the
laboratory facilities and major equipment that will be required for the study. For field studies, describe the field area including its
size, population, and means of communications.
Hospitals:
The hospitals are selected maintaining the wide geographical diversity and government and non-government
ratio. Based upon this assumption, the following hospitals are initially selected for the surveillance activity, as shown
in the following table.
24
Table: Selected hospitals with location, types and number of beds
Hospitals
District
Administration #of beds*
Jahurul Islam Medical College Hospital, Kishorganj
Kishorganj
Non-government
400
Rajshahi Medical College Hospital
Rajshahi
Government
650
Bangabandhu Memorial Hospital, USTC
Chittagong
Non-government
350
Khulna Medical College Hospital
Khulna
Government
250
Jalalabad Ragib-Rabeya Medical College Hospital
Sylhet
Non-government
Sher-e-Bangla Medical College Hospital
Barisal
Government
750
500
* Source: Government hospitals: MIS Division, DGHS; Non-government hospitals: Personal communication with the respective hospitals
Table: laboratory facilities in selected hospitals
Hospital
Refrigerator
Blood culture
Microscopic diagnosis of slide
ICT for malaria
ELISA for dengue
CBC,TC,DC,ESR
JIMCH,
Kishorganj
√
√
√
√
x
√
BBMH,
Chittagong
√
x
√
x
x
√
RMCH, KMCH,
Rajshahi Khulna
√
√
√
x
√
√
x
x
x
x
√
√
SBMCH,
Barisal
x
x
√
x
x
√
JRRMCH,
Sylhet
√
√
√
√
x
√
Laboratory:
At ICDDR,B laboratory we will do microscopic diagnosis for malaria parasite and IgM MAC ELISA for
dengue antibody detection. US CDC will do the laboratory analysis for chikungunya, leptospira, bartonella and
ricettsial pathogen.
Data Safety Monitoring Plan (DSMP)
All clinical investigations (biomedical and behavioural intervention research protocols) should include the Data and Safety
Monitoring Plan (DSMP) to provide the overall framework for the research protocol’s data and safety monitoring. It is not
necessary that the DSMP covers all possible aspects of each elements. When designing an appropriate DSMP, the following
should be kept in mind.
a)
b)
c)
d)
All investigations require monitoring;
The benefits of the investigation should outweigh the risks;
The monitoring plan should commensurate with risk; and
Monitoring should be with the size and complexity of the investigation.
Safety monitoring is defined as any process during clinical trails that involves the review of accumulated outcome data for groups
of patients to determine if any treatment procedure practised should be altered or not.
This is not a clinical trial so we can exclude this issue.
Data Analysis
Describe plans for data analysis. Indicate whether data will be analyzed by the investigators themselves or by other professionals.
Specify what statistical software packages will be used and if the study is blinded, when the code will be opened. For clinical
trials, indicate if interim data analysis will be required to monitor further progress of the study.
25
The investigators themselves will analyze the data. We will record personal and illness related informations
from the patients. Data will be double entered. We will use a unique ID for each individual. We will code the data and
secure the master list linking the code to the subject identifier. We will maintain the data in a locked environment.
This will ensure confidentiality. Statistical software SPSS, STATA and Epi-Info will be used for data analysis. We
will calculate the descriptive statistics, including cases of malaria, cases of dengue fever, cases of leptospirosis, cases
of chikungunya fever, cases of rickettsia and cases of bartonellosis. We will report the estimated percentage of patients
presenting with fever of unknown origin at the 6 mentioned facilities with these specific etiologies of interest. As data
accumulates, additional analysis will provide understanding of age and geographic distribution of some unknown
etiologies of febrile illness.
Ethical Assurance for Protection of Human Rights
Describe in the space provided the justifications for conducting this research in human subjects. If the study needs observations on
sick individuals, provide sufficient reasons for using them. Indicate how subject’s rights are protected and if there is any benefit or
risk to each subject of the study.
Risks: The risk to enrolled subjects from participation in study activities is minimal. Specimens will be collected only
after informed consent is obtained from the patient or from the patient’s legal guardian. Collection of the specimen
may cause bruise or mild discomfort for the subject during the procedure.
Benefits: Patients will be directly benefited by participating in this study because, rapid malaria test results will be
provided to the attending physician. Also, we will report confirmation test from ICDDR,B in a timely manner . But we
will not get the results of other tests in hand in time so other testing will not have any direct impact on patient care
Therefore, patients will receive routine medical care until the test results become available to inform patient care.
However, the information collected will be valuable to understand the etiology of fever in these sentinel sites.
Adverse events: Adverse medical events are not anticipated from the sample and data collection procedures involved
in study activities. We have trained lab technicians, health assistant and surveillance physician so we will do our best
effort to manage any untoward outcomes ranging from anxiousness to multiple pricks. In routine diagnostic purpose
blood is collected frequently but no adverse outcome occurs usually. However, we anticipate that there will be no
adverse event due to technical fault as we are going to recruit very expert personnel from our parasitology lab for
blood collection. The ethical review committee of ICDDR,B will be notified of any adverse events and deviations
from protocol.
Informed consent process: All potential subjects will be informed of the purposes and intent of the study, as well as
the procedures involved. Participation in the study will be voluntary; those agreeing to participate must provide
written informed consent. If the subject is an adult (age ≥ 18 years), consent will be obtained from the subject
himself/herself; if the subject is between 7-17 years of age, assent will be sought from him/her and from his/her
parents or legal guardian; if the subject is a child of less than 7 years, consent will be sought from his/her parents or
legal guardian.
Subject confidentiality: All data and specimens collected will be kept confidential. Any data not stripped of
identifiers will be stored in a locked file to which only study personnel will have access. All biological specimens will
be assigned a unique identification number to ensure confidentiality throughout the study. The identification number
will only be linked to the subjects identifying information in data maintained confidentially by the study coordinator.
Use of Animals
Describe in the space provided the type and species of animals that will be used in the study. Justify with reasons the use of
particular animal species in the experiment and the compliance of the animal ethical guidelines for conducting the proposed
procedures.
No animal will be used in this surveillance.
26
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Literature Cited. There is no page limit for this section, however exercise judgment in assessing the “standard” length.
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1083-5.
Dissemination and Use of Findings
Describe explicitly the plans for disseminating the accomplished results. Describe what type of publication is anticipated: working
papers, internal (institutional) publication, international publications, international conferences and agencies, workshops etc.
Mention if the project is linked to the Government of the People’s Republic of Bangladesh through a training programme.
Data and findings will be disseminated through several channels. Significant findings will be reported to
Government of Bangladesh through IEDCR, when a public health response is necessary. Additionally, the researchers
will write a Health and Science Bulletin (HSB) by ICDDR, B article summarizing the results at the end of the study
including all the descriptive statistics. As data accumulates, significant findings will also be reported in peer-reviewed
publications. In addition, there will be national dissemination of study findings at the end of the study.
HSB bulletins are sent to clinicians periodically. This bulletin includes Bengali translation besides original
English text. So it is comfortable and convenient for the clinicians to go through the study findings. From ICDDR,B,
6006 copies are sent through posted mails to the specific addresses of the medical professionals; most of them are
physicians and engage in clinical practice. We will do the study in the hospital settings in close contact with the
clinicians so we can disseminate the study finding to them very easily. Clinicians especially the medicine specialists
themselves will be very interested about the study as they very often clinically diagnose these diseases without the
scope of laboratory confirmation.
Collaborative Arrangements
Describe briefly if this study involves any scientific, administrative, fiscal, or programmatic arrangements with other national or
international organizations or individuals. Indicate the nature and extent of collaboration and include a letter of agreement
between the applicant or his/her organization and the collaborating organization.
Funding for this study is provided by the Centers for Disease Control and Prevention (CDC) through their
cooperative agreement with ICDDR,B). There will be an active collaboration between International Centre for
Diarrhoeal Disease Research, Bangladesh (ICDDR,B) and Institute of Epidemiology, Disease Control and Research
(IEDCR) for the implementation of the study. IEDCR and ICDDRB have developed a memorandum of understanding
to formalize their collaboration with each of the 12 surveillance hospitals for the ongoing hospital based influenza
surveillance activities. From these 12 hospitals, 6 hospitals are going to be considered for this study using the same
infra-structure and similar set-up.
The study will not provide any additional incentives to study physicians as they are already offered by
influenza surveillance. But their sample collection activities in existing surveillance will be reduced for this extraeffort.
31
Biography of the Investigators
Give biographical data in the following table for key personnel including the Principal Investigator. Use a photocopy
of this page for each investigator.
(Note: Biography of the external Investigators may, however, be submitted in the format as convenient to them)
1. Name:
Stephen P Luby
2. Present position:
Head, Programme on Infectious Diseases and Vaccine Sciences
3. Educational background:
(last degree and diploma & training
relevant to the present research proposal)
University of Texas Southwestern Medical School at Dallas
MD 1986
University of Rochester Strong Memorial Hospital
Internship and residency in Internal Medicine.
Centers for Disease Control -- Epidemic Intelligence Service 1990
Completed Preventive Medicine Residency 1993
4. List of ongoing research protocols
(start and end dates; and percentage of time)
4.1. As Principal Investigator
Protocol Number
2005-026
2006-043
2003-024
2007-003
2007-002
2007-010
2007-030
Starting date
1 Oct 2005
1 Nov 2006
1 Sep 2003
1 Feb 2007
March 2007
June 2007
Sep 2007
End date
31 Dec 2007
31 July 2007
31 Dec 2007
12 Dec 2008
Sep 2009
Sep 2008
Sep 2008
Percentage of time
5
5
5
3
5
3
5
4.2. As Co-Principal Investigator
Protocol Number
Starting date
End date
Percentage of time
5 Publications
Types of publications
a) Original scientific papers in peer-review journals
b) Peer reviewed articles and book chapters
Numbers
113
9
32
c) Papers in conference proceedings
d) Letters, editorials, annotations, and abstracts in peer-reviewed
journals
1
3
e) Working papers
0
0
f) Monographs
6 Five recent publications including publications relevant to the present research protocol
1) Luby S, Carmichael S, Shaw G, Gamble W, Jones J. A nosocomial outbreak of Mycobacterium tuberculosis
from an unrecognized case. The Journal of Family Practice. June 1994, 39(1):21-25.
2) Siddiqui R, Luby S. Discitis following surgery for prolapsed intervertebral discs at a hospital in Pakistan.
Infection Control and Hospital Epidemiology. 1998, 19(7):526-529.
3) Altaf A, Luby S, Ahmed AJ, Zaidi NA, Khan AJ, Mirza S, McCormick J, Fisher-Hoch S. Outbreak of
Crimean-Congo haemorrhagic fever in Quetta, Pakistan: Contact tracing and risk assessment. Tropical Medicine and
International Health. 1998, (11):878 82.
4) Luby S, Zaidi N, Rehman S, Northrup R. Improving private practitioner sick-child case management in two
urban communities in Pakistan. Tropical Medicine & International Health. 2002 March; 7(3):210-219.
5) Luby SP, Agboatwalla M, Feikin DR, Painter J, Billhimer W, Altaf A, Hoekstra RM. Effect of handwashing
on child health: a randomised controlled trial. Lancet. July 15, 2005; 366:225-33.
------------------------------------------------------------------------------------------------------------
33
Biography of the Investigators
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
Rahman Mahmudur
Director
eRA COMMONS USER NAME
EDUCATION/TRAINING
INSTITUTION AND LOCATION
DEGREE
YEAR(s)
MBBS
1983
Medicine
ASEAN Institute of Health Development, Mahidol
University, Bangkok, Thailand
MPH
1988
Public Health
University of Cambridge, UK
Ph.D
1996
Epidemiology
Chittagong Medical College, Bangladesh
FIELD OF STUDY
A. Positions
October 2004 to date, Director.Institute of Epidemiology, Disease Control and Research. Mohakhali, Dhaka,
Bangladesh
June 2004 – October, 2004, Professor & Head, Department of Cancer Epidemiology,
National Institute of Cancer Research and Hospital, Dhaka, Bangladesh
2002-2004, Professor & Head, Department of Epidemiology, NIPSOM- National Institute of Preventive and Social
Medicine, Dhaka, Bangladesh
1988-2002, Associate Professor/Assistant Professor /Medical Officer (Lecturer), Department of Epidemiology,
NIPSOM, Dhaka, Bangladesh.
1984-1987, Medical Officer & Resident Medical Officer in different Thana Health Complexes & Urban Dispensaries (Rural and urban Health Centres in Bangladesh.
B. Selected peer reviewed publication and book chapters
1. Begum, J Ara and Rahman, M. A study on Nurses attitude and practices towards patient care JOPSOM, 1990,
4-9: 42-46.
2. Hussain, T. Rashiduzzaman, M. Hossain, M A. Rahman, M. and Banu F A. Prevalence of intestinal parasite in
the context of Socio-economic status in the rural community. The hygeia, 1990; Vol 4; No.3: 105-109.
3. Akhtaruzzaman, K M and Rahman, M. A study on spectrum of surgical intervention in the two upazila health
complexes, Medicine Today; 1991; Vol.3, No.11: 54-56.
4. Rahman M. Measurements in Epidemiology. Textbook of Community Medicine and Public Health. Rashid,
Khabir and Haider ed. RKH publishers, Dhaka, Bangladesh, 1992: 77-81.
5. Rahman, M. Investigation of an Epidemic. Textbook of Community Medicine and Public Health. Rashid, Khabir
and Haider ed. RKH Publishers, Dhaka, Bangladesh, 1992: 81-84.
6. Rahman, M. and Rahman, M. Communicable Diseases - Bacterial. Textbook of Community Medicine and
Public Health. Rashid, Khabir and Haider ed. RKH Publishers, Dhaka, Bangladesh, 1992: 196-203.
7. Das, A M and Rahman, M. (1992). An assessment of the quality of patient care in three government health
facilities; JOPSOM, 1992; Vol.11;No.2: 49-53.
34
8. Rahman, M. Rahman, M. and Ahmed, S. Occurrence of Otitis Media as a cause of tetanus amongst the patients of
Infectious Disease Hospital. JOPSOM, 1992; Vol. 11; No. 2: 59-62.
9. Rahman, M. and Rahim, A. Factors influencing early removal of Copper-T; JOPSOM:1993; Vol.12 No.4: p 103107.10.
10. Das, A M. and Rahman, M. Health care quality assurance: Concept, Strategy and issues; JOPSOM: 1993; Vol. 12
No. 3: 92-97.
11. Rahman, M. and Rahman, M. (1994). Socio-demographic characteristics of tetanus patients admitted in IDH;
JOPSOM: 1994; Vol. 13 No. 2-4: p 89-92.
12. Rahman, M. Weekly cleaning practices of hospitals. In: Khan F, Khan AW, Begum RA, and Shahidullah M, eds.
Case studies in Health Management. NIPSOM, Dhaka, 1995: p 61-62.
13. Rahman, M. Half-hearted supervision by higher authority. In: Khan F, Khan AW, Begum RA, and Shahidullah
M, eds. Case studies in Health Management. NIPSOM, Dhaka, 1995: p 70.
14. Rahman M., Mascie-Taylor C.G.N & Rosetta L (2001) The Duration of Lactational Amenorrhoea in Urban
Bangladeshi Women. J. biosoc. Sci. 34. 75-89.
15. Kabir I, Rahman M, Flora M S & Azad A K (2001) Arsenicosis and Body Mass Index (BMI) in a Selected Area
of Bangladesh; JOPSOM: 20 (1):6-12.
16. Hasan Z., Rahman M., Shaikh A. & Sarder A.R. (2001). Hearing Loss Among Autorickshaw Drivers of Dhaka
City. Journal of Bangladesh College of Physicians and Surgeons. 19(1), 19-23.
17. Yasmeen F & Rahman M. (2001). Are the Student’s of Institute of Health Technology (Dhaka) Getting Proper
Teaching ? JOPSOM: 20 (2): 83-86.
18. Begum F, Shamsuddin L, Hussain MA, Chowdhury TA, Rahman M & Das TR (2001). Effect of Oestrogen
Replacement Therapy on Bone Mass in Post-Menopausal Bangladeshi Women. Bangladesh Med. Res. Counc.
Bull. 2001: 27 (3): 103-111.
19. Textbook of Community Medicine and Public Health (2004). Edited by Rashid KM, Rahman M, Hyder S (561).
35
Biography of the Investigators
Give biographical data in the following table for key personnel including the Principal Investigator. Use a photocopy
of this page for each investigator.
(Note: Biography of the external Investigators may, however, be submitted in the format as convenient to them)
1
Name: Labib Imran Faruque
2
Present Position: Research Fellow, PIDVS, ICDDR,B
3
Educational background:
(last degree and diploma & training
relevant to the present research proposal)
Dhaka Medical College, MBBS, 2004
4.0 List of ongoing research protocols
(start and end dates; and percentage of time)
4.3.
As Principal Investigator
Protocol Number
4.4.
End date
Percentage of time
Starting date
End date
Percentage of time
Starting date
End date
Percentage of time
As Co-Principal Investigator
Protocol Number
4.5.
Starting date
As Co-Investigator
Protocol Number
5 Publications
Types of publications
a. Original scientific papers in peer-review journals
b. Peer reviewed articles and book chapters
Numbers
36
c.
d.
e.
f.
6
Papers in conference proceedings
Letters, editorials, annotations, and abstracts in peer-reviewed journals
Working papers
Monographs
Five recent publications including publications relevant to the present research protocol
1)
2)
3)
4)
5)
.
37
Detailed Budget for New Proposal
Project Title: Hospital based selected febrile illness study in Bangladesh
Name of PI: Stephen P Luby
Protocol Number: 2008-025
Funding Source: CDC
Name of Division: HSID
Amount Funded (direct): $ 41,000
Starting Date: 01 October 2008
Total: $ 41,000
Closing Date: 31 September 2009
Strategic Plan Priority Code(s): 4.1
38
Budget Justifications
Please provide one page statement justifying the budgeted amount for each major item. Justify use of human
resources, major equipment, and laboratory services.
This study is being funded by CDC. Fund is available for 1 year. Salaries for some of the key personnel are covered
under other projects; therefore their salaries are not included in the budget. A research fellow will coordinate the entire
study with full time effort. Six surveillance physicians recruited for hospital based influenza surveillance from the
existing staffs of the selected hospitals will conduct the field activities. Minimum financial incentive will be provided
to the surveillance physicians from the influenza surveillance budget. We will also recruit two laboratory technicians
for blood collection, centrifugation, specimen preparation and package for transportation. They will also prepare the
slides for malaria and perform the microscopic diagnosis for malaria. They will be paid 349 dollars each per month,
therefore both of them in total 698 $ per month with a total of 8376 $ for 1 year contractually. The field assistants
appointed from the influenza surveillance will help in collection, transportation and storage of the samples.
Extensive local traveling will be required for regular active surveillance. Therefore $3144 is allocated for per diem
and transportation for local travel.
Substantial amount of budget ($ 17880) is allocated for supplies and materials. The study will fund the reagents and
supplies required for pathogen detection and isolation. Yearly 720 specimens will be collected from regular active
surveillance. Cost for each specimen collection supplies will be $ 1.5. That is $ 1125 will be required for this purpose.
Laboratory reagent and supplies are going to cost the bulk amount. This surveillance aims to collect 720 specimens
and each of these specimens will require $ 2 each that is $ 1500 in total. For malaria rapid kit test of $ 2 totally $ 1500
will be needed. For IgM MAC ELISA test of $ 12.50 totally $ 9375 will be needed. Again for centrifugation in the
field we need centrifuge machine $300 each, at least 6 in number which may cost $1800 and will be kept in the local
hospital. Furthermore we have to preserve leptospira samples in incubator that may be $2500. 4 cold boxes of $20 will
add $80 to the subtotal.
Shipment for specimens costs in total 10,600 US dollars. For one shipment of 2 ml cryovials of 750 bartonella
specimens at −70°C 2200 US dollars have been aloocated. If we estimate about 500 chikungunya specimens then for
one shipment at −70°C it will cost 1300 US dollars. We will have 750 serum samples and 750 EDTA blood cryovials
for rickettsial specimens for which in total we have allocated 2900 US dollars for one shipment at −70°C. On the other
hand, for leptospira samples we have to send 120 specimens more frequently every 2 months provided that these
samples will be required to run in the laboratory within 3 months. One shipment costs 700 US dollars, so in total for 6
shipments we have allocated 4200 US dollars.
For other contractual services, $ 800 has been allocated. This includes the procurement of mobile phone sets for the
study activities, monthly mobile phone bills and cost of fax, telephone, postage, trainings, workshops, seminars and
printings and publications.
The interdepartmental services will cost $ 200, which includes data entry services, routine laboratory tests, printing
and photocopying.
Contributions of the co-investigators:
Emily Gurley was involved with the protocol from the very early stage. She has reviewed most of the drafts and has
given valuable comments. She also helped in budget section of the protocol. Her scientific contribution makes the
draft solid and perfect.
Rashidul Haque, head of parasitology laboratory, will give the necessary logistics and laboratory support for specimen
storage. He proposed us to assist in recruitment of two laboratory technicians for specimen collection and diagnosis of
39
malaria. He agreed to involve the technicians in training for blood collection, serum separation, microscopic slide
observation and other necessary works. He agreed to provide a space for an incubator in his lab for storing of
leptospira specimens. He will try his best to accommodate the collected specimens until shipment.
Rashid Zaman was involved with the protocol from the early stage. We are going to perform this study on the existing
infrastructure of Hospital Based Influenza Surveillance program which is implemented and co-ordinated by
Rashid.Zaman. He was involved in the Pilot study, site selection, follow-up meetings related to the protocol. He gives
valuable feedback of drafts very often.
Renee Galloway has reviewed the protocol and sent important feedback. She will perform leptospira culture in her
laboratory.
Michael Kosoy and Ying Bai have reviewed the protocol and give valuable comments. They will perform culture for
bartonella species.
Ann Powers have reviewed the protocol and she gave advice regarding the chikungunya specimen separation. Her
laboratory will perform chikungunya laboratory test.
Robert Massung and William L. Nicholson have reviewed the protocol. They gave suggestions regarding laboratory
analysis for rickettsial pathogen identification. They will perform IFA assay and PCR for rickettsia in their laboratory.
A. S. M. Alamgir will act as focal collaborator on behalf of IEDCR as he is now performing for Hospital Based
Influenza Surveillance program. Besides, being a virologist he has given helpful advice regarding virology section of
the protocol.
Other Support
Describe sources, amount, duration, and grant number of all other research funding currently granted to PI or under
consideration.
Stephen P Luby
Protocol Title
Burden of Pneumococcal Disease in children in Bangladesh
Surveillance for hospitalization and death due to pneumonia and meningitis
in Dhaka, Bangladesh
Nipah Virus transmission in Bangladesh
Assessing three assays for early detection of active turberculosis -- a pilot
study
Hospital based human influenza surveillance in Bangladesh
Poultry Influenza Surveillance in Bangladesh
Risk factors for meningococcal disease in Bangladesh
Protocol
number
2003-024
$
amount
1,077,917
2005-023
2005-026
$
$
157,037
99,993
4111336
4111351
2006-043
$
11,370
4211533
pending
pending
pending
$
$
$
412,000
135,000
7,926
4211533
4211533
4211533
budget code
4110691
Appendix A
40
International Centre for Diarrhoeal Disease Research, Bangladesh
Voluntary Consent Form
[Flesch-Kincaid Grade Level = 7.0]
Title of the Research Project: Hospital based selected febrile illness study in Bangladesh
Principal Investigator: Stephen P Luby
Name of the patient
ID Code
Introduction & Purpose:
We are from International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) and Institute of Epidemiology
Disease Control and Research of Bangladesh Government. In Bangladesh there are microbial agents cause substantial
morbidity and mortality. Their contribution to febrile illness remains unknown due to lack of systematic observation.
Therefore, this study will investigate the relative importance of pathogens among patients who present with fever across
Bangladesh. We invite you in this study because you have some symptoms of different causes of fever. If we find you with
any of these diseases, we may contact with you. We may visit your home to find out where do you live and collect other
information.
Procedures:
If you agree to be part of this study, we will take about 20 minutes of your time today. We will ask you few questions. You
may refuse to answer any questions you do not want to answer. We will also collect one and half teaspoonful of blood sample
from your arm. We will do rapid kit test for malaria. It will take about 15 minutes. The result will be shared with you and your
physician. We will also test your blood for several other infections that can cause fever. Some testing will be done at
ICDDR,B in Dhaka and some testing will be done at the Centers for Disease Control in the USA. However, those tests will
take much longer time. We will not get the results in hand for your present treatment.
Storage of specimen:
We will preserve serum for long-term storage. A new pathogen can be discovered with a new diagnostic at any time.
We will have the option for further tests of that sample for new pathogen. In future we may look for other infections
that can cause fever.
Risks or Discomforts:
Risks to you from participation in this study are minimal. Skilled laboratory personnel will collect blood specimen from your
hand. This will not be comfortable and may cause bruise. But it is a safe procedure and will cause no harm.
Benefits:
This study might help your care today. We will inform the result of the malaria test to your physician. The results of this study
will help us to find the better ways. It will prevent people from infection with different causes of fever in the future.
Confidentiality:
We will keep confidential what we talk about today and your test results. We will keep the records and the sample under a
code number rather than by your name. Only study staffs will be allowed to look at your questionnaire and test results. The
code that links a number to your name will be kept by study staff in locked files. Your name will not appear when we talk
about this study or publish its results.
Cost or Payment:
There is no cost, nor payment, for participation in this study.
Right to Refuse:
41
You may not join the study but will receive the same care. If you decide to leave, you can change your mind at any time for
any reason.
Persons to contact:
If you have questions during the procedure, ask at any time.
If you have any more questions related to study, please contact:
Dr. Labib Imran Faruque
Research Fellow
Programme on Infectious Diseases and Vaccine Sciences (PIDVS)
ICDDR,B
Mohakhali, Dhaka 1212
Phone: 8860523-32 # 2538 (Available from 8:30 AM to 5.00 PM except holydays),
01819252576
If you have questions about your rights in regard to being part of this study or if you think some harm has been done to you
because of the study, you may contact:
Mr. M. A. Salam Khan
Assistant Coordination Manager, IRB
Research Administration, Executive Director's Division
ICDDR,B
Mohakhali, Dhaka 1212,
Phone: 8860523-32 # 3206
Your signature or thumb print on this form mean that you understand all the information. You understand that participation is
voluntary. You may withdraw from the study at any time. Now if you agree to participate in the study, please sign or give your
thumb impression at the space indicated below.
Signature of Investigator/agents
Date:
Signature / thumb impression of Subject/ Guardian
Date:
You may participate in the study and give samples only to test for once. You may refuse to store the samples and to test the
samples in future. If you allow us to store the samples for long time and to test the samples in future, please sign or give your
thumb impression at the space indicated below.
Signature of Investigator/agents
Date:
Signature / thumb impression of Subject/ Guardian
Date:
Voluntary Consent Form (Bangla)
42
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44
Appendix B
International Centre for Diarrhoeal Disease Research, Bangladesh
Voluntary Assent Form (for 7-17 years)
[Flesch-Kincaid Grade Level = 4.8]
Title of the Research Project: Hospital based selected febrile illness study in Bangladesh
Principal Investigator: Stephen P Luby
Name of the patient
ID Code
Introduction:
We are from Cholera hospital and from IEDCR. Please ask questions if you do not understand. If you give your phone
number, we can contact with you later.
Purpose:
This study will help us to know some unknown causes of fever in this country.
Procedure
We will take 20 minutes to ask you some questions. We will collect one and half teaspoonful of blood from you. We
will do blood tests to find out what infections are present in your body. We will do malaria test now. We will test your
blood for several other infections as well that can cause fever. Those tests will take much longer time. We will not get
the results in hand for your present treatment.
Storage of specimen:
We will store serum for long-term. We will have the option for further tests of that sample for any new pathogen. We
may look for other infections that can cause fever.
Voluntary
You may or may not attend in our survey, will receive the same care. It is voluntary.
Benefits:
We will inform the malaria test result to your doctor now. This study will help other people and children. They can keep away
from infection with different causes of fever in the future.
Harms
You may feel discomfort and bruise may occur during blood collection. But this is a safe procedure. This will not
affect your health.
45
Confidentiality:
We will keep the records and the sample under a code number. Only study staffs will look at your questionnaire and test
results. Your name will not appear when we talk or publish this study.
Cost/Payment:
There is no cost, nor payment, for participation in this study.
Persons to Contact:
If you have questions during the procedure, ask at any time.
If you have any more questions regarding the study, please contact:
Dr. Labib Imran Faruque
Research Fellow,
Programme on Infectious Diseases and Vaccine Sciences (PIDVS)
ICDDR,B
Mohakhali, Dhaka 1212,
Phone: 8860523-32 # 2538 (Available from 8:30 AM to 5.00 PM except holydays),
01819252576
If you have questions about your rights in regard to being part of this study or if you think some harm has been done to
you because of the study you may contact:
M. A. Salam Khan
Assistant Coordination Manager, IRB
Research Administration, Executive Director's Division
ICDDR,B
Mohakhali, Dhaka 1212,
Phone: 8860523-32 # 3206
If you agree to participate in the study, please sign or give your thumb impression below.
Signature of Investigator/agents
Date:
Signature / thumb impression of Subject
Date:
Signature / thumb impression of Guardian
Date:
You may give samples only to test for once. You may refuse to store the samples and to test the samples in future. If
you allow us to store the samples for long time and to test the samples in future, please sign or give your thumb
impression at the space indicated below.
Signature of Investigator/agents
Signature / thumb impression of Subject
46
Date:
Date:
Signature / thumb impression of Guardian
Date:
Voluntary Consent Form (Bangla)
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47
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48
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Symptoms
Fever
Headache
Bodyache
Muscle pain
Joint pain
Rash
Bleeding from
any site
Date of onset
(dd/mm/yy)
Y
N
Y
Y
Y
Y
Y
Y
N
N
N
N
N
N
Symptoms
Retro-orbital
pain
Eye-redness
Jaundice
Neck stiffness
Reduced
Urinary output
others
Patient’s
name
Household
head
Address
Date of onset
(dd/mm/yy)
Y
N
Y
Y
Y
Y
Y
Y
N
N
N
N
N
N
Appendix C:
Standardize
d assessment
form
Age (ie3 month=0003,
12 years=1200)
Sex (male=1,female=2)
Village
Union
Upazilla
District
Occupation
Mobile number of self
Neighbourhood number
Cell phone shop number
2
2
Unique ID
Patient
ID
Pediatric
Outpatient
Hospital
ID
1
1
year
Department
Medicine
Unit
Inpatient
Surveillance personnel
Date
(dd/mm/yyyy)
month
Hospital name
Time in 24 hours format
(ie 0900,1300)
49
Signs
Pulse
Temperature
Blood pressure
Respiratory rate
Others
anemia
jaundice
/
Yes=1
No =2
edema
Rash
Neck rigidity
Dehydration
Yes
=1
No
=2
Yes
=1
No
=2
Description of fever pattern
Description of rash
Clinical diagnosis at admission
Clinical diagnosis at discharge
Laboratory investigation
WBC
Platelet
ESR
Nutrophil (%)
Hb %
Lymphocyte (%)
S.bilirubin
(gm/dl)
S.creatinine
(gm/dl)
Specimen collection documentation (yes=1, no=2)
Blood
Centrifuge
collected
done
ICT for Malaria
Blood clot
positive result
taken
EMJH tube
Serum
inoculation
aliquoting
Slide prepared
Treatment received
EDTA tube
Comments
Signature of the study physician
Appendix D: Laboratory report form
Hospital based selected febrile illness study in Bangladesh
The International Centre for Diarrhea Disease Research, Bangladesh (ICDDR,B) and the Institute of Epidemiology Disease
Control and Research (IEDCR) of the government of Bangladesh are doing a research study to learn more about febrile illness
in Bangladesh. As a part of this study the malaria rapid kit test “Immuno-chromatography test” is performed free of cost and
the result is mentioned below.
Date (dd/mm/yyyy)
Hospital name
Unique ID
Medicine
Department
Patient’s name
Address
Pediatrics
Unit
Inpatients
Outpatients
Year
Hospital Patient
Month ID
ID
Age
Sex
50
Name of the investigation: Immunochromatography test (ICT) for Malaria
Result of the test:
Signature and name of the laboratory technician:
Signature of the surveillance physician:
Appendix E: Follow-up questionnaire
When he was discharged
When he get recovery from illness
Specify, if there is any disability
Does he need any medication
Is there anyone in area got same illness
and if yes then address
Number of Family members
Whether they had similar attacks
What are the assets they have
Patient
ID
Hospital
ID
Unique ID
year
Date(dd/mm/yyyy)
Time in 24 hours format (ie 0900)
Interview personnel
month
Patient’s name
Occupation
Monthy income
Amount of land
Type of fuel use for cooking
Pure drinking water facility
Sanitation facility
Main material of roof
Main material of floor
Main material of wall
51
Symptoms
Fever
Joint pain
Rash
Nodular skin
lesion
Bleeding from
any site
Date of onset
(dd/mm/yy)
Y
Y
Y
N
N
N
Y
N
Y
N
Symptoms
Date of onset
(dd/mm/yy)
Cognitive
dysfunction
Jaundice
Edema
Y
Y
Y
Y
N
N
N
N
others
Y
N
Comments
Signature of the interviewer
Appendix F:
Specimen collection procedures
Venous blood samples
Materials for collection
· Skin disinfection: 70% alcohol (isopropyl alcohol, ethanol) or 10% povidone iodine, swabs, gauze pads, band aid
· Disposable latex or vinyl gloves
· Tourniquet, Vacutainer, or similar vacuum blood collection devices, or disposable syringes and needles
· Vacutainer or sterile screw-cap tubes, blood culture bottles with appropriate media
· Labels and indelible marker pen.
Method of collection
· Place a tourniquet above the venepuncture site.
· Palpate and locate the vein. It is critical to disinfect the venepuncture site meticulously with 10% povidone iodine or
70% isopropyl alcohol by swabbing the skin concentrically from the centre of the venepuncture site outwards. Let the
disinfectant evaporate. Do not repalpate the vein again. Perform venepuncture.
· Withdrawal of 7-10 ml of whole blood from the study participants with conventional disposable syringes.
· Remove the tourniquet. Apply pressure to site until bleeding stops, and apply sticking plaster.
· Using aseptic technique, transfer the specimen to relevant cap transport tubes and culture bottles. Secure caps tightly.
Be sure to follow the instructions on the correct amount and method for inoculation of blood culture bottles.
· Label the tube, including the unique patient identification number, using indelible marker pen.
52
· Do not recap used sharps. Discard directly into the sharps disposal container.
· Complete the case assessment and the laboratory report forms using the same identification number.
Appendix G: External reviewer’s comments [1]
Reviewed by: Sonja J. Olsen , PhD
Lead, Global Activities
Division of Emerging Infections and Surveillance Services
National Center for Preparedness, Detection, and Control of Infectious Diseases .
(NCPDCID)
Centers for Disease Control and Prevention
1600 Clifton Road, NE, Mailstop C-14, Atlanta, GA 30333
Phone: (404) 639-3534, (800) 311-3435, 404-639-2945 (fax)
Dated On: 11th February, 2008
This protocol is designed to be a 6 site, one-year study of select causes of febrile illness in
Bangladesh. The sites are already part of a sentinel flu surveillance network so have clinical and
surveillance staff that can also assist on this project. The pathogens of interest in this study are
dengue, malaria, Bartonella, chikungunya and leptosiprosis.
General comments
1. The protocol was a little confusing to follow at times and has grammatical errors throughout.
I think the addition of a few flow charts could help clarify the process. See below for
specifics.
2. Did the investigators consider any additional pathogens, such as Rickettsia?
53
3. Blood culture would obviously pick up a variety of pathogens that cause fever. I assume it is
not feasible to do blood culture in these hospitals? Probably worth mentioning in the
background section of the protocol.
Specific comments
Methods
1. It would be nice to have some additional information on the specific tests to be run,
specifically the PCR and ELISA.
2. Did the investigators consider obtaining a convalescent serum sample? For the antibody
tests, I suspect it will be difficult to interpret the findings without an acute and convalescent
sample. Even specimens taken after day 5-7 of illness may not have antibodies present. If
convalescent specimens are not possible to take that should be stated in the protocol.
3. What is meant by indoor or outdoor patients? Is this supposed to be inpatient and
outpatient?
4. It is not clear, but is the approach to seek inpatients first and if there are none to then go to
the outpatient clinics? This section needs to be clarified. Perhaps a flow chart would be
helpful to explain how cases will be identified. Are the first patients of the day enrolled? I
think it also needs to be clear that this is a convenience sample.
5. Exclusion criteria. Why is a patient with diarrhea considered someone with a known case of
fever? Usually ‘known cause’ implies laboratory confirmation of some kind. Do you really
expect that any fever causes will really be identified in these sites? Perhaps the exclusion
should be exclusion of patients with other clinical syndromes not of interest in this study
(e.g., respiratory or gastrointestinal symptoms)?
6. Surveillance personnel. I think the intent is to use personnel already engaged in ongoing
influenza surveillance activities. If this is the case then the description just needs to be a
little clearer on this point. Also, are you sure these people have enough time to add on
additional duties? At the end of the protocol you imply that their influenza duties will be
decreased.
7. Specimen sections. These sections are not very clear on the process for collecting,
aliquoting, transporting, storing, and shipping specimens. For example, it is not clear what
happens to the blood inoculated into the EMJH media. Is that frozen for 3 months before
being shipped to US CDC or is the incubation done at ICDDR,B? Again, a flow chart might
help clarify the process.
8. Malaria testing at ICDDR.B. Will this test be done by technicians blinded to the rapid test
result? This would be preferable so that the results can be presented as an unbiased
comparison.
9. Why is serum not also stored at -70oC? Since you are able to do this for the blood clots I
think it is preferable for serum to avoid degradation of antibodies.
10. What serologic and PCR tests will be used? Also, probably best to just refer to US CDC and
not specify Atlanta or Ft. Collins since the sites are not always correctly references as it is
currently written.
11. There should be some information on data and how it goes from the paper form to a
database. Will it be double entered? How will the personal identifiers be dealt with? What
will be done to insure confidentiality.
12. Sample size. It is not clear how the sample size was derived. Even for descriptive
epidemiology it would be nice to think through what you might expect. What percent
positive do you expect from similar studies? I would guess less than 50%. Also, do you
want to be able to say anything about the frequency by site since you have specifically gone
after geographic diversity? If yes, you sample size in each of these groups is getting small.
Alternatively, perhaps the sample was chosen based on the number of specimens that can
be processed?
13. Facilities. Can you add a brief description of the current hospitals in terms of laboratory
equipment? Do they all have a refrigerator for storage of the blood? I assume none have the
capacity for blood culture.
54
14. Ethics. I think the section on benefits needs to be re-worded. The only testing that is of
immediate benefit to the patients is the rapid malaria test. I think it should be clearly stated
that the other testing will not have any direct impact on patient care. Also, this study is not
going to tell you anything about burden. Burden implies you have data on incidence and
cost, neither of which you will have here. Instead, I think the study will provide insight into
the etiology of fever in these sentinel sites.
15. Adverse events. Does everyone already know what the “routine practices” are in the event
of an adverse event?
16. Dissemination of findings. What about the dissemination of findings to clinicians? Do they
usually read the Bulletin or are there other ways to reach them?
17. Collaborative arrangements. The last sentence implies that the influenza surveillance
activities will be cut back so that the staff can take on these added responsibilities for the
fever study. Is this true? Does this have implications?
Consent
I think the language is too advanced. For example, do you really think most people will know what
‘manifestations’ and ‘etiologies’ mean? I think the consent forms need to be compared to a reading
scale to make sure they are at a low enough reading level.
Appendix H: Responses to external reviewer’s comments [1]
This protocol is designed to be a 6 site, one-year study of select causes of febrile illness in
Bangladesh. The sites are already part of a sentinel flu surveillance network so have clinical and
surveillance staff that can also assist on this project. The pathogens of interest in this study are
dengue, malaria, Bartonella, chikungunya and leptosiprosis.
General comments
1. The protocol was a little confusing to follow at times and has grammatical errors throughout.
I think the addition of a few flow charts could help clarify the process. See below for
specifics.
We have added 2 flowcharts and tried to correct the grammatical errors.
2. Did the investigators consider any additional pathogens, such as Rickettsia?
We have added Rickettsia to be included in our study.
3. Blood culture would obviously pick up a variety of pathogens that cause fever. I assume it is
not feasible to do blood culture in these hospitals? Probably worth mentioning in the
background section of the protocol.
55
Conventional blood culture would allow us to explore the role of less esoteric pathogens.
But in most of the study hospitals there is no facility for standard blood culture for example in
SBMCH and JRRMCH. Moreover ELISA for dengue and ICT for malaria are not possible in
some places as in BBMH and JRRMCH. So besides large volume of blood collection and huge
expenses to perform blood culture and other tests, it will create inconsistency in the study
results. In addition, actually this is a study to look at under-considered causes of febrile
illness in Bangladesh as a fishing expedition for some specific organisms of interest. As this is
the primary rationale for this study, thus we are not going to perform routine blood culture to
diagnose typhoid disease or brucellosis. Moreover considering the health seeking behaviour
and listed characteristics of pathogens in the study it will not feasible to perform blood
culture. Therefore, the following section has been added in the background:
“Other potential causes of febrile illness in Bangladesh include typhoid fever and
brucellosis. Typhoid fever is a common cause of fever in Bangladesh [72-78]. Brucella is
another potential pathogen that can be transmitted either through contact with infected
animals or from incompletely pastereurized dairy products. Because the confirmed diagnosis
of these two pathogens requires a substantial volume of blood in a separate blood culture, we
have chosen not to include them in the present study. Indeed, we are not attempting to
conduct a comprehensive study of all causes of febrile illness in Bangladesh. Instead, we are
focusing on a handful of pathogens that there is very little data on to assess if they are
important or occasional causes of fever. The list of pathogens that we are assessing is
limited by cost and logistics.”
Specific comments
Methods
1. It would be nice to have some additional information on the specific tests to be run, specifically
the PCR and ELISA.
page.
This section has been added in the Research Design and Methods section in the 18th
“Description of Laboratory methods with sensitivity and specifity of the tests
The sensitivity and specificity of the Dengue IgM Capture ELISA are reported to be 94.7
% (with CI 85.4 - 98.9 %) and 100% (with CI 95.7 - 100%), respectively. The dengue IgM
Capture ELISA determines the level of IgM antibodies to dengue in a patient’s serum. A
positive result (> 11 Panbio units) is indicative of either an active primary or secondary
dengue infection. < 9 Panbio units will be counted as negative results. (Dengue IgM Capture
ELISA package insert. Rev 28/01/03.,E-DEN01M, www.panbio.com)
We will have the option to do PCR for dengue for the specimen collected during the
earlier period of illness. But with the specimen collected after that period ELISA can be done.
Therefore, we will perform ELISA test for all the specimens. Through labeling the specimen
during collection, we will mark samples of those cases which become ELISA negative but
collected within first 5 days. In that case we will have the option to perform more reliable test
such as PCR. Multiplex Reverse Transcriptase-PCR (MRT-PCR) for dengue is highly sensitive &
specific compare to ELISA and confirms the diagnosis with serotype identification. Gel based
PCR will be used. The reaction mixture will contain 50 mM KCl, 10 mM Tris (pH 8.5), 0.1%
56
TritonX-100, 0.01% gelatin. Dengue RNA can be detected by using serotype specific primers,
reverse transcriptase and thermostable polymerase (Typing of Dengue Viruses in Clinical
Specimens and Mosquitoes by Single-Tube Multiplex Reverse Transcriptase PCR, Journal of
Clinical Microbiology, Sept. 1998, p. 2634–2639 Vol. 36, No. 9). Multiplex Reverse
Transcriptase-PCR (MRT-PCR) is highly sensitive, specific along with cost-effective and less
time consuming. Without proper precaution, contamination can lead to false-positive results
(Source: Dengue hemorrhagic Fever diagnosis, treatment, prevention and control. WHO
Geneva 1997).”
Malaria will be diagnosed by rapid diagnostic test (RDT) based on the detection of
P.falciparum-specific antigen and plasmodium vivax-specific antigen. The trade name of this
Rapid Diagnostic Test is “FalciVax” and it is being produced by Zephyr Biomedicals, India and
“BinaxNow” produced by Inverness medical, USA. Falcivax is rapid self-performing,
qualitative, two-site sandwith immunoassay utilizing whole blood for the detection of
P.falciparum specific histidine rich protein-2 (pf, HRP-2) and P.vivax specific pLDH. The test
can be used for specific detection and differentiation of P.falciparum and P.vivax malaria. The
standardization of this test has already been done by Zephyr Biomedicals. Sensitivity of the
RDT is similar to that commonly achieved by good field microscopy. Sensitivity and specificity
of the Rapid Diagnostic Test used for the detection of P.falciparum and P.vivax will be more
than 95 % and now been recommended for use in the malaria control program by the WHO
(Singh N et al. 2002; Moody A. 2002; WHO, 2004)
Both thick and thin film will be done for diagnosis of malaria by microscopy. The blood
films will be stained with Giemsa stain in phosphate buffer saline and examined under the
microscope at a magnification of x 1000 for the presence of malaria parasites. Blood films
were defined as negative if no parasite were observed in 300 oil immersion fields
(magnification, x 1000) on thin film by an experienced microscopist (Warhurst DC and
William JE, 1996).
Microscopy diagnosis is fairly sensitive and highly specific. False negative results may
be seen in conditions of very low parasitemia, maturation of sequestered parasites in the
broods, partially treated with antimalarials or on chemoprophylaxis, and may be due to
technical factors; (poorly prepared slides, poorly stained slides, poor quality microscope,
examination of only thin films, inexperienced technician etc). False positive results are seen
due to stained particles, which may be confused for malarial parasite by an inexperienced
microscopist. In a study in Bangladesh it has been shown that both types of malaria the
sensitivity was 94% (95% CI 71.3–99.9), and the specificity was 93% (95% CI 90.0–98.5)
(Failure of national guidelines to diagnose uncomplicated malaria in Bangladesh. am. j. trop.
med. hyg., 67(4), 2002, pp. 396–399).
In ICDDR,B laboratory we will perform microscopic diagnosis for malaria for all the
samples to confirm that malaria and also able to identify the specific species. If there is any
discrepancy between these two malaria test results, we will inform the results to the
attending physician. Therefore, we will get more reliable results with specific spices
identification by performing both of the tests. We will classify plasmodium vivax and
plasmodium falciparum. The degree of parasitaemia in each case will be reported as scanty
(1-10 parasites per 100 high-power fields), moderate (10-100 parasites per 100 high-power
fields), or heavy (>100 parasites per 100 high-power fields). (Concurrent malaria and enteric
fever in Pakistan, Singapore Med J 2005; 46(11): 635)
Leptospira culture is the optimum way to confidently identify infection, but a negative
culture does not exclude Leptospira infection. Serology should be sought for identification of
57
the infecting serovar or serogroup prior to typing of the isolate.
(http://www.health.gov.au/internet/main/publishing.nsf/Content/cda-phlncdleptospirosis.htm)
Indirect immunofluorescence antibody (IFA) assay is the “gold standard” technique and
is used as a reference technique in most laboratories. There is no other serologic test is
better than the sensitivity and specificity of these assays. Serum antibodies bind to fixed
antigens on a slide and are detected by a fluorescein-labeled conjugate. The sensitivity of the
IFA assay is substantially dependent on the period of sample collection. As the illness
progresses to 7–10 days, the sensitivity of IFA serology increases. The IFA is expected to be
94%–100% sensitive after 14 days and sensitivity is increased if paired samples are tested
(Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted
Fever, Ehrlichioses, and Anaplasmosis — United StatesMarch 31, 2006 / Vol. 55 / No. RR-4
Prepared by Alice S. Chapman, DVM1 in collaboration with the Tickborne Rickettsial Diseases
Working Group and Brouqui P, Bacellar F, Baranton G, et al. Guidelines for the diagnosis of
tick-borne bacterial diseases in Europe. Clin Microbiol Infect Dis 2004;10:1108–32). For the
detection of R. rickettsii responsible for Rocky Mountain spotted fever (RMSF), sensitivity, as
tested with 60 paired serum specimens, including specimens with stationary titers (5%) and
fourfold rising titers (95%), was 100% ( Kleeman, K. T., J. L. Hicks, R. L. Anacker, R. L.
Philip, E. A. Casper, K. E. Hechemy, C. M. Wilfert, and J. N. MacCormack. 1996. Early
detection of antibody to Rickettsia rickettsii: a comparison of four serological methods:
indirect hemagglutination, indirect fluorescent antibody, latex agglutination, and complement
fixation, p. 171–178. In J. Kazar (ed.), Rickettsiae and rickettsial diseases. Veda, Publishing
House of the Slovak Academy of Sciences, Bratislava, Slovakia). In a study with patients
with no rickettsial diseases, a titer of ≥ 1:64 had a specificity of 100% and a sensitivity of
84.6%, and a titer of ≥ 1:32 had a specificity of 99.8% and a sensitivity of 97.4%
(Newhouse, V. F., C. C. Shepard, M. D. Redus, T. Tzianabos, and J. E. McDade. 1979. A
comparison of the complement fixation, indirect fluorescent antibody, and microagglutination
tests for the serological diagnosis of rickettsial diseases. Am. J. Trop. Med. Hyg. 28:387–
395). For scrub typhus, the sensitivity of IFA is low if high specificity is required that can be
explained as follows: for a titer of ≥ 1:100, sensitivity is 84% and specificity is 78%, for a
titer of ≥ 1:200, sensitivity is 70% and specificity is 92%, and for a titer of ≥ 1:400,
sensitivity is 48% and specificity is 96% (Brown, G. W., A. Shirai, C. Rogers, and M. G.
Groves. 1983. Diagnostic criteria for scrub typhus: probability values for immunofluorescent
antibody and Proteus OXK agglutinin titers. Am. J. Trop. Med. Hyg. 32:1101–1107). A
fourfold increase to a titer of ≥ 1:200 is 98% specific and 54% sensitive (Laboratory
Diagnosis of Rickettsioses: Current Approaches to Diagnosis of Old and New Rickettsial
Diseases Bernard La Scola and Didier Raoult Journal Of Clinical Microbiology, 00951137/97/$04.0010 Nov. 1997, p. 2715–2727 vol. 35, no. 11). Usually the indirect
microimmunofluorescence assay is not positive when the patient is acutely unwell, but it has
been the most sensitive and specific of the traditional serological tests (Graves, S.R., et al.,
Flinders Island spotted fever: a newly recognised endemic focus of tick typhus in Bass Strait,
Part 2: Serological investigations.Med J Aust 1991. 154: p. 99–104). Culture techniques are
very sensitive but can take up to 2 months to get a positive result that may limit their clinical
usefulness (B, U.N., et al., Not only ‘Flinders Island’ spotted fever. Pathology, 2005. 37: p.
242–245). Real-time PCR is both highly specific and extremely sensitive for the diagnosis of
rickettsioses and may offer the advantages of speed, reproducibility, quantitative capability,
and low risk for contamination, compared with conventional PCR (Fenollar F, Raoult D.
Molecular genetic methods for the diagnosis of fastidious microorganisms. APMIS 2004;
112:785–807 and Stenos, J., s.r. Graves, and n.b. Unsworth, A highly sensitive and specific
real-time pcr assay for the detection of spotted fever and typhus group rickettsiae. Am. J.
Trop. Med. Hyg., 2005. 73(6): p. 1083–1085).
58
Bartonella culture is considered confirmatory diagnosis for bartonella species. If
Bartonella-like colonies are seen on an agar plate, material from one colony will be streaked
onto a new agar plate. Presumptive Bartonella cultures will be used for the extraction of
Bartonella DNA and PCR.”
2. Did the investigators consider obtaining a convalescent serum sample? For the antibody tests,
I suspect it will be difficult to interpret the findings without an acute and convalescent sample.
Even specimens taken after day 5-7 of illness may not have antibodies present. If convalescent
specimens are not possible to take that should be stated in the protocol.
We have added this section in the Research Design and Methods section under the subsection of case definition in the 19th page.
“We performed a pilot study in Rajshahi Medical College Hospital and consulted with
our focal collaborators for Hospital Based Influenza Surveillance in other hospitals about this
coming protocol. They have informed us that in most cases febrile illness of 10 days will
present in the outpatient department. So in most cases we will get samples from outpatients
who usually take consultation for once and will be difficult to track them within 7 days unless
their illnesses become complicated. Therefore, it will not be feasible to collect convalescent
sample.”
3. What is meant by indoor or outdoor patients? Is this supposed to be inpatient and outpatient?
In South Asia, and Indoor refers to inpatients and outdoor refers to outpatients. To
assist our North American readers we have changed the terminology.
4. It is not clear, but is the approach to seek inpatients first and if there are none to then go to the
outpatient clinics? This section needs to be clarified. Perhaps a flow chart would be helpful to
explain how cases will be identified. Are the first patients of the day enrolled? I think it also
needs to be clear that this is a convenience sample.
Your understanding is correct. At first we will seek inpatients. If we have not identified
total 10 cases from both adult medicine and paediatric ward that meet our case definition
then we will go to outpatient clinic to take the rest of the samples.
We have added a flow chart to the methods section.
5. Exclusion criteria. Why a patient with diarrhea is considered someone with a known case of
fever? Usually ‘known cause’ implies laboratory confirmation of some kind. Do you really
expect that any fever causes will really be identified in these sites? Perhaps the exclusion
should be exclusion of patients with other clinical syndromes not of interest in this study (e.g.,
respiratory or gastrointestinal symptoms)?
Exclusion criteria needs to have explicit decision rules on which to exclude, otherwise
different clinicians will make different exclusions which will reduce the comparability of the
data from different sites.
15:
We have re-written the section as below in the subsection of exclusion criteria in page
“The Surveillance physicians will be instructed to exclude the following symptoms:
 Fever for more than 10 days.
59
 Cough with productive sputum.
 Urgency, frequency, hesitancy during micrurition.
 Cellulites/abscess/boil/local skin infection in the body.”
6. Surveillance personnel. I think the intent is to use personnel already engaged in ongoing
influenza surveillance activities. If this is the case then the description just needs to be a little
clearer on this point. Also, are you sure these people have enough time to add on additional
duties? At the end of the protocol you imply that their influenza duties will be decreased.
We have added the following section in the surveillance personnel sub-section in the
16th page.
“They don’t need to collect samples from the patients of both influenza and febrile
illness surveillance. This will be performed by our newly recruited laboratory research officer
for the study.”
7. Specimen sections. These sections are not very clear on the process for collecting, aliquoting,
transporting, storing, and shipping specimens. For example, it is not clear what happens to the
blood inoculated into the EMJH media. Is that frozen for 3 months before being shipped to US
CDC or is the incubation done at ICDDR,B? Again, a flow chart might help clarify the process.
Again thanks for this advice. We have added a flow chart in the research design and
method section in 20th page for the clarification of the whole process.
8. Malaria testing at ICDDR.B. Will this test be done by technicians blinded to the rapid test result?
This would be preferable so that the results can be presented as an unbiased comparison.
We have mentioned this paragraph in Specimen transportation to ICDDR,B laboratory
subsection at the 21st page.
“We have single laboratory personnel for both rapid and microscopic tests. Therefore,
we have an alternative approach to make the technician blinded about the rapid test results.
The approach is stated below.
When research officer will prepare the slides in the field he will assign a unique ID
number to the respective slide. Then he will cover that ID number with a translucent scotch
tap like the electric tap. At ICDDR,B he will bring the slides and observe under the
microscopy. Thereafter, he will point out the microscopic results and finally remove the scotch
tap. But he has to write with the permanent ink.”
9. Why is serum not also stored at -70oC? Since you are able to do this for the blood clots I think it
is preferable for serum to avoid degradation of antibodies.
Your concerned are well considered. We have talked with our laboratory co-workers.
Though they assured us about dengue and chikungunya serum preservation at -20oC but they
also recommend that it will be better to seek for -70oC freezer. Therefore we will try to
preserve the 720 samples at -70 oC.
10. What serologic and PCR tests will be used? Also, probably best to just refer to US CDC and not
specify Atlanta or Ft. Collins since the sites are not always correctly references as it is currently
written.
60
We will perform Dengue IgM Capture ELISA and Multiplex Reverse Transcriptase-PCR
(MRT-PCR) for Dengue virus. For chikungunya viral fever, only those samples with history of
joint pain will be sent to US CDC to perform ELISA or PCR. For rickettsial antibody detection
Indirect fluorescent antibody (IFA) assay will be done along with Real time PCR duplex essay.
We have cited the term US CDC according to your suggestion.
11. There should be some information on data and how it goes from the paper form to a database.
Will it be double entered? How will the personal identifiers be dealt with? What will be done to
insure confidentiality?
We have cited this section at Data Analysis section in the 26th page.
“We will record personal and illness related informations from the patients. Data will be
double entered. We will use a unique ID for each individual. We will Code the data and secure
the master list linking the code to the subject identifier. We will maintain the data in a secure
environment. This will ensure confidentiality.”
12. Sample size. It is not clear how the sample size was derived. Even for descriptive epidemiology
it would be nice to think through what you might expect. What percent positive do you expect
from similar studies? I would guess less than 50%. Also, do you want to be able to say
anything about the frequency by site since you have specifically gone after geographic
diversity? If yes, you sample size in each of these groups is getting small. Alternatively,
perhaps the sample was chosen based on the number of specimens that can be processed?
We have re-written the sample size section. The sample size is based on overall
prevalence, and not site specific prevalence.
13. Facilities. Can you add a brief description of the current hospitals in terms of laboratory
equipment? Do they all have a refrigerator for storage of the blood? I assume none have the
capacity for blood culture.
We have contacted individual staffs in each study hospital to get the glimpse of the
laboratory facilities which we have mentioned below and add under the facilities section in the
25th page.
Hospital
Refrigerator
Blood culture
Microscopic diagnosis of slide
ICT for malaria
ELISA for dengue
CBC,TC,DC,ESR
JIMCH,
Kishorganj
√
√
√
√
X
√
BBMH
Chittagong
√
x
√
x
x
√
RMCH
Rajshahi
√
√
√
x
x
√
KMCH
Khulna
√
x
√
x
x
√
SBMCH
Barisal
x
x
√
x
x
√
JRRMCH
Sylhet
√
√
√
√
x
√
14. Ethics. I think the section on benefits needs to be re-worded. The only testing that is of
immediate benefit to the patients is the rapid malaria test. I think it should be clearly stated that
the other testing will not have any direct impact on patient care. Also, this study is not going to
tell you anything about burden. Burden implies you have data on incidence and cost, neither of
which you will have here. Instead, I think the study will provide insight into the etiology of fever
in these sentinel sites.
61
The comments are well taken. We have mentioned about the result of malaria rapid test
which we can inform the patient immediately. We will have the contact number of the patients. So
we can inform the microscopic test results to the patients or attending physician to benefit their
treatment. But we have also mentioned in the consent form that the results of other test results will
not be available during treatment. So these tests will not have any impact on their treatment. We
have also omitted the phrase of burden. We re-write that the results of this study will help us to find
the better ways. It will prevent people from infection with different causes of fever in the future.
15. Adverse events. Does everyone already know what the “routine practices” are in the event of an
adverse event?
Such an activity is rare in our hospital settings so people do not know the “routine
practice” during untoward effects of blood collection hazards. We have added the section in
26th page:
“We have trained lab officer, health assistant and surveillance physician so we will do
our best effort to manage any untoward outcomes ranging from anxiousness to multiple
pricks. In routine diagnostic purpose blood is collected frequently but no adverse outcome
occurs usually. However, we strongly anticipate that there will be no adverse event due to
technical fault as we are going to recruit very expert personnel from our parasitology lab for
blood collection.”
16. Dissemination of findings. What about the dissemination of findings to clinicians? Do they
usually read the Bulletin or are there other ways to reach them?
We have added the following paragraph in 31st page:
Yes, HSB bulletins are sent to clinicians periodically. This bulletin includes Bengali
translation besides original English text. So it is comfortable and convenient for the clinicians
to go through the study findings. From ICDDR,B, 6006 copies are sent through posted mails
to the specific addresses of the medical professionals; most of them are physicians and
engage in clinical practice. Besides as we will do the study in the hospital settings in close
contact with the clinicians so we can disseminate the study finding to them very easily. In
addition clinicians especially the medicine specialists themselves will be very interested about
the study as they very often clinically diagnose these diseases without the scope of laboratory
confirmation.
17. Collaborative arrangements. The last sentence implies that the influenza surveillance activities
will be cut back so that the staff can take on these added responsibilities for the fever study. Is
this true? Does this have implications?
We have omitted this sentence. This sentence is clarified in the response of 6th
comment.
Consent
I think the language is too advanced. For example, do you really think most people will know what
‘manifestations’ and ‘etiologies’ mean? I think the consent forms need to be compared to a reading
scale to make sure they are at a low enough reading level.
The points are well taken. We have omitted the words and phrases which are more
technical in terms. For making lower index of readability scale we have made the complex
62
sentience into simple ones and rephrased the complex words. Finally, Flesch-Kincaid Grade
Level for the consent form came to 7.0.
Appendix I: External reviewer’s comments [2]
Reviewed by: Frank J. Mahoney,
Epidemiologist and past director
Disease Surveillance Program
Naval Medical Research Unit Number 3, Cairo, Egypt.
To: Science Review Board, ICDDR,B
Date: 2/25/2008
Subject: Comments on protocol entitled: Hospital based febrile illness study in Bangladesh
This is a well-written protocol which outlines a plan to conduct surveillance for patients with acute
febrile illness in 6 hospitals in Bangladesh. Briefly, patients meeting the case definition will undergo
a standardized clinical and laboratory evaluation.
Specific comments:
General comments: The authors propose to study 5 specific pathogens as a cause of AFI. It is
unclear why other bacterial causes of disease (typhoid fever, brucellosis…) are not addressed. Are
these diseases not common in Bangladesh? Will the results be reliable without ruling out these
diseases? What background data is available to compare these diseases in terms of public health
63
importance with the proposed list to be studied? Will patients with a clinical diagnosis of these
infections be included or excluded? A review of the epidemiology of these diseases in the
background section would be helpful to explain why they are not included.
It would be helpful to provide more background on the hospitals in the study including whether
they are referral hospitals, infectious disease hospitals, district hospitals, etc. It would be
particularly useful if information is available on background rates of disease are available (eg
number of patients seen each year with dengue or malaria by age group) to compare proposed
sample size with number of patients being seen in these facilities.
Case definition: On page 14, the section on the case definition needs to be rewritten: The case
definition is mixed with numerous details related to the laboratory investigation. Fever should be
defined as T0 > xx.x taken by a specific method (eg history alone, versus documented by oral,
axillary…). Will a patient who is not febrile but with just a history of fever be enrolled?
The investigators need to define what is a pediatric patient by age group and ensure that this age
cut-off is applied across facilities.
Laboratory methods: The authors should provide more background on sensitivity and specificity of
different laboratory assays and how patients will be classified based on laboratory results. How will
malaria be classified based on smear results? How will smear positive patients be classified if the
ELISA is positive? Please comment on ELISA by pathogen tested, IgM versus IgG?? More
information should be provided on PCR methods (primers that will be used, methods (real time
versus gel-based), sensitivity, specificity …)
Sample size: Sample size calculations should be based on objectives of the study. The stated
objective is: “To assess the contribution of dengue, malaria, leptospira, chikungunya and bartonella
to febrile illness from the hospital patients of Bangladesh.”
The word “relative” is missing from this objective. Is this intentional? If the objective of he study is
to assess relative contribution, it will be difficult to do with the limited number of patients that will
be included in the study, particularly since the authors are only evaluating 2 pediatric patients per
month per facility and mixing outpatients and inpatients in the study. A more useful design would
be to evaluate all patients meeting the case definition on the selected days.
I assume the authors are restricting the analysis to 10 patients per month based on costs.
However, the current design will greatly limit ability to analyze the epidemiology of these diseases
with relation to disease burden, seasonality, age distribution of disease, etc. and it will be
challenging to make definitive conclusions regarding the relative public health importance of the
pathogens.
There is a reason more children are seen with febrile illness than adults and it is usually because
adults have acquired these diseases as a child and are no longer susceptible.
Disease burden: The investigators should consider broadening the data collection methods to
provide the ability to comment on disease burden. This could be done if the authors collected data
on the total number of patients admitted, total meeting the case definition and total evaluated.
Logistics: It is challenging to get multiple samples from the same patient. The investigators need to
outline how they will do this. Are the authors going to provide incentives for patients to come back
and get convalescent serum samples.
64
Appendix J: Responses to external reviewer’s comments [2]
This is a well-written protocol which outlines a plan to conduct surveillance for patients with acute
febrile illness in 6 hospitals in Bangladesh. Briefly, patients meeting the case definition will undergo
a standardized clinical and laboratory evaluation.
Specific comments:
General comments: The authors propose to study 5 specific pathogens as a cause of AFI. It is
unclear why other bacterial causes of disease (typhoid fever, brucellosis…) are not addressed.
We have added the following paragraph in the background section:
“Other potential causes of febrile illness in Bangladesh include typhoid fever and
brucellosis. Typhoid fever is a common cause of fever in Bangladesh [72-78]. Brucella is
another potential pathogen that can be transmitted either through contact with infected
animals or from incompletely pastereurized dairy products. Because the confirmed diagnosis
of these two pathogens requires a substantial volume of blood in a separate blood culture, we
have chosen not to include them in the present study. Indeed, we are not attempting to
conduct a comprehensive study of all causes of febrile illness in Bangladesh. Instead, we are
focusing on a handful of pathogens that there is very little data on to assess if they are
important or occasional causes of fever. The list of pathogens that we are assessing is
limited by cost and logistics.”
Are these diseases not common in Bangladesh?
65
Typhoid is a common disease and we have a good number of studies on this disease.
In this study, we are focusing on a set of diseases that we have less information on. We have
also considered Brucella initially but we chose not to include it for reasons of cost and the
volume of blood required.
Will the results be reliable without ruling out these diseases?
The study is not designed to find all causes of febrile illness, but rather some important
causes of fever of which we have very little data.
What background data is available to compare these diseases in terms of public health importance
with the proposed list to be studied?
We have very little data on the pathogens we have identified to study further
compared to typhoid. This is one of the reasons we want to take a first step and investigate
them further.
Will patients with a clinical diagnosis of these infections be included or excluded?
If any case meets our inclusion and exclusion criteria then we will enroll the patient
regardless of the clinical diagnosis performed by the clinicians in the surveillance hospitals.
Different clinicians will make clinical diagnosis based on different standard of inclusion and
exclusions which will reduce the comparability of the data from different sites. Therefore,
enrollment of patient needs to have explicit decision rules on which to include and exclude.
However, the inclusion and exclusion criteria are optimized to identify the particular
pathogens of interest and exclude the other pathogens.
A review of the epidemiology of these diseases in the background section would be helpful to
explain why they are not included.
We have now explained the reason for their exclusion in the background.
It would be helpful to provide more background on the hospitals in the study including whether
they are referral hospitals, infectious disease hospitals, district hospitals, etc.
The comments are well taken. These hospitals are tertiary medical college hospitals,
mostly represent as referral hospitals for each division. A table showing their laboratory
facilities is provided in the facilities section in the 25th page.
It would be particularly useful if information is available on background rates of disease are
available (eg number of patients seen each year with dengue or malaria by age group) to compare
proposed sample size with number of patients being seen in these facilities.
We have scarcity of such data, so we are better motivated for this study to gain this
information. In most cases disease register in these facilities is not strictly maintained with
confirmed laboratory diagnosis.
If we assume that 200,000 people seek care for febrile illness at these hospitals per
year, and the real prevalence of infection with one of these organisms is 0.75%, then a
sample of 675 will provide a 95% probability of identifying point prevalence between 0.1%
and 1.4%. Or stated another way with a sample of 675 study subjects we have a 95% chance
of not missing a pathogen that is causes 0.75% of the febrile illnesses in this population. We
will collect 10 specimens from each hospital in each month. This will provide 720 specimens
in a year. The additional specimens provide some additional power.
66
Case definition: On page 14, the section on the case definition needs to be rewritten: The case
definition is mixed with numerous details related to the laboratory investigation. Fever should be
defined as T0 > xx.x taken by a specific method (eg history alone, versus documented by oral,
axillary…).
used.
I am grateful to get this advice. Case definition has been rewritten. Only history will be
Will a patient who is not febrile but with just a history of fever be enrolled?
Yes, we will enroll febrile patients based on the history. We have added the following
paragraph in the subsection of number 4 of inclusion criteria.
“We will enroll febrile patients based on the history. But if we have the opportunity to
select the patients of documented fever we always appreciate to get that case.”
The investigators need to define what a pediatric patient by age group is and ensure that this age
cut-off is applied across facilities.
Actually government medical colleges maintain up to 14 years as pediatric age groups
and from emergency or ticket counter of the hospitals they are separated, under 14 ages will
be sent to pediatrics inpatient wards or outpatient department.
Laboratory methods: The authors should provide more background on sensitivity and specificity of
different laboratory assays and how patients will be classified based on laboratory results.
We have added the following paragraphs in the research design and methods section
under the subsection of objective in the 18th page:
“The sensitivity and specificity of the Dengue IgM Capture ELISA are reported to be
94.7 % (with CI 85.4 - 98.9 %) and 100% (with CI 95.7 - 100%), respectively. The dengue
IgM Capture ELISA determines the level of IgM antibodies to dengue in a patient’s serum. A
positive result (> 11 Panbio units) is indicative of either an active primary or secondary
dengue infection. < 9 Panbio units will be counted as negative results.
Multiplex Reverse Transcriptase-PCR (MRT-PCR) is highly sensitive & specific compare
to ELISA and confirms the diagnosis with serotype identification.
Malaria will be diagnosed by rapid diagnostic test (RDT) based on the detection of
P.falciparum-specific antigen and plasmodium vivax-specific antigen. The trade name of this
RDT is “FalciVax” and it is being produced by Zephyr Biomedicals, India and “BinaxNow”
produced by Inverness medical, USA. Falcivax is rapid self-performing, qualitative, two-site
sandwith immunoassay utilizing whole blood for the detection of P.falciparum specific
histidine rich protein-2 (pf, HRP-2) and P.vivax specific pLDH. The test can be used for
specific detection and differentiation of P.falciparum and P.vivax malaria. The standardization
of this test has already been done by Zephyr Biomedicals. Sensitivity of the RDT is similar to
that commonly achieved by good field microscopy. Sensitivity and specificity of the RDT used
for the detection of P.falciparum and P.vivax will be more than 95 % and now been
recommended for use in the malaria control program by the WHO (Singh N et al. 2002;
Moody A. 2002; WHO, 2004)
67
Both thick and thin film will be done for diagnosis of malaria by microscopy. The blood
films will be stained with Giemsa stain in phosphate buffer saline and examined under the
microscope at a magnification of x 1000 for the presence of malaria parasites. Blood films
were defined as negative if no parasite were observed in 300 oil immersion fields
(magnification, x 1000) on thin film by an experienced microscopist (Warhurst DC and
William JE, 1996).
Microscopy diagnosis is fairly sensitive and highly specific. False negative results may
be seen in conditions of very low parasitemia, maturation of sequestered parasites in the
broods, partially treated with antimalarials or on chemoprophylaxis, and may be due to
technical factors; (poorly prepared slides, poorly stained slides, poor quality microscope,
examination of only thin films, inexperienced technician etc). False positive results are seen
due to stained particles, which may be confused for malarial parasite by an inexperienced
microscopist. In a study in Bangladesh it has been shown that both types of malaria the
sensitivity was 94% (95% CI 71.3–99.9), and the specificity was 93% (95% CI 90.0–98.5).
(Failure of national guidelines to diagnose uncomplicated malaria in Bangladesh. am. j. trop.
med. hyg., 67(4), 2002, pp. 396–399)
In context of test sensitivity and specificity, Leptospira culture is the gold standard for
detection of the organism but a negative culture does not exclude an infection with the agent.
Negative Predictive value does not exclude leptosiprosis for the diagnosis. Positive Predictive
value confirms the diagnosis of leptosiprosis but serology should be sought for identification
of the infecting serovar or serogroup prior to typing of the isolate.
(http://www.health.gov.au/internet/main/publishing.nsf/Content/cda-phlncdleptospirosis.htm)
Indirect immunofluorescence antibody (IFA) assay is the “gold standard” technique and
is used as a reference technique in most laboratories. There is no other serologic test is
better than the sensitivity and specificity of these assays. Serum antibodies bind to fixed
antigens on a slide and are detected by a fluorescein-labeled conjugate. The sensitivity of the
IFA assay is substantially dependent on the period of sample collection. As the illness
progresses to 7–10 days, the sensitivity of IFA serology increases. The IFA is expected to be
94%–100% sensitive after 14 days and sensitivity is increased if paired samples are tested
(Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted
Fever, Ehrlichioses, and Anaplasmosis — United StatesMarch 31, 2006 / Vol. 55 / No. RR-4
Prepared by Alice S. Chapman, DVM1 in collaboration with the Tickborne Rickettsial Diseases
Working Group and Brouqui P, Bacellar F, Baranton G, et al. Guidelines for the diagnosis of
tick-borne bacterial diseases in Europe. Clin Microbiol Infect Dis 2004;10:1108–32). For the
detection of R. rickettsii responsible for Rocky Mountain spotted fever (RMSF), sensitivity, as
tested with 60 paired serum specimens, including specimens with stationary titers (5%) and
fourfold rising titers (95%), was 100% ( Kleeman, K. T., J. L. Hicks, R. L. Anacker, R. L.
Philip, E. A. Casper, K. E. Hechemy, C. M. Wilfert, and J. N. MacCormack. 1996. Early
detection of antibody to Rickettsia rickettsii: a comparison of four serological
methods:indirect hemagglutination, indirect fluorescent antibody, latex agglutination, and
complement fixation, p. 171–178. In J. Kazar (ed.), Rickettsiae and rickettsial diseases. Veda,
Publishing House of the Slovak Academy of Sciences, Bratislava, Slovakia). In a study with
patients with no rickettsial diseases, a titer of ≥ 1:64 had a specificity of 100% and a
sensitivity of 84.6%, and a titer of ≥ 1:32 had a specificity of 99.8% and a sensitivity of
97.4% (Newhouse, V. F., C. C. Shepard, M. D. Redus, T. Tzianabos, and J. E. McDade. 1979.
A comparison of the complement fixation, indirect fluorescent antibody, and
microagglutination tests for the serological diagnosis of rickettsial diseases. Am. J. Trop. Med.
Hyg. 28:387–395). For scrub typhus, the sensitivity of IFA is low if high specificity is required
68
that can be explained as follows: for a titer of ≥ 1:100, sensitivity is 84% and specificity is
78%, for a titer of ≥ 1:200, sensitivity is 70% and specificity is 92%, and for a titer of ≥
1:400, sensitivity is 48% and specificity is 96% (Brown, G. W., A. Shirai, C. Rogers, and M.
G. Groves. 1983. Diagnostic criteria for scrub typhus: probability values for
immunofluorescent antibody and Proteus OXK agglutinin titers. Am. J. Trop. Med. Hyg.
32:1101–1107). A fourfold increase to a titer of ≥ 1:200 is 98% specific and 54% sensitive
(Laboratory Diagnosis of Rickettsioses: Current Approaches to Diagnosis of Old and New
Rickettsial Diseases Bernard La Scola and Didier Raoult Journal Of Clinical Microbiology, 00951137/97/$04.0010 Nov. 1997, p. 2715–2727 vol. 35, no. 11). Usually the indirect
microimmunofluorescence assay is not positive when the patient is acutely unwell, but it has
been the most sensitive and specific of the traditional serological tests (Graves, S.R., et al.,
Flinders Island spotted fever: a newly recognised endemic focus of tick typhus in Bass Strait,
Part 2: Serological investigations.Med J Aust 1991. 154: p. 99–104). Culture techniques are
very sensitive but can take up to 2 months to get a positive result that may limit their clinical
usefulness (B, U.N., et al., Not only ‘Flinders Island’ spotted fever. Pathology, 2005. 37: p.
242–245). Real-time PCR is both highly specific and extremely sensitive for the diagnosis of
rickettsioses and may offer the advantages of speed, reproducibility, quantitative capability,
and low risk for contamination, compared with conventional PCR (Fenollar F, Raoult D.
Molecular genetic methods for the diagnosis of fastidious microorganisms. APMIS 2004;
112:785–807 and Stenos, J., s.r. Graves, and n.b. Unsworth, A highly sensitive and specific
real-time pcr assay for the detection of spotted fever and typhus group rickettsiae. Am. J.
Trop. Med. Hyg., 2005. 73(6): p. 1083–1085).
Bartonella culture is considered confirmatory diagnosis for bartonella species. If
Bartonella-like colonies are seen on an agar plate, material from one colony will be streaked
onto a new agar plate. Presumptive Bartonella cultures will be used for the extraction of
Bartonella DNA and PCR.”
How will malaria be classified based on smear results?
We have added the following paragraph in the research design and methods section
under the subsection of objective in the 18th page:
In ICDDR,B laboratory we will perform microscopic diagnosis for malaria to confirm
that malaria and also able to identify the specific species. We will classify plasmodium vivax
and plasmodium falciparum. The degree of parasitaemia in each case will be reported as
scanty (1-10 parasites per 100 high-power fields), moderate (10-100 parasites per 100 highpower fields), or heavy (>100 parasites per 100 high-power fields). (Concurrent malaria and
enteric fever in Pakistan, Singapore Med J 2005; 46(11): 635)
How will smear positive patients be classified if the ELISA is positive?
ways:
These points have addressed great issue. For this issue we can approach in three
Firstly: We will be able to correlate the lab-results with clinical and other laboratory
parameters as we have a date collection form mentioning the brief history with clinical exam
and other laboratory & chemical parameters
Secondly: We can get the impression of co-infection as both the malaria and dengue
are prevalent in Bangladesh.
Finally: We can state that smear positive test indicate confirmed diagnosis for malaria
but ELISA positive does not confirm a dengue case. In that case if we want to specify the
diagnosis, we have to exclude Dengue by performing PCR test.
69
Please comment on ELISA by pathogen tested, IgM versus IgG??
Your comments are well taken. We have added the following paragraph in research
design and method section in 21st page.
“For dengue virus detection we will do MAC ELISA for IgM antibody in the ICDDR, B
laboratory. In cases where a single specimen is available, detection of anti dengue IgM
permits the diagnosis of recent dengue virus infection even in primary cases where the level
of heamagglutination-inhibition antibody will not be diagnostic. (Source: Dengue hemorrhagic
Fever diagnosis, treatment, prevention and control. WHO Geneva 1997).”
More information should be provided on PCR methods (primers that will be used, methods (real
time versus gel-based), sensitivity, specificity …)
Your comments are well taken. We have added the following paragraph in the research
design and method section in the 18th page.
“We will have the option to do PCR attaching labels to the samples. Multiplex Reverse
Transcriptase-PCR (MRT-PCR) for dengue is highly sensitive & specific compare to ELISA and
confirms the diagnosis with serotype identification. Gel based PCR will be used. The reaction
mixture will contain 50 mM KCl, 10 mM Tris (pH 8.5), 0.1% TritonX-100, 0.01% gelatin.
Dengue RNA can be detected by using serotype specific primers, reverse transcriptase and
thermostable polymerase (Typing of Dengue Viruses in Clinical Specimens and Mosquitoes by
Single-Tube Multiplex Reverse Transcriptase PCR, Journal of Clinical Microbiology, Sept. 1998,
p. 2634–2639 Vol. 36, No. 9). Multiplex Reverse Transcriptase-PCR (MRT-PCR) is highly
sensitive, specific along with cost-effective and less time consuming. Without proper
precaution, contamination can lead to false-positive results (Source: Dengue hemorrhagic
Fever diagnosis, treatment, prevention and control. WHO Geneva 1997).”
Sample size: Sample size calculations should be based on objectives of the study. The stated
objective is: “To assess the contribution of dengue, malaria, leptospira, chikungunya and bartonella
to febrile illness from the hospital patients of Bangladesh.”
We have revised the sample size estimation and framed it within the study question.
The word “relative” is missing from this objective. Is this intentional? If the objective of the study is
to assess relative contribution, it will be difficult to do with the limited number of patients that will
be included in the study, particularly since the authors are only evaluating 2 pediatric patients per
month per facility and mixing outpatients and inpatients in the study. A more useful design would
be to evaluate all patients meeting the case definition on the selected days.
Yes, it is intentional. In this study relative prevalence is not a primary objective of the
study. The primary objective is to detect the presence of the pathogens of our interest in the
study facilities. Besides we have increased our pediatric samples from 2 to 5 cases per facility
per month.
70
I assume the authors are restricting the analysis to 10 patients per month based on costs.
However, the current design will greatly limit ability to analyze the epidemiology of these diseases
with relation to disease burden, seasonality, age distribution of disease, etc. and it will be
challenging to make definitive conclusions regarding the relative public health importance of the
pathogens.
It is not the objective of the study to make definitive conclusions regarding the relative
public health importance of the pathogens, but rather to take an initial step to explore their
presence and distribution. It is efficient to look for all these pathogens together rather than
single pathogen in multiple single pathogen studies.
There is a reason more children are seen with febrile illness than adults and it is usually because
adults have acquired these diseases as a child and are no longer susceptible.
We agree with your comment. Out of the 10 specimens, we will collect 5 specimens
from the adult medicine clinics and 5 from the pediatric clinics through purposive sampling
technique. The objective of the study is not particularly focused on any age group; rather
circulating etiologies will be identified for any age group. Therefore no age group will be
excluded for collecting samples from the inpatients or outpatient departments. But the
commonality of fever in young children risks over-sampling. So using purposive sampling we
will collect 5 from adult and same from pediatrics. The medical college hospitals included in
the study usually define 14 years as the higher limit of pediatric age groups.
Disease burden: The investigators should consider broadening the data collection methods to
provide the ability to comment on disease burden. This could be done if the authors collected data
on the total number of patients admitted, total meeting the case definition and total evaluated.
These points are well taken. This will be done in the following approach. We have
added the following sections in the protocol in page no 15 and 16.
When out team will visit each hospital they will also collect these data from the study
physician. Study physicians will maintain a register book to organize these data for the whole
month and provide these data to the study team. Most of the patients may have a cell phone
number. If it is not possible, then they will name someone in their village or neighborhood,
with whom we can contact. If that is also impossible then they may provide the contact
number of those who rents cell phone services in their locality. Thus, if we find number of
patients with a disease of interest, we will have the option to reconnect with them
immediately and collect Global Positioning System (GPS) reading and potentially other
information from them. We will do a follow-up of all patients after two months. We will have a
way to contact them through their cell phone numbers. Therefore, it would be informative to
collect additional information and put on a short questionnaire together.
Logistics: It is challenging to get multiple samples from the same patient. The investigators need to
outline how they will do this. Are the authors going to provide incentives for patients to come back
and get convalescent serum samples.
The following section is added in the 19th page under the section of research design
and method of the protocol.
“A pilot study was done in Rajshahi Medical College Hospital and we also consulted with
our focal collaborators for Hospital Based Influenza Surveillance in other hospitals about this
coming protocol. They have informed us that in most cases febrile illness of 10 days will
present in the outpatient department. So in most cases we will get samples from outpatients
71
who usually take consultation for once and will be difficult to track them within 7 days unless
their illnesses become complicated. Therefore, it will not be feasible to collect convalescent
sample.”
Appendix K : External reviewer’s comments [3]
Reviewer:
Henry (Kip) Baggett, MD, MPH
Chief, Epidemiology Section
International Emerging Infections Program
Thailand MOPH-US CDC Collaboration (TUC)
Department of Disease Control, 3rd Floor, Building 7
Ministry of Public Health, Tivanon Road, Nonthaburi 11000 Thailand
Tel: (66 2) 591-1294 Ext. 314, Mobile: (66 89) 810-8992
Fax: (66 2) 580-0911
External Protocol Review: Hospital based febrile illness study in Bangladesh
Date: 25 February 2008
Overview
The protocol describes a 1-year surveillance project to determine the relative frequency of selected
pathogens as causes of undifferentiated febrile illness in 6 hospitals in 6 different districts in
Bangladesh. The diseases of interest are malaria, dengue fever, leptospirosis, chikungunya fever,
and bartonellosis. The project will enroll 10 patients per month primarily from the inpatient wards of
each hospital. Outpatients will be enrolled if there are not enough inpatients to meet the targeted
enrollment each month. Patients will be excluded if they have a respiratory or gastrointestinal
72
illness or have a known etiology for their illness other than one of the five diseases of interest.
Written consent will be obtained from all participants.
I have no major concerns about this study protocol and no major suggestions. The project is built
on the infrastructure of existing sentinel surveillance for influenza, which presumably is working
well.
Major Comments
 No major comments.
Minor Comments
 Seasonality. One of the stated objectives of the study is to describe potential seasonal
patterns of the diseases of interest. It will be difficult to make a strong case for seasonality
with only 1 year of data. Having only 60 specimens per month might also limit conclusions
about seasonality. It would be nice if the project could be continued past 1 year.
 Pathogens. Is there any interest locally in testing for rickettsial diseases, especially scrub
typhus? I am not familiar with the frequency of ricksettsial diseases in Bangladesh or the
Indian subcontinent. You will already be collecting the specimens and it might be a good
opportunity to assess. A quick Pubmed search for “scrub typhus and Bangladesh” revealed
only one article: Miah MT, Rahman S, Sarker CN, Khan GK, Barman TK. Study on 40 cases of
rickettsia. Mymensingh Med J. 2007 Jan;16(1):85-8. PMID: 17344787
 Bartonella. For the information of the investigators, there was a recent publication
describing a new species of Bartonella from Thailand. Kosoy et al. Bartonella tamiae sp.
nov., a newly recognized pathogen isolated from three human patients from Thailand.J Clin
Microbiol. 2008 Feb;46(2):772-5.
 Study hospitals. The selection of the 6 study hospitals makes sense given the ongoing
influenza surveillance in these hospitals. It is worth considering the balance of factors in the
populations served by these hospitals that could affect the frequency of certain diseases
(e.g., rural vs. urban, occupational exposures in the region, proximity to borders).
 Inclusion criteria. Consider including only patients with documented fever. These hospitals
seem large enough that you would be able to enroll 10 patients with documented fever each
month.
 Lab testing for chikungunya virus and dengue virus. Consider conducting both PCR and
ELISA on all specimens. It will often be difficult to determine the exact timing of the fever,
and the timing of antibody rise and detectable antigen will vary between patients. Especially
for chikungunya, you don’t want to miss even a single case.
 Leptospirosis testing. Culture takes a very long time. Have you considered microscopic
agglutination testing (MAT)? I think this is still time consuming but could give results faster
than culture, I believe.
 Timing of specimen collection each month. I suggest trying to vary the days of specimen
collection for each hospital each month to avoid any confounding from factors that affect
when patients come to hospital. For example, patients who present to the hospital on
Mondays may differ from other days of the week.
 Sampling adults and children. I worry that data from children will be of limited utility with
only 12 specimens per month (i.e., 2 specimens per month per hospital). The concern about
the frequency of fever in children is understandable. However, you will be excluding patients
with respiratory or GI illness as well as patients with a known cause of illness. The remaining
pediatric patients should be similar to the adults in clinical characteristics. These diseases
are often more common (e.g., dengue) and more severe in children (e.g., malaria), so I would
suggest increasing pediatric enrollment to at least 4 per month.
 Exclusion criteria. Not exactly sure what is meant by “symptoms of hepatitis”. I am not sure
if this would include elevated transaminases, but liver enzymes can be elevated in several of
the diseases of interest.
73



Under “Specimen collection, handling, and aliquoting”. Last paragraph. Testing for
chikungunya and dengue will presumably be done at CDC, Fort Collins and Puerto Rico, not
CDC, Atlanta.
Timeline. Keep in mind that the bartonella and leptospirosis testing will be time consuming
when you are estimating when you will have data for summary presentation.
Ethical considerations
o Assent. Do you need to get verbal assent from patients aged 7 years or older? Your
local ethics board will provide guidance on this.
o English version of the consent form is at a fairly high reading level, but this may be
irrelevant for the Bengali version
o Specimen storage. It is mentioned that specimens will be kept for the duration of the
study, but long-term storage is not addressed. Will the specimens be destroyed after
the study?
Appendix L: Responses to external reviewer’s comments [3]
External Protocol Review: Hospital based febrile illness study in Bangladesh
25 February 2008
Reviewer:
Henry (Kip) Baggett, MD, MPH
Chief, Epidemiology Section
International Emerging Infections Program
Thailand MOPH-US CDC Collaboration (TUC)
Department of Disease Control, 3rd Floor, Building 7
Ministry of Public Health, Tivanon Road
Nonthaburi 11000 Thailand
Tel: (66 2) 591-1294 Ext. 314
Mobile: (66 89) 810-8992
Fax: (66 2) 580-0911
Overview
The protocol describes a 1-year surveillance project to determine the relative frequency of selected
pathogens as causes of undifferentiated febrile illness in 6 hospitals in 6 different districts in
Bangladesh. The diseases of interest are malaria, dengue fever, leptospirosis, chikungunya fever,
and bartonellosis. The project will enroll 10 patients per month primarily from the inpatient wards of
each hospital. Outpatients will be enrolled if there are not enough inpatients to meet the targeted
enrollment each month. Patients will be excluded if they have a respiratory or gastrointestinal
illness or have a known etiology for their illness other than one of the five diseases of interest.
Written consent will be obtained from all participants.
74
I have no major concerns about this study protocol and no major suggestions. The project is built
on the infrastructure of existing sentinel surveillance for influenza, which presumably is working
well.
Major Comments
 No major comments.
Minor Comments
 Seasonality. One of the stated objectives of the study is to describe potential seasonal
patterns of the diseases of interest. It will be difficult to make a strong case for seasonality
with only 1 year of data. Having only 60 specimens per month might also limit conclusions
about seasonality. It would be nice if the project could be continued past 1 year.
The points are well taken. As we are not considering the study to continue for further 1
year so we have omitted the concept of seasonality.

Pathogens. Is there any interest locally in testing for rickettsial diseases, especially scrub
typhus? I am not familiar with the frequency of ricksettsial diseases in Bangladesh or the
Indian subcontinent. You will already be collecting the specimens and it might be a good
opportunity to assess. A quick Pubmed search for “scrub typhus and Bangladesh” revealed
only one article: Miah MT, Rahman S, Sarker CN, Khan GK, Barman TK. Study on 40 cases of
rickettsia. Mymensingh Med J. 2007 Jan;16(1):85-8. PMID: 17344787
Your points are well taken. We have added the rickettsial species in our protocol.
“Rickettsial pathogens are highly specialized gram-negative, obligate intracellular
Bacterium causing rickettsial fever, and rash usually transmitted to man by the contamination
of the bite site or skin abrasions with Rickettsia-containing f lea feces or by direct bite of
ticks.
Rickettsial diseases are widely distributed throughout the world and recent studies in
(Rangoon) Myanmar and Nepal reveals their continued presence in several parts of the Indian
subcontinent, particularly that of scrub typhus (Spotted fevers & typhus fever in Tamil
Nadu.Indian J Med Res 126, August 2007, pp 101-103 and Rickettsial Pathogens and their
Arthropod Vectors Abdu F. Azad and Charles B. Beard†, Vol. 4, No. 2, April–June 1998 179
Emerging Infectious Diseases). High infectivity, low index of suspicion and the lack of
diagnostic facility associated with significant morbidity and mortality (Rickettsial Infections
Around the World, Part 2: Rickettsialpox, the Typhus Group, and Bioterrorism, J Cutan Med
Surg 2005; 105–115) (Spotted fevers & typhus fever in Tamil Nadu.Indian J Med Res 126,
August 2007, pp 101-103) (Rickettsial Infections in South India – How to Spot the Spotted
Fever Indian Pediatrics 2001; 38: 1393-1396). Suburban expansion and environmental
modifications through destruction and reduction of natural habitats and commensal rats such
as R. rattus and R. norvegicus,and their fleas, in particular the oriental rat flea X. cheopis.are
present in Bangladesh (Flea-borne Rickettsioses:Ecologic Considerations, Vol. 3, No. 3, July–
September 1997 319 Emerging Infectious Diseases(Epidemiology Of Murine Typhus,Annu.
Rev. Entomol. 1990.35:553-570). Recently 40 ricketttsial cases have been detected in a
study in Bangladesh in Mymensingh Medical College Hospital. But this was a small study with
small population size. The study area was limited to only one district. Only suspected cases
were selected. No routine surveillance system or standard case definition was followed for
clinical diagnosis. Sampling design was not representative. Laboratory diagnostic test was not
reliable and confirmatory. (Mymensingh Med J. 2007 Jan;16(1):85-8., Study on 40 cases of
rickettsia.Miah MT, Rahman S, Sarker CN, Khan GK, Barman TK.)”
75

Bartonella. For the information of the investigators, there was a recent publication
describing a new species of Bartonella from Thailand. Kosoy et al. Bartonella tamiae sp.
nov., a newly recognized pathogen isolated from three human patients from Thailand.J Clin
Microbiol. 2008 Feb;46(2):772-5.
As Michael Kosoy is one of our co-investigator so we had a great opportunity to get his
review on this protocol earlier and share the information of that study.

Study hospitals. The selection of the 6 study hospitals makes sense given the ongoing
influenza surveillance in these hospitals. It is worth considering the balance of factors in the
populations served by these hospitals that could affect the frequency of certain diseases
(e.g., rural vs. urban, occupational exposures in the region, proximity to borders).
We are very grateful to get the comments of inclusion these factors for the hospital
selection. We have added the following section in the 13th page under research design and
method section and settings sub-section:
“Kishorganj is located in rural settings and Khulna, Barisal & Chittagong are located
near the Bay of Bengal. Therefore occupational exposures responsible for leptospirosis are
well observed in these regions. Khulna, Rajshahi and Sylhet are quite closer to the Indian
border to be a factor for chikungunya affected region. Sylhet, Barisal & Chittagong are
metropolitan cities so dengue cases may be higher there due to mosquito breeding places and
congested areas. On the other hand, hilly areas like Chittagong and Sylhet can present
malaria cases more than any other region.”

Inclusion criteria. Consider including only patients with documented fever. These hospitals
seem large enough that you would be able to enroll 10 patients with documented fever each
month.
According to the pilot study in Rajshahi Medical College Hospital and consulted with our
focal collaborators for Hospital Based Influenza Surveillance in other hospitals we can
conclude that we will get most patients from outpatient department. In most cases they will
just provide the history of fever without any documentation of fever.

Lab testing for chikungunya virus and dengue virus. Consider conducting both PCR and
ELISA on all specimens. It will often be difficult to determine the exact timing of the fever,
and the timing of antibody rise and detectable antigen will vary between patients. Especially
for chikungunya, you don’t want to miss even a single case.
For dengue we will perform IgM MAC ELISA for all samples in our ICDDR,B laboratory
but for samples with fever of first 5 days we will mark the specimen for PCR tests. Again for
chikungunya we will send only those samples to the US CDC with the history of joint pain. As
we have budget constraint so that we can not perform both the tests for dengue and
chikungunya.

Leptospirosis testing. Culture takes a very long time. Have you considered microscopic
agglutination testing (MAT)? I think this is still time consuming but could give results faster
than culture, I believe.
MAT testing is difficult to interpret especially in the setting of unknown serovars. Our
collaborators in US CDC are interested in doing culture. Besides we have mentioned in the
76
informed consent form that we will not get the results of these tests in time to influence the
treatment procedure of the patients so that we can perform time consuming tests.

Timing of specimen collection each month. I suggest trying to vary the days of specimen
collection for each hospital each month to avoid any confounding from factors that affect
when patients come to hospital. For example, patients who present to the hospital on
Mondays may differ from other days of the week.
This will be a great approach. We will always try to maintain the system during field
visits. We have added the approach in the 15th page under filed operation sub-section.

Sampling adults and children. I worry that data from children will be of limited utility with
only 12 specimens per month (i.e., 2 specimens per month per hospital). The concern about
the frequency of fever in children is understandable. However, you will be excluding patients
with respiratory or GI illness as well as patients with a known cause of illness. The remaining
pediatric patients should be similar to the adults in clinical characteristics. These diseases
are often more common (e.g., dengue) and more severe in children (e.g., malaria), so I would
suggest increasing pediatric enrollment to at least 4 per month.
The comments are well accepted. We are going to increase the number of pediatric
specimens up to 5 samples along with 5 adult sample which in total 10 samples per month in
each facility. Then the sample size will be proportionate to the number of children in
Bangladesh and hopefully prevent them from overwhelming the study.

Exclusion criteria. Not exactly sure what is meant by “symptoms of hepatitis”. I am not sure
if this would include elevated transaminases, but liver enzymes can be elevated in several of
the diseases of interest.
The critiques are well taken. “Symptoms of hepatitis” has been omitted. The sentence
has been re-written under the section of exclusion criteria as below:
“The Surveillance physicians will be instructed to exclude the following symptoms:
 Fever for more than 10 days.
 Cough with productive sputum.
 Urgency, frequency, hesitancy during micrurition.
 Cellulites/abscess/boil/local skin infection in the body. ”

Under “Specimen collection, handling, and aliquoting”. Last paragraph. Testing for
chikungunya and dengue will presumably be done at CDC, Fort Collins and Puerto Rico, not
CDC, Atlanta.
According to the Dr. Olsen’s advice the phrase has been changed to US CDC to clarify
the confusion.

Timeline. Keep in mind that the bartonella and leptospirosis testing will be time consuming
when you are estimating when you will have data for summary presentation.
We are highly glad to get the points and we have corrected it.
Ethical considerations
o Assent. Do you need to get verbal assent from patients aged 7 years or older? Your
local ethics board will provide guidance on this.
77
years.
Your comments are well taken. We have prepared a separate assent form for 7-17
o
English version of the consent form is at a fairly high reading level, but this may be
irrelevant for the Bengali version
Your comments are well taken. By assessing the Flesch-Kincaid Grade Level, we have
simplified the language and now the grade is 7.0.
o
Specimen storage. It is mentioned that specimens will be kept for the duration of the
study, but long-term storage is not addressed. Will the specimens be destroyed after
the study?
We have added the following section in the Storage and shipment of specimen to CDC,
Atlanta in 22nd page:
“We will separate the serum in cryovials for the specific laboratory tests mentioned in
our protocol. We will also preserve the rest of the serum for the long-term storage. A new
pathogen can be discovered with a new diagnostic at any time. Therefore the left over
aliquots of specimens will give us an opportunity to test the sample quickly and efficiently.”
Checklist
CHECK-LIST FOR SUBMISSION OF RESEARCH PROTOCOL
FOR CONSIDERATION OF RESEARCH REVIEW COMMITTEE (RRC)
[Please check (X) appropriate box]
1.
Has the proposal been reviewed, discussed and cleared at the Division level?
Yes
No
If No, please clarify the reasons:
2.
Has the proposal been peer-reviewed externally?
Yes
No
If the answer is ‘No’, please explain the reasons:
If yes, have the external reviews’ comments and their responses been attached
Yes
3.
No
Has the budget been cleared by Finance Department?
Yes
No
78
If the answer is ‘No’, reasons there of be indicated:
4.
Does the study involve any procedure employing hazardous materials, or equipments?
Yes
No
If ‘Yes’, fill the necessary form.
_________________________________
Signature of the Principal Investigator
__________________
Date
79