Optimization of Mass Drug Administration With Existing Drug Regimens for Lymphatic Filariasis and Onchocerciasis
Conditions: Lymphatic Filariasis, Soil Transmitted Helminth Infections
Sponsor: Washington University School of Medicine

i 
TITLE 
 
Optimization of Mass Drug Administration with existing drug regimens for Lymphatic 
Filariasis and Onchocerciasis 
(‘DOLF’ project, Death to onchocerciasis and lymphatic filariasis) 
 
Sponsored by: 
Bill and Melinda Gates Foundation: GH 5342 
Principal Investigators: 
Gary J. Weil, M.D., Peter U. Fischer, Ph.D., and Taniawati Supali, Ph.D.  
 
Draft or Version Number: 5 
 
Day Month Year: 31 December 2013 
 
 
 
Statement of Compliance 
This study will be carried out in accordance with Good Clinical Practice (GCP) as required by the: 
• 
U.S. Code of Federal Regulations applicable to clinical studies (45 CFR 46) 
http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.htm#46 
ICH GCP E6 Completion of Human Subjects Protection Training 
http://grants.nih.gov/grants/guide/notice-files/NOT-OD-0 1-061 .html 
• 
Bill and Melinda Gates Foundation “grant agreement” terms and conditions  

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SIGNATURES 
The signature below documents the approval of this protocol and the attachments, and provide 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality and according to local legal and 
regulatory requirements and to the principles outlined in applicable U.S. federal regulations and ICH 
guidelines. 
Signed: 
Date (dd/mm/yyyy): 
 
__________________________ 
________________________ 
 
 
Peter U. Fischer, Ph.D. - Washington University School of Medicine  
Title: Principal Investigator, Community MDA Studies, DOLF Project, Objective 1  
 
 
Taniawati Supali, Ph.D. University of Indonesia 
Title: Country Principal Investigator, DOLF Project, Objective 1 
 
 
 
Gary J. Weil, M.D. - Washington University School of Medicine  
Title: Principal Investigator, DOLF Project  

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Table of Contents 
page 
Statement of Compliance .......................................................................................................................... i 
Signatures .................................................................................................................................................... ii 
Table of Contents ...................................................................................................................................... iii 
List of Abbreviations ................................................................................................................................. v 
Protocol Summary .................................................................................................................................... vi 
1 
 Key Roles............................................................................................................................................. 1 
2 
Background Information and Scientific Rationale ..................................................................... 2 
2.1 Background Information .................................................................................................................. 2 
2.2    Rationale ............................................................................................................................................. 2 
2.3    Potential Risks and Benefits / Potential Risks ......................................................................... 3 
2.3.1 Potential Risks .................................................................................................................................. 3 
2.3.2 Known Potential Benefits.............................................................................................................. 4 
3 
Objectives ............................................................................................................................................. 5 
4 
Study Design ....................................................................................................................................... 5 
5 
Study Populations .............................................................................................................................. 7 
6 
Study Procedures / Laboratory Evaluations ............................................................................... 7 
6.1 Laboratory Evaluations .................................................................................................................... 7 
6.2 Instructions for Specimen Preparation, Handling, and Storage ............................................ 8 
6.3 Specimen Shipment ............................................................................................................................ 9 
7 
Statistical Considerations .............................................................................................................. 10 
7.1 Study Outcome Measures ............................................................................................................. 10 

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7.2 Sample Size Considerations ........................................................................................................... 10 
7.3 Participant Enrollment and Follow-Up........................................................................................ 10 
7.4 Statistical Analysis Plan ................................................................................................................. 10 
8 
Subject Confidentiality .................................................................................................................... 13 
8.1 Future Use of Stored Specimens ................................................................................................. 14 
9 
Informed Consent Process ............................................................................................................ 14 
9.1 Informed Consent/Assent Process (in Case of a Minor or Others Unable to Consent for 
Themselves. ............................................................................................................................................... 14 
10   Literature References ...................................................................................................................... 15 
Supplements / Appendices .................................................................................................................... 16 
   Standard Operating Procedure (SOP) Summary of Laboratory Work ..................................... 16 
   Standard Operating Procedures Summary (SOP) for Data Collection and Field Work ...... 17 
   Informed Consent Script to Be Read in Population-Based Field Surveys ............................. 18 
 
 
 
 

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List of Abbreviations 
Alb 
Albendazole  
APOC 
African Programme for Onchocerciasis Control 
CFR 
Code of Federal Regulations  
CRF 
Case Report Form 
DEC 
Diethylcarbamazine  
DMID 
Division of Microbiology and Infectious Diseases, NIAID, NIH  
DSMB 
Data Safety Monitoring Board 
ELF 
Elimination of Lymphatic Filariasis  
FWA 
Federal-Wide Assurance 
GCP 
Good Clinical Practice  
GPELF Global Programme to Eliminate Lymphatic Filariasis 
IATA 
International Air Transport Association 
ICH 
International Conference on Harmonisation 
ICIDR 
International Collaboration for Infectious Disease Research  
ICT 
Immunochromatography Test  
IntTrans Interruption of transmission 
IRB 
Institutional Review Board  
IVER 
Ivermectin 
LF 
Lymphatic Filariasis 
MDA 
Mass Drug Administration  
MF 
Microfilaria(e) 
OCP 
Onchocerciasis Control Programme 
OHRP 
Office for Human Research Protection 
ONCHO Onchocerciasis 
OSHA 
Occupational Safety and Health Administration 
PELF 
Program to Eliminate Lymphatic Filariasis 
PI 
Principal Investigator 
SAE 
Severe adverse events 
SOP 
Standard Operating Procedure 
STH 
Soil transmitted helminths 
WHO 
World Health Organization 
 

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1.  We will compare the relative impact and cost effectiveness of annual vs. twice 
yearly mass drug administration (MDA) for elimination of lymphatic filariasis (LF). 
2. We will also study the impact of annual vs. semiannual MDA on soil transmitted 
helminth (STH) infection in these populations. 
Remarks on the project structure: The Project “Optimization of Mass Drug Administration 
with existing drug regimens for Lymphatic Filariasis and Onchocerciasis” has 3 major 
Objectives (Objective 1-Community MDA Trials, Objective 2-Randomized Clinical Trials 
and Objective 3-Pilot Flubendazole Studies). Objective 1 will be conducted in 4 countries 
in Africa and 2 countries in the Asia. Although the overall objectives are similar, there are 
country-specific modifications. This protocol was developed for Indonesia.
Protocol Summary 
Title: Optimization of Mass Drug Administration with existing drug regimens for 
Lymphatic Filariasis and Onchocerciasis  
Population: 
Number of Sites: 
Study Duration: 
Subject Duration: 
Objectives: 
Approximately 6,400 people will participate per year. The study 
population will include females and males over 5 years of age who 
live in filariasis endemic areas. Subject selection will not be based 
on health status.  
Two sites in Indonesia (in separate B. timori and W. bancrofti 
endemic areas) 
4 years 
Participants will be studied only once in cross-sectional 
surveys. Some subjects may be included in more than one 
annual population survey, but this is not a longitudinal study. 
To evaluate different mass drug administration (MDA) 
regimens for lymphatic filariasis and its impact on soil 
transmitted helminthes.  MDA is administered by others (e.g., 
Ministry of Health) and may enhance efforts to control and 
eliminate these important neglected tropical diseases. 

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1  KEY ROLES 
 
Principal Investigator: Peter U. Fischer, Ph.D. Community MDA Studies 
(DOLF Objective 1)  
 
DOLF project Principal Investigator: Gary J. Weil, M.D.  
 
Project Statistician: Professor Philip Miller 
 
Indonesian collaborating scientist: Taniawati Supali, Ph.D. (Country 
Principal Investigator, DOLF Objective 1)  
 
Institutions:   
Infectious Diseases Division, Washington University School of Medicine, 
Campus Box 8051, 660 S. Euclid Avenue, St. Louis, Missouri, 63110, 
USA. Contact: Peter U. Fischer, Ph.D. Tel. +1 314 454 7876. Fax. 314 454 
5293. Email: pufische@dom.wustl.edu 
Department of Parasitology, Faculty of Medicine, University of Indonesia,  
Salemba 6, Jakarta 10430, Indonesia. Contact: Taniawati Supali, Ph.D. 
Tel. + 62 812 9355870, Email: taniawati@yahoo.com 
  

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2 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
 
2.1 Background Information 
Lymphatic filariasis (LF) is a deforming and disabling infectious disease that causes 
elephantiasis and genital deformity (especially hydroceles). The infection affects some 120 million 
people in 81 countries in tropical and subtropical regions with well over 1 billion people at risk of 
acquiring the disease [1]. LF is caused by Wuchereria bancrofti and Brugia spp. (B. malayi and 
B.timori), nematode parasites that are transmitted by mosquitoes. The World Health Organization 
(WHO) developed a plan for LF elimination that is based on using novel approaches to rapidly 
map endemic areas and 4 to 6 annual rounds of mass drug administration (MDA) with antifilarial 
medications [2, 3]. A recent report from WHO reported that more than 1.9 billion doses of MDA 
were distributed between 2000 and 2007 [4]. Thus, the Global Programme to Eliminate Lymphatic 
Filariasis (GPELF) is the largest infectious disease intervention program attempted to date based 
on MDA. MDA has worked better in some areas than others. A recent publication reviewed 
challenges faced by GPELF [5]. The limited macrofilaricidal activity of current MDA regimens 
(especially Ivermectin/Albendazole) that necessitate repeated annual rounds of MDA, and the 
difficulty of achieving high compliance rates for MDA over a period of years. It is clear that new 
dosing schedules for MDA have the potential to greatly improve the number of countries 
that will successfully eliminate LF by the WHO target date of 2020.  
 
 
Drugs used for LF MDA are also active against soil transmitted helminths (STH, e.g., Ascaris, 
Hookworm, and Trichuris). The estimated numbers of infected individuals (in millions) for Ascaris, 
hookworm and Trichuris range from 807-1227, 576-740, and 604-795 respectively [6]. De-
worming campaigns using anthelminthics usually target special groups of the population, such as 
schoolchildren, and have limited impact on the transmission. Treatment of the total population 
and semiannual treatments may reduce re-infection considerably and will most likely lead to 
reduced infection densities and infection prevalences. Suppression of STH is an important 
ancillary benefit of MDA programs for filarial infections [2]. 
  
2.2    Rationale 
This study is based on the assumption that currently used MDA regimens and schedules 
are not optimal for achieving elimination of LF. These regimens (either annual Alb 400 mg 
plus DEC 6 mg/kg or Alb 400 mg plus Iver 200 µg/kg for LF) were developed more than 10 years 
ago [3, 7].  
WHO (WHA 50.29) has targeted LF for global elimination by the year 2020. GPELF 
recommends 4 to 6 years of annual MDA [2]. However, the likelihood of this strategy to succeed 
depends on a number of factors [5]. While some national MDA programs have succeeded, others 

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are struggling to sustain MDA with high coverage rates for 5 to 7 years according to WHO 
guidelines, and others have not completed mapping or initiated MDA. Annual DEC/Alb may be 
sufficient to eliminate LF in endemic areas with low to moderate endemicity when high MDA 
coverage (at least 75%) can be sustained for 5 or more years. However, in a few countries in 
South East Asia the endemicity is high in some areas and annual DEC/Alb may not be optimal. In 
addition, the Iver/Alb regimen used for LF in Africa is less active against adult worms than 
DEC/Alb, and annual Iver/Alb may not be sufficient to eliminate LF in Africa. We assume that 
more effective and efficient MDA regimens might tip the balance toward LF elimination in areas 
that are failing or at risk of failing to reach elimination targets with current regimens. Therefore, 
the goal of Objective 1 is to compare intensive (twice per year, 2X) MDA with conventional annual 
MDA (1X) to determine whether improved efficacy of the 2X regimens justifies increased 
resources required for this more intensive treatment. These trials are based on two major 
assumptions: 
 
A. 
We assume that 2X MDA regimens will reduce the time needed to reach LF elimination 
targets. 2X MDA should shorten the time it takes to interrupt transmission (preventing infections 
that would otherwise have to be treated in later years); depending on the drug combination used 
and parasite species targeted, 2X treatment should also lead to faster clearance of adult worms. 
Current MDA treatment strategies are based on interrupting transmission for the reproductive life 
span of the adult worms (estimated to 5-7 years for species that cause LF).  However, these 
durations may be overly conservative, because they do not factor in the partial macrofilaricidal 
activity of repeated rounds of treatment. Accelerated MDA programs may improve compliance 
and reduce costs for social mobilization and training expenses for health personnel. These factors 
may reduce the time required to reach elimination targets. In addition, more intensive LF 
elimination programs may be more attractive for public health officials to undertake than programs 
that require 6 years or longer. 
  
B. 
Suppression of STH is an important ancillary benefit of MDA programs for LF [2]. We 
assume that 2X MDA will be more effective for this purpose than annual MDA. The impact of 
MDA on STH varies for different parasite species and drug regimens [8, 9]. For example, Ascaris 
and hookworm are more susceptible to Albendazole than Trichuris. Persistence of Ascaris eggs 
in soil limits the ability of MDA to reduce Ascaris transmission.    
 
2.3    Potential Risks and Benefits / Potential Risks 
 
2.3.1 Potential Risks 
This research includes risks associated with collection of blood by finger prick  
 

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Likely 
Less Likely 
Collection of blood causes minor pain 
Collection of blood can cause a bruise, bleeding or infection at the 
collection site. To minimize risks, sterile disposable lancets will be used for 
blood collection. Blood will be collected by experienced technicians after 
disinfecting the skin with alcohol. Subjects will be instructed to apply pressure to 
finger prick sites to minimize bleeding and hematoma formation. The amount of 
blood being collected (less than 1 ml) is not enough to cause anemia. 
 
There are no risks associated with collection of stool samples.  
 
Long range risks: None 
• 
Rationale for the necessity of such risks: Blood is required for diagnosis 
of filarial infections. Procedures in this project meet the definition of 
“minimal risk”. 
• 
Alternative data gathering procedures that have been considered or will be 
considered: None. Blood is needed for diagnosing filarial infections 
which are the subject of this project.  
• 
Why the value of the information to be gained outweighs the risks involved. 
The potential benefits to individuals, society and science are 
considerable. The risks are minimal. 
 
2.3.2 Potential Benefits 
 
• 
To subjects: 
The study will screen participants for filarial infections and refer infected subjects 
for free treatment at their local government health center.  
 
• 
To society: 
Participation will provide information on the impact of MDA on filarial and STH 
infections that affect the public health in the community. 
  
 

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3 
OBJECTIVES 
To conduct population-based field studies to determine the relative cost and efficacy of 
annual vs. semiannual MDA for elimination of lymphatic filariasis.  
 
1. 
We will test the hypothesis that twice annual MDA is superior to annual 
MDA for eliminating lymphatic filariasis. 
  
2. 
We will test the hypothesis that twice annual MDA is superior to annual 
MDA for controlling soil transmitted helminth infections. 
  
4 
STUDY DESIGN 
(Please read this section together with Section 5, “Study Population”) 
The project is comprised of repeated annual cross-sectional surveys in sentinel communities 
before and after initiation of MDA for LF. MDA (standard regimens recommended by WHO) will 
be provided by government health officials. Data collection will include demographic data, history 
of lymphedema, scrotal swelling (hydrocele), acute filarial fever or adenolymphangitis, prior 
treatment for LF. Blood samples (400 µL per person) will be collected from systematically 
sampled households. Blood will be tested for filarial antigenemia with the Binax Now Filariasis 
Card Test and Alere Filariasis Strip Test (which detects Wuchereria bancrofti antigen in blood, 
manufactured by Alere, Portland ME, USA). Blood from persons with positive filarial antigen tests 
will also be tested for microfilaremia (see Section 6 below for methods). The Brugia Rapid 
(BmR1) test will be used to detect antibodies to Brugia parasites antibodies in serum or blood 
samples. Stool examinations will also be performed for diagnosis of STH infections. Note not all 
test will be performed on blood and stool samples. 
Safety oversight: Study personnel will monitor subjects for adverse events related to blood 
collection. No medical monitor or DSMB is needed for this minimal risk, non-intervention study. 
Time to collect specimens: Most participants in the community will only be studied once 
(demographic data, blood, and stool samples).  Thus, the duration of subject participation will be 
one day. 
Parameters and criteria for assessing the impact of MDA: 
Blood tests for Wuchereria bancrofti infection, Binax Now Filariasis Card Test and Alere 
Filariasis Strip Test (with MF testing of antigen positive subjects): We are using these tests 
as a measure of LF endemicity. The rapid tests detect antigen released from living adult filarial 
worms potentially capable of resuming MF production in the future. The antigen tests are much 
more sensitive for W. bancrofti infection than tests for microfilaremia. However, antigen  tests may 

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remain positive years after treatment has cleared microfilaremia. The antigen tests will be 
performed in individual residents over 5 years of age. This will be done by sampling 
approximately 100 randomly selected households per sentinel site (village) to produce N of 400. 
There will be 4 sentinel sites for each treatment zone (annual vs twice annual MDA). The target 
number of samples per village village is close to the minimum sample size of 335 needed to show 
that the rate of antigenemia (card test) positivity is less than 2% (with 95% confidence and 80% 
power). In post-MDA populations, this corresponds to a MF rate of less than 0.5%, which is an LF 
elimination target. We expect to collect 4 to 5 blood samples per household. Subjects with 
positive card tests will be tested for microfilaremia with 60 µl night blood thick smears. Our 
decision to detect MF by thick smears of finger prick blood rather than membrane filtration of 
venous blood is because thick smears are recommended for LF elimination program guidelines. 
In addition, our prior studies in Egypt have shown that L3 production by Culex mosquitoes that 
feed on MF carriers with negative thick smears is negligible. The justification for collecting blood at 
night for MF testing is that microfilariae of W. bancrofti and B. timori in Indonesia are nocturnally 
periodic with highest counts in peripheral blood between 9 p.m. and 2 a.m.  
Blood tests for Brugia timori infection B. timori infections will be detected by two methods, 
namely microscopic examination and the BrugiaRapid Test (BmR1 antibody) Cassette (Cellabs, 
Malaysia). Microscopic examination of thick blood smears prepared with 60 µl of blood collected 
between the hours of 9 p.m. and 2 a.m. The Brugia Rapid test is a rapid diagnostic test kit for 
detecting antibodies to filarial parasites using serum or whole blood.  
Blood samples will collected from randomly selected households per sentinel site (village) to 
produce N of 400. There will be 4 sentinel sites for each treatment zone (annual vs twice annual 
MDA). This sample size number exceeds the minimum target sample size of 335 needed to show 
that the MF rate is less than 2% (with 95% confidence and 80% power, assuming an actual rate 
of 0.5%, which is an LF elimination target). We expect to collect 4 to 5 samples per household.  
Stool examination for infection with soil transmitted helminths. We will examine stool 
samples of the study population mentioned above for the presence and the density of STH eggs. 
We will use the FLOTAC method to examine a single stool sample from each subject [10]. This 
quantitative method has an excellent sensitivity and will provide relatively accurate data on both 
prevalence and density of infection. We will use community egg loads as the primary endpoint for 
assessing the impact of MDA on STH infections, because this combines both prevalence and 
density of infection.  
Hypotheses, outcomes, and methods for statistical testing are provided in Section 7, 
Analysis plan. 
 

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5 
STUDY POPULATIONS 
Description of human subject involvement in research: 
We will perform repeated annual cross-sectional surveys in study communities. 
Eligibility: This project will study people who live in communities that are endemic (or that were 
endemic at the beginning of the study) for LF.  The endemicity rate (MF rate) at baseline should 
be at least 10% and study areas should have limited or no prior experience with MDA for LF.  
Gender, minority and child inclusion: Males and females will be included in population-based 
village studies without regard to race, religion, or ethnic group. There is no reason to exclude 
pregnant women and older children from population-based studies. Exclusion of children less 
than 6 years of age from community studies is justified because prevalence rates for filariasis 
tend to be very low in young children and because of difficulties associated with collecting clinical 
specimens from this population.  
 
6 
STUDY PROCEDURES / LABORATORY EVALUATIONS 
Study procedures include collection of blood and testing for microfilaremia, filarial antigenemia 
and anti-filarial antibodies. We will perform stool examination to detect STH infections.  
All assays will be performed in Indonesia (filarial antigen and antibody tests, MF smears, 
stool examinations). Some samples will be tested in parallel at Washington University 
for QC. 
6.1 Laboratory Evaluations 
The rationale for performing these tests is described in some detail in Section 4 (study design) 
above. Briefly, the study calls for collection of blood that will be tested for microfilaremia (60 µl 
thick blood smear) and filarial antigenemia (75-100 µl blood, Binax Now Filariasis Card Test 
and/or Alere Filariasis Strip Test) and anti-filarial antibodies ((25-35 µl), BrugiaRapid (BmR1) 
cassette test-Sikka district only) and for collection stool (at least 1g of stool) that will be tested 
for the presence of STH infections.  
Test methods: 
Filarial antigen testing: The Binax Now Filariasis Card Test and/or Alere Filariasis Strip Test ( 
Alere, Portland, Maine) will be performed according to manufacturer’s instructions. Briefly, the 
subject’s finger is cleaned with an alcohol wipe, dried with sterile cotton, and stuck with a sterile 
lancet. Finger blood is collected with a 75-100 µl capillary tube and placed on a sample pad on the 

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one or both filariasis tests.  The result is read at 10 minutes. If desired, test results can be fixed by 
adding a drop of ethanol or methanol to the nitrocellulose strip of either tests..  
Anti-Filarial antibody testing:  In addition to rapid antigen tests, Brugia Rapid (BmR1) antibody 
test (Cellabs, Malaysia) will be performed according to manufacturer’s instructions but only in the 
Sikka district where W. Bancrofti and Brugia infections are co-endemic.  Blood will be collected by 
finger prick into EDTA anticoagulated microcontainer tubes. Sera may be separated by 
centrifuge. Sera (25 µl) will be tested on the BrugiaRapid Test (BmR1). Results are provided 
within 15 minutes for sera and 20 minutes for whole blood.  
Microfilaria (MF) detection: For both filarial infections three-line thick smears are prepared with 
a measured total 60 µl of finger prick blood collected between 9 pm and 2 am. Slides are fixed, 
stained with Giemsa, and examined by microscopy with a 10x objective (higher magnification for 
suspicious objects). The species of MF will be determined by morphological criteria. 
Testing stool for ova of soil transmitted helminths. Stool will be collected in 25 ml containers 
and preserved in formalin (final concentration 5%). One gram of stool will be examined using the 
FLOTAC method [10]. This method provides a sensitive means of detecting and quantifying 
helminth eggs. Some stools will also be tested with traditional parasitological methods currently 
used in Indonesia for comparison with FLOTAC results. In Indonesia Ascaris lumbicoides, 
hookworms and Trichuris trichiura are the mosty prevalent STH. Eggs/larvae of other STH will be 
recorded, but not statistically analyzed.  
 
6.2 Instructions for Specimen Preparation, Handling, and Storage 
Finger prick blood will be collected in Eppendorf or microtainer tubes with EDTA anticoagulant and 
diagnostic tests will be performed within 48h following collection. rapid LF antigen tests (Binax Now 
Filariasis Card Test or Alere Filariasis Strip Test) will be read in day light 10 minutes after finishing 
the procedure as required for the current test. BmR1 will be tested any time after blood collection. 
All persons will be tested for blood microfilaremia by thick smear examination (60 µl). Blood 
samples will be stored at RT or 4°C (if available) and away from direct sunlight. 
Stool samples will be preserved with formalin (final concentration 5%) and stored at room 
temperature for later examination for helminth eggs. Samples will be labeled with barcode 
numbers that can be linked to unique identifiers (ID numbers).  
 
Safety: Project personnel will treat all human blood and stool specimens as if they were 
infectious, and we will comply with OSHA safety regulations (29CFR part 1910,1030). Field 
personnel will wear gloves, and laboratory personnel will wear gloves and lab coats for 
protection while working with blood or stool samples.  

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6.3 Specimen Shipment 
All samples will be tested in Indonesia. Some preserved stool samples and questionable blood 
smears will be tested in parallel in the reference laboratory at Washington University in St. 
Louis for quality control. This will be performed together with UI scientists. Shipment will 
comply with University of Indonesia rules and with IATA regulations.  

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7 
STATISTICAL CONSIDERATIONS 
7.1 Study Outcome Measures 
The following variables will be treated as binary variables: Microfilaremia, antigenemia, anti-
filarial antibody, stool parasites (Ascaris, Hookworm, and Trichuris). Microfilaria counts will 
also be recorded (MF/60 µl smear), and stool egg counts per species will be recorded as 
eggs/gram of feces.  
 
7.2 Sample Size Considerations 
Except as otherwise noted, all sample sizes in this protocol were calculated using a binomial 
power calculator (using alpha = .05, two-tailed tests, and power = .80). These sample sizes were 
chosen to provide confidence that measured prevalence rates will be below specified limits based 
on assumptions regarding true prevalence rates. The sample size analysis considered various 
contingencies to assess robustness of the sample size estimates (see Appendix). 
Note: Sample size considerations for community surveys for demonstrating that 
prevalence rates are below pre-defined targets for antigenemia, microfilaremia, antifilarial 
antibody and STH infections are provided in Section 4, above, and this is not repeated 
here. 
 
7.3 Participant Enrollment and Follow-Up 
This project aims to enroll approximately 6,400 subjects per year for two study areas (total for W. 
bancrofti and B. timori sub-projects). Each of the two study areas (one for Brugia and Wuchereria 
(Sikka) and one for Wuchereria (Java)) will have cluster surveys of approximately 400 people per 
village for 4 villages from annual MDA areas and 400 people per village for 4 villages from twice 
per year MDA areas.  
The community/household surveys are cross-sectional with no follow-up planned. 
 
 7.4 Statistical Analysis Plan 
The following outcome variables will be collected and treated as binary variables: 
Microfilaremia, antigenemia with the Binax Now Filariasis Card Test and/or the Alere 
Filariasis Strip Test, anti-filarial antibody (Brugia endemic area only) and stool parasites 
(separate analysis for Ascaris, Trichuris, and Hookworm). Microfilaria counts (recorded as 

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MF/60 µl smear) and stool egg counts per species (recorded as eggs/gram of feces) will be 
taken as count variables.  
Data will also be collected for demographic characteristics and covariates including age, 
gender, history of lymphedema, scrotal swelling (hydrocele), and prior treatment for LF.  
For demographic characteristics variables, descriptive statistics will be examined for the 
individuals who participate in the trials by sentinel site (village) and over years of observation. 
Frequency will be tabulated for categorical variables. Descriptive statistics (mean, median, range 
and standard deviation) will be obtained for continuous variables.  
Impact of MDA on study outcome measures 
1. Testing primary hypothesis 
The primary hypothesis is that twice annual MDA is superior to annual MDA for eliminating LF. The 
binary outcome variables used to test this hypothesis include microfilaremia and antigenemia and 
anti-filarial antibody (Brugia endemic area only). The model which is being used will fit a linear 
trend over time within each village. The mean slope across villages within each treatment group 
will then allow estimates of time to target levels for control (microfilaremia < .5% and antigenemia 
< 2%) for a selection of baseline prevalences. To illustrate the analysis strategy, we use 
antigenemia as an example.  
      The antigenemia prevalence is expressed as filarial antigenemia rate (proportion of examined 
individuals with filarial parasite antigenemia detected with the Binax Now Filariasis Card Test). To 
estimate the effect of MDA on antigenemia prevalence, we will tabulate mean rates of 
antigenemia across the four sentinel sites from each treatment zone (annual vs. twice annual 
MDA) over years of observation including the baseline. A generalized linear mixed model (GLMM) 
with a logistic link function will be used to account for the correlation of observations clustered 
within the same household from the same sentinel site. The model to estimate the difference in 
the odds of antigenemia includes treatment regimen and year of observation, and interaction 
between treatment regimen and year of observation. Some covariates that will be adjusted for in 
the analysis include sentinel sites, history of lymphedema, scrotal swelling (hydrocele) and prior 
treatment for LF as well as demographic variables of interest. Modeling will be performed with use 
of PROC GLIMMIX in SAS 9.2. CONTRAST statements will be used to test the significance of the 
log odds of antigenemia between the two treatment regimens at each year of observation. The 
difference of slope of rate decrease between the two regimens over the years of observations will 
also be tested with CONTRAST statements in PROC GLIMMIX. The resulting estimates will be 
converted to odds ratios with 95% confidence interval calculated from the robust estimator of 
variance. The other binary outcome variables (microfilaremia and anti-filarial antibody) will be 
analyzed with the same technique.  

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With microfilaria (per 60 µl of blood) measured as counts, we will display mean counts of 
microfilaria across the four sentinel sites from each treatment zone (annual vs. twice annual 
MDA) over years of observation including the baseline. To accommodate zero counts in 
modeling, we will use Geometric mean intensity (GMI) of circulating microfilaria. GMI is 
calculated as antilog [(Σlog(x+1))/n] – 1, where x is the number of microfilaria per 60 mm3 of 
blood and n is number of subjects. A generalized linear mixed model (GLMM) (implemented 
in PROC GLIMMIX) will be used to account for correlation of observations resulted from 
clustering. CONTRAST statements will be used to test the significance of differences in 
GMI of antigenemia between the two treatment regimens at each year of observation. The 
resulting estimates of GMI will be presented with 95% confidence interval. All statistical 
tests will be two-sided at a significance level of 0.05, but one-sided p-value will be reported 
according to the hypothesis. Similar analytic models will be done for community Mf loads 
and community egg load for each of three intestinal worm eggs. 
The cost analysis comparing the two treatment regimens will be conducted by Ann 
Goldman of the George Washington University School of Public Health and Health 
Services. The model will also allow cost effectiveness analysis when combined with the 
efficacy analysis. 
2. Testing secondary hypothesis 
The secondary hypothesis is that twice annual MDA is superior to annual MDA in controlling 
STH. The primary endpoint for assessing the impact of MDA on STH infections is 
community egg load (average stool egg count per species). The analysis strategy for these 
end points is the same for microfilaria count in testing the primary hypothesis.  
Adverse event 
Since this is a minimal risk and nonintervention study, adverse events are not expected to be an 
issue. However, subjects will be monitored for adverse events related to blood collection. 
Proportion of subjects with an adverse event will be tabulated with 95% confidence interval for 
each sentinel site at each year of observation.  
Treatment coverage rate 
Treatment coverage rate of the eligible population will be reported from the repeated cross-
sectional surveys for the sentinel sites (villages) during different rounds of treatment. Differences 
in treatment coverage rates will be evaluated between the two treatment regimens (annual vs. 
twice annual MDA) over time with the use of Chi-square test.   
  

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8 
SUBJECT CONFIDENTIALITY 
Subject confidentiality will be strictly held in trust by the participating investigators and 
staff. This confidentiality is extended to cover testing of biological samples in addition 
to the clinical information relating to participating subjects. This project does not include 
genetic testing. The study protocol, documentation, data and all other information 
generated will be held in strict confidence. No information concerning the study or the data 
will be released to any third party without prior written approval of the DOLF Principal 
Investigator.  
Study monitors or sponsor representatives may inspect all documents and records 
required to be maintained by the investigator at any time during or after the completion of 
the study. 
 
Data privacy control: 
1- Every individual will have one unique bar code identifier. 
2- Every sample collected in the field will be labeled with barcode stickers linked to ID 
number.  
3- The Laboratory will test and report results using sample numbers, not names or ID. 
4- Person names and identifying information will be directly entered into Smart phone, and 
the information will be synchronized daily from these devices to a laptop with a flash drive 
backup. Names will be removed automatically from the standard project database and will 
be kept in a separate database that will be password protected and accessed only by the 
Indonesian Project Data Manager (PDM) to be identified in response to a written request 
from the DOLF PI (Dr. Weil), the Objective 1 PI (Dr. Fischer) or country PI (Dr. Supali). 
This is not expected to be a common event and will only be necessary if there are 
questions about discrepancies between serial/sample numbers and ID numbers. 
5- The Data Management Team will be responsible for ensuring that no one in the project 
can link the the unique bar code identifier with the enrolled person except the PDM and 
others listed in #4 above. Person ID’s will not be released for use in other projects. 
6- The data server will keep a log file/data audit trail that will indicate the time/date and 
identify for every access to the data files. No one outside the Data Management Group 
will be able to modify any data on the server, and unauthorized persons will not be able to 
access any of the data files. 

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7- Each computer, Smartphone with project data will have a separate password. Also, 
each database will have an access system with user names and passwords that will 
control the level of access and permissions for each user. The PDM is responsible for 
controlling these permissions, and she/he is the only one that can change this. 
 
8.1 Future Use of Stored Specimens 
Residual specimens (serum, blood smears) will be maintained after the study is 
completed. Samples will be stripped of unique identifiers and stored in endemic country 
laboratories or in the PI’s laboratory at Washington University.  . Sequencing will be 
performed with DNA isolated from a small number of stool or blood samples to detect and 
characterize worm parasite DNA (blood or stool) and bacterial DNA (stool). The samples will be 
deidentified prior to any sequencing procedures. In addition, all human DNA sequences will be 
filtered out prior to bioinformatic analysis and no genetic testing of humans will be performed. 
9 
INFORMED CONSENT PROCESS 
We will follow procedures outlined in the DHHS Regulation on Informed Consent 45 CFR Part 
46 - Subpart A, 46.116 (http://www. hhs.gov/ohrp/humansubjects/guidance/45cfr46. htm#46. 116). 
Informed consent (IC) is a process that is initiated prior to the individual’s agreeing to participate 
in the study and continuing throughout the individual’s study participation. Study physicians will 
read the consent script (with potential risks and benefits) in a locally understood language to 
subjects and their families and leave a copy of this with each family. The consent document will 
be reviewed and approved by project IRB’s prior to initiation of the study. Study physicians will 
discuss risks and possible benefits of participation in this study with subjects and their families. 
Study physicians will explain the purpose of the study to subjects and answer any questions that 
may arise. The subjects may withdraw consent at any time during the course of the study. The 
rights and welfare of the subjects will be protected by emphasizing to them that the quality of their 
medical care will not be adversely affected if they decline to participate in this study.  
 
9.1 Informed Consent/Assent Process (in Case of a Minor or Others Unable to 
Consent for Themselves.  
Inclusion of minor children in community surveys will require consent from at least one parent 
plus assent of the child.  

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Human studies for this project are pending at FWA registered IRBs at Washington University (Dr. 
Fischer) and at IRB of the Faculty of Medicine of the University of Indonesia (Dr. Supali); no human 
studies will be conducted prior to approval by both IRBs. 
  
10 LITERATURE REFERENCES 
1. 
Michael, E., D.A. Bundy, and B.T. Grenfell, Re-assessing the global prevalence 
and distribution of lymphatic filariasis. Parasitology, 1996. 112 (Pt 4): p. 409-28. 
 
2. 
Ottesen, E.A., Lymphatic filariasis: Treatment, control and elimination. Adv 
Parasitol, 2006. 61: p. 395-441. 
 
3. 
Ottesen, E.A., et al., Strategies and tools for the control/elimination of lymphatic 
filariasis. Bull World Health Organ, 1997. 75(6): p. 491-503. 
 
4. 
Ottesen, E.A., et al., The global programme to eliminate lymphatic filariasis: health 
impact after 8 years. PLoS Negl Trop Dis, 2008. 2(10): p. e317. 
 
5. 
Kyelem, D., et al., Determinants of success in national programs to eliminate 
lymphatic filariasis: a perspective identifying essential elements and research 
needs. Am J Trop Med Hyg, 2008. 79(4): p. 480-4. 
 
6. 
Bethony, J., et al., Soil-transmitted helminth infections: ascariasis, trichuriasis, and 
hookworm. Lancet, 2006. 367(9521): p. 1521-32. 
 
7. 
Remme, J.H., The African Programme for Onchocerciasis Control: Preparing to 
launch. Parasitology Today, 1995. 11(11): p. 1995. 
 
8. 
Oqueka, T., et al., Impact of two rounds of mass drug administration using 
diethylcarbamazine combined with albendazole on the prevalence of Brugia timori 
and of intestinal helminths on Alor Island, Indonesia. Filaria J, 2005. 4: p. 5. 
 
9. 
Albonico, M., et al., Intervention for the control of soil-transmitted helminthiasis in 
the community. Adv Parasitol, 2006. 61: p. 311-48. 
 
10. 
Cringoli, G., et al., FLOTAC: new multivalent techniques for qualitative and 
quantitative copromicroscopic diagnosis of parasites in animals and humans. Nat 
Protoc, 2010. 5(3): p. 503-15. 
 
 

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SUPPLEMENTS / APPENDICES 
Standard Operating Procedure (SOP) Summary of Laboratory Work   
Project laboratories will receive, process, and archive biological samples. They will 
perform assays and report results to the Data Management Team and Project scientists. 
The Laboratory will perform the following procedures and assays.  
 
Blood samples: The laboratory will use finger blood samples to perform several tests 
(filarial antigen test and thick smears for MF detection).  
Microfilaria testing: Thick blood smears (60 µl in 3 parallel lines of 20 µl each) will be 
stained with Giemsa and examined with a microscope for MF.  
Filarial antigen tests: Binax Now Filariasis Card Test and/or Alere Filariasis Strip Test 
using finger prick blood (75-100 µL) will be performed in the laboratory according to the 
kit protocol and read at 10 minutes.  
Antifilarial Antibody Testing: The Brugia Rapid Test detects antibodies to filarial parasite 
with finger prick blood (25 µL for serum/ 35 µL for whole blood). This test will be 
performed in field sites or in a laboratory according to the kit protocol and read at 15 
minutes for serum and 20 minutes for whole blood. 
Stool samples for STH examination:  Stool samples will be collected in 25 ml 
containers and preserved in formalin (final concentration 5%). One gram of stool will be 
examined using the FLOTAC method [10]. This method provides a sensitive means of 
detecting and quantifying helminth eggs.  
Specimen archive: In addition to new samples generated during this study, the 
Laboratory will be responsible for maintenance of archived samples (used Binax 
Now Filariasis Card Test, preserved stool samples, etc.) collected during the DOLF 
project.  
QC activities: DOLF investigators will visit study sites to review procedures and 
results. They will also keep in close contact with Indonesian counterparts by email, 
telephone, and during visits by collaborating scientists from Indonesia to the USA. All 
samples will be tested in the endemic country laboratories. Some preserved stool 
samples and questionable blood smears will be tested in parallel in the reference 
laboratory at Washington University.  
 

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Standard Operating Procedures Summary (SOP) for Data Collection and 
Field Work 
Field work will be conducted by trained physicians and paramedical personnel who 
will be trained for data and blood collection. 
All field team members will be trained by staff physicians with assistance from other 
project personnel: 
1. All field epidemiology personnel (physicians, technicians and field workers) will 
be trained in GCP topics for research involving human subjects. 
 
2. Field and laboratory personnel will also be trained regarding proper procedures 
for collecting blood, for preparing blood smears, for performing filarial antigen 
tests, and for labeling specimens (barcode stickers). 
 
3. Field data will be entered in the field using electronic questionnaires on PDAs or 
smart telephones. Finger-prick blood (400 µl) will be collected by physicians or 
trained technicians for laboratory studies. All samples will be labeled in the field. 
 
4. Technicians will be responsible for transferring blood samples to the Laboratory 
immediately after field trips (same night of collection). 
 
5. Stool samples will be collected by the field epidemiological personnel during the 
survey or on the next day. 
 
 

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Informed Consent Script to Be Read in Population-Based Field Surveys 
 
Project title:   
Optimization of chemotherapy for control and elimination of 
onchocerciasis and lymphatic filariasis  
Project Director:  
Dr. Taniawati Supali (country PI) and Dr. Peter Fischer (PI, 
DOLF Project Objective 1) 
Sponsored by:  
Bill and Melinda Gates Foundation (USA) 
 
1.  Invitation:  You (and your family members) are being invited to participate in a project 
to test the impact of a public health program on parasite infections that are common in this 
area.  
2.  Purpose:  The purpose of this project is to assess effects of mass drug administration 
on filariasis and other worm infections in Indonesia. This project is being conducted by the 
University of Indonesia in Jakarta in cooperation with scientists at Washington University 
in St. Louis USA.    
3.  Procedures:  Your finger will be cleaned with alcohol, and a few drops of blood will be 
collected by finger prick. We will keep samples collected now to do other research studies 
of infectious diseases in the future. We will also collect information (such as name, age, 
and address or house location). The screening procedure will take a few minutes of your 
time for collecting information and the blood sample. We will also collect stool samples to 
test for intestinal worm infections. 
4.  Risks:  There are no significant risks.  Collection of blood samples (a few drops by 
finger prick) causes minor pain and occasionally a small bruise. You will be asked to keep 
pressure with cotton on your finger for 2 minutes to stop bleeding. The small amount of 
blood being collected will not harm your health.   
5.  Number of participants and duration of the project:  Approximately 6,400 people 
will participate in the project per year; the surveys will be repeated annually for 4 years.  
6.  Benefits:  Participation in this health project will benefit you and your family by 
providing a free screening test for early detection of parasitic worm infections. If you or 
any of your family members are found to have worm infections, you will be referred to a 
nearby primary health center for treatment. The project will also help the Ministry of Health 
in Indonesia by providing information on the effects of their mass drug administration 
program on filariasis and other worm infections in your community.  

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7.  Confidentiality:  Results of any abnormal blood or stool tests will be provided to you 
and also to local health authorities. All project records may be reviewed by the sponsors 
of the program. Otherwise, any information linked to your name will be strictly confidential.  
8.  Questions:  You are free to ask any question regarding this health study now. If 
questions arise later, please contact Dr.Taniawati Supali, who can be reached by 
telephone at 62-8129355870.  
9.  Participation is voluntary:  Participation by you and your family members in this 
public health assessment project is entirely voluntary. Children should not be forced to 
participate against their will. Refusal to participate will not penalize you in any way, and 
you are free to withdraw at any time.  
If you willingly agree to participate, you will provided a copy of this information. 
----------------------------------- 
Note:  Program personnel must mark each computer record to verify that they have read 
this script to all participants and answered any questions. A copy of this information form 
should be left in each house included in the program.