Optimization of Mass Drug Administration With Existing Drug Regimens for Lymphatic Filariasis and Onchocerciasis for Liberia
Conditions: Lymphatic Filariasis, Onchocerciasis, Soil Transmitted Helminth (STH) 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 Investigator: 
Gary J. Weil, M.D. 
 
Version Number: 6.2 
NCT Number: NCT01905436 
 
Day Month Year: 09 May 2019 
 
 
 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
2 
 
 
1. 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  
 
2. 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: 8 May, 2019 
 
 
Peter U. Fischer, Ph.D. - Washington University School of Medicine  
Title: Principal Investigator, Community MDA Studies (DOLF Objective 1)  
 
 
Gary J. Weil, M.D. - Washington University School of Medicine  
Title: Principal Investigator, DOLF Project

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
iii 
 
3. Table of Contents 
Statement of Compliance ................................................................................................................................ 2 
SIGNATURES .................................................................................................................................................... 2 
Table of Contents ........................................................................................................................................... iii 
List of Abbreviations ....................................................................................................................................... iv 
Protocol Summary .......................................................................................................................................... vi 
1 
KEY ROLES ................................................................................................................................................ 7 
2 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE .................................................................. 7 
2.1 
Background Information ..................................................................................................................... 7 
2.2 
Rationale .............................................................................................................................................. 9 
2.3 
Potential Risks and Benefits / Potential Risks ................................................................................... 11 
2.3.1 
Potential Risks ............................................................................................................................... 11 
2.3.2 
Known Potential Benefits .............................................................................................................. 12 
3 
OBJECTIVES ............................................................................................................................................ 12 
4 
STUDY DESIGN ....................................................................................................................................... 13 
6 
STUDY PROCEDURES / LABORATORY EVALUATIONS ............................................................................ 16 
6.1 
Laboratory Evaluations ...................................................................................................................... 16 
6.2 
Instructions for Specimen Preparation, Handling, and Storage ...................................................... 17 
6.3 
Specimen Shipment ........................................................................................................................... 17 
7 
STATISTICAL CONSIDERATIONS ............................................................................................................. 17 
7.1 
Study Outcome Measures ................................................................................................................. 18 
7.2 
Sample Size Considerations ............................................................................................................... 18 
7.3 
Participant Enrollment and Follow-Up .............................................................................................. 18 
7.4 
Analysis Plan ...................................................................................................................................... 18 
8 
SUBJECT CONFIDENTIALITY ................................................................................................................... 20 
8.1 
Future Use of Stored Specimens ....................................................................................................... 21 
9 
INFORMED CONSENT PROCESS ............................................................................................................. 22 
9.1 
Informed Consent/Assent Process (in Case of a Minor or Others Unable to Consent for Themselves.
 
22 
10 
LITERATURE REFERENCES .................................................................................................................. 22 
Annex 1: Protocol for the Survey on resumption of mass treatment for parasitic infections in Foya district 
following the recent Ebola oubreak .............................................................................................................. 24 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
iv 
 
SUPPLEMENTS / APPENDICES ....................................................................................................................... 26 
 
4. List of Abbreviations 
ALB 
Albendazole 
APOC 
African Programme for Onchocherciasis Control 
CFR 
 
Code of Federal Regulations 
DEC 
Diethylcarbamazine 
DEC/Alb 
Diethylcarbamazine and Albendazole 
DOLF 
Death to Onchocerciasis and Lymphatic Filariasis 
DSMB  
Data Safety Monitoring Board 
EDTA 
Ethylenediaminetetra-acetic Acid 
ELF 
Elimination of Lymphatic Filariasis 
FWA 
Federal-Wide Assurance 
GPELF 
Global Program to Eliminate Lymphatic Filariasis 
GCP 
Good Clinical Practice 
IATA 
International Air Transport Association 
ICH 
International Conference on Harmonisation 
ICT 
Immunochromatography Test 
IRB 
Institutional Review Board 
Iver 
Ivermectin 
KK 
Kato-Katz 
LIBR 
Liberia 
LF 
Lymphatic Filariasis 
MDA 
Mass Drug Administration 
MF 
Microfilaria(e) 
OCP 
Onchocerciasis Control Programme 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
v 
 
OHRP 
Office of Human Research Protection 
Oncho 
Onchocerciasis 
OSHA 
Occupational Safety and Health Administration 
PDM 
Project Data Manager 
PI 
Principal Investigator 
SAE 
Serious Adverse Events 
SOP 
Standard Operating Procedure 
STH 
Soil Transmitted Helminth 
WHO 
World Health Organization 
µl 
Microlitre 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
vi 
 
5. Protocol Summary 
 
Title: 
Optimization of Mass Drug Administration with existing drug regimens for Lymphatic Filariasis 
and Onchocerciasis  
 
Population: 
Approximately 5,200 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.    
Number of Sites: 
Two sites in Liberia 
Study Duration: 
7 years 
Subject Duration: 
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. 
Objectives: 
 
A. To evaluate different mass drug administration (MDA) regimens for lymphatic filariasis and 
onchoceriasis as well as 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. 
B. We will compare the relative impact and cost effectiveness of annual vs. twice yearly mass drug 
administration (MDA) for elimination of lymphatic filariasis (LF) and control of onchocerciasis. 
C. We will also study the impact of annual vs. semiannual MDA on soil transmitted helminth (STH) 
infection in the population. 
D. Following conclusive results on the first objectives that annual MDA is as effective as 
semiannual MDA for LF, oncho and STH, we will conduct an additional survey to determine long 
term (7 year) impact of continued MDA on onchocerciasis, LF, and STH in two areas.  
 
Remarks on the project structure:  The Project “Optimization of Mass Drug Administration with existing 
drug regimens for Lymphatic Filariasis and Onchocerciasis” has 2 major Objectives (Objective 1-
Community MDA Trials, Objective 2-Randomized Clinical Trials). Objective 1 will be studied in 4 countries 
in Africa and 2 countries in South East Asia. Although the overall objectives are similar there are country 
specific modifications. This protocol was developed for Liberia.

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
7 
 
6. 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 
 
Country Principal Investigator: Fatorma Bolay, Ph.D. (DOLF Objective 1 Liberia)  
 
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 
The Liberian Institute for Biomedical Research (LIBR), Director Dr. Fatorma Bolay, 
P.O. Box 31, Charlesville, Margibi County, LIBERIA, Contact: Fatorma Bolay. Tel. 
+2316516302 / +2316516803/ +231886513040. Email:  director.libr@gmail.com 
 
7. 2 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
8. 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]. These include (among others) inability to conduct 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
8 
 
MDA programs in areas of Africa where Loa loa is coendemic because of the unacceptable risk of Serious 
Adverse Events (SAE’s) with Ivermectin in persons with heavy L. loa infections, 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. 
 
Onchocerciasis (“Oncho”) is similar in some ways to LF in that it is a vector-borne nematode parasitic 
disease that causes severe disability. Oncho affects approximately 33 million people, mostly in 30 
countries in sub-Saharan Africa (with small foci in Latin America and Yemen) [5]. In contrast to LF, this 
disease causes blindness and severe skin disease rather than elephantiasis, and it is spread by black flies 
instead of mosquitoes. O. volvulus adult worms live in subcutaneous nodules while the adult worms of 
the LF parasites live in lymphatic vessels. O. volvulus adult worms are larger and less sensitive to 
available drug treatments than those of the species that cause LF. They also have a longer lifespan 
(approximately 14 years rather than the estimated 7 years for LF parasites). Several programs and 
developments have greatly improved the Oncho situation since the 1970’s when the Onchocerciasis 
Control Programme (OCP) in West Africa (green countries in the map) was initiated. OCP relied 
exclusively on vector control in its early years. However, following the appearance of Ivermectin 
(Mectizan) on the scene in the late 1980’s, OCP transitioned to become a drug distribution program with 
annual Ivermectin MDA in 11 countries. OCP ended in 2002. This was replaced by the African 
Programme for Onchocherciasis Control (APOC) which coordinates community directed distribution of 
Ivermectin MDA in 28 African countries (including the former OCP countries).  OCP and APOC have done 
a good job of reducing parasite infection intensities and Oncho disease rates in many endemic countries. 
Unfortunately, there is no real end in sight for the APOC approach (apart from a funding endpoint in 
2015); while it may be possible to eliminate Oncho in selected areas by MDA with Ivermectin (alone, or 
combined with vector control), disease control programs in most African countries will require active 
maintenance for many years to come. While Ivermectin has good activity against the parasite larvae that 
cause disease in the skin and eye (microfilariae or MF), it does not kill O. volvulus adult worms, and they 
resume production of MF that can lead to transmission of new Oncho cases by black flies after a few 
months. APOC activities are focused on areas with high infection rates (where disease risks are highest). 
However, extensive areas in Africa where fewer than 20% of adult men have Oncho nodules detectable 
by palpation are not receiving interventions for onchocerciasis at this time. These areas are not disease 
free (Oncho dermatitis can be severe in hypoendemic areas), and they also may serve as a source for 
reintroduction of the parasite into previously controlled areas after interventions stop.  More effective 
drugs or dosing schedules for MDA against Oncho could shorten the number of years needed to 
interrupt Oncho transmission in areas that previously had high disease rates. Improved treatments 
should also make it feasible to extend MDA into areas that are currently not being helped. These 
changes have the potential to completely change the game to make global elimination of onchocerciasis 
a feasible goal.   
  
 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
9 
 
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]. Taken together these worm 
infections cause more disability adjusted life years than malaria. 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]. Increasingly control programs for filariasis and STH are being integrated with programs for 
other parasitic diseases such as schistosomiasis. For this reason, participants in Foya district will also be 
tested for schistosomiasis. Results from this survey will help the Liberian Ministry of Health implement 
schistosomiasis control programs in the study area and in other parts of the country.  
Results of Previous Work  
The result of the studies conducted under this protocol produced evidence that annual 
treatment is superior to semiannual treatment for reduction of mf (both LF and oncho), and of STH 
infection intensities (multiple species).  
In Maryland, at the last survey in 2016, O. volvulus microfilaria (mf) prevalence was 11% in 
the semiannual villages and 17% in the annual villages. Positive ICT for Wb antigen was 13% in the 
semiannual group and 5% in the annual group. In the semiannual villages, Ascaris spp. prevalence 
was 40%, hookworm was 17%, and Trichuris was 79%.  In the annual group, Ascaris spp. prevalence 
was 50%, hookworm was 9%, and Trichuris was 7%.  
In Lofa County, at the last survey in 2017, O. volvulus mf prevalence was 5% in the 
semiannual villages and 4% in the annual villages. Positive ICT for Wb antigen was 0% in the 
semiannual group and 0% in the annual group. Ascaris spp. prevalence was 2%, hookworm was 11%, 
and Trichuris was 1% in the annual group. In the semiannual group, Ascaris spp. was 2%, hookworm 
was 10%, and Trichuris was 2%.   
 
9.  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]. Annual Iver (150 µg/kg) is 
effective for prevention of blindness, but it is unlikely to lead to elimination in most endemic areas. The 
highly successful African Programme for Onchocerciasis Control (APOC) is focused on morbidity 
prevention rather than disease elimination, and this explains its focus on hyperendemic and 
mesoendemic areas where Oncho blindness and dermatitis are significant public health problems [7]. 
The relatively small Onchocerciasis Elimination Program for the Americas (OEPA) provides a proof of 
principal that Oncho can be eliminated in some ecological settings [8]. Computer simulations suggest 
that Iver MDA may be sufficient to eliminate Oncho in some areas in Africa. However, the very long 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
10 
 
duration of MDA and coverage rates required for this outcome [9] suggest that different strategies may 
be needed to improve chances for elimination in a reasonable period of time. 
 
WHO (WHA 50.29) has targeted lymphatic filariasis 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 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 and 10-15 years for O. 
volvulus).  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.   
 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
11 
 
DOLF studies in Liberia have provided some of the most complete data on the impact of 
community MDA regimens for LF and other NTDs. These studies have shown that 3 years of MDA had 
significant impacts on LF, STH and onchocerciasis prevalence in Liberia and other study sites. However, 
three years of MDA was not sufficient to meet elimination targets for those infections. An additional 
survey in the Maryland district will provide further data on the rate of decline of filarial antigenemia 
prevalence following MDA in Africa and on the effects of additional rounds of MDA on onchocerciasis 
and on STH prevalence and intensities. Three of these study areas had very high STH prevalence at 
baseline (two in Liberia and one in DRC), and continuation of measuring the impact MDA through 7 
years after initiation will prepare these areas as potential sites for later studies of the efficacy of 
school-based deworming or other maintenance strategies to prevent the rebound of STH following 
cessation of MDA for LF. 
In the last three years, annual MDA has been administered by the the NTD team of the 
Ministry of Health (MOH) in Lofa and by MOH/COUNTDOWN in Maryland. At a follow-up visit in 2019 
by  Liberian DOLF team members, all 16 study villages had received MDA. In Lofa County, MDA was 
conducted through the MOH alone, and the DOLF team recorded that not all of the 26 study villages 
in Lofa received MDA in 2018.   
The proposed additional survey will provide data on long-term impact of MDA and support 
endgame research to understand how many rounds of MDA are needed to achieve elimination of LF 
and oncho and control of STH infections. The elimination targets for LF are <2% prevalence of antigen 
(assessed with FTS) and <1% prevalence of microfilaria (assessed with thick blood smear). The 
elimination target for onchocerciasis is <5% microfilaria in the skin (assessed by skin snip). The 
proposed survey will determine if these targets have been reached, and will answer the question of 
how many additional rounds of MDA are needed to achieve elimination targets. The answer to this 
question is critical for global elimination programs and may inform global policies.  
 
10. 2.3 
Potential Risks and Benefits / Potential Risks 
Immediate risks 
11. 2.3.1 
Potential Risks 
This research includes risks associated with collection of blood by finger prick and of skin snips 
(superficial skin biopsies collected by corneoscleral biopsy punch). 
Likely  
Collection of blood and skin snips causes minor pain. 
Less Likely 
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 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. 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
12 
 
Collection of skin snips can cause a bruise, bleeding or infection at the collection site. 
To minimize risks, skin snips will be collected after disinfecting the skin with alcohol 
using a sterile skin snip punch. Skin snip punches will be cleaned and soaked in 4% 
Mucocit-P for 5 minutes and then rinsed with 70% ethanol between subjects. Subjects 
will be instructed to apply pressure to skin snip sites for two minutes and to keep these 
areas clean.  
 
 
There are no risks associated with collection of stool or urine samples.  
 
Long range risks: None 
• 
Rationale for the necessity of such risks: Blood and skin snips are required for 
diagnosis of filarial infection. Procedures in this project meet the definition of 
“minimal risk”. Stool and urine samples are needed for diagnosis of other STH 
and schistosomiasis infections.  
• 
Alternative data gathering procedures that have been considered or will be 
considered: None. Blood and skin snips are needed for diagnosing filarial 
infections (microfilariae) 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. 
12. 2.3.2 
Known Potential Benefits 
• 
To subjects: 
The study will screen participants for parasitic infections and refer infected subjects for 
treatment at their local government health center. We will also provide data on infection 
rates to district health officials. This information will help the Liberian Ministry of Health 
to plan public health programs for villages where our study subjects live.  
• 
To society: 
Participation will provide information on the impact of MDA on filarial and STH infections 
that affect the public health in the community. 
  
3 
OBJECTIVES 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
13 
 
To conduct population-based field studies to determine the relative cost and efficacy of annual vs. 
semiannual MDA for control and elimination of onchocerciasis and 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 and onchocerciasis. 
3. Following conclusive results on the first objectives that annual MDA is as effective as 
semiannual MDA for LF, oncho and STH, we will conduct an additional survey to determine 
long term (7 year) impact of continued MDA on onchocerciasis, LF, and STH in two areas.  
  
13. 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 mass drug administration 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. Information on the presence of onchocercal nodules as well as on onchocercal skin and eye disease 
will be also recorded. Nodules will be assessed by superficial palpation. Blood samples will be collected 
from systematically sampled households. Blood will be tested for filarial antigenemia with one or more 
of the following: the BinaxNow Filariasis card test (also known as ICT), the Alere Filariasis Test Strip, and 
the TropBio test. Blood will also be tested for onchocerciasis (serology testing). Blood from persons with 
positive filarial antigen tests will also be tested for microfilaremia (W. bancrofti MF in the blood, see 
Section 6 below for methods). Two skin snips will be examined for microfiladermia (O. volvulus MF in the 
skin, see Section 6 below for methods). Stool examinations will be performed for diagnosis of STH 
infections and schistosomiasis (S. mansoni), and urine will be tested for S. hematobium. Note that not all 
tests will be performed on all samples (blood, stool and/or urine). 
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, skin snips, stool and urine 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: The Binax Now Filariasis card test, the Alere Filariasis 
Test Strip, and the TropBio antigen test (with MF testing of antigen positive subjects). We are using 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
14 
 
these tests as measures of LF endemicity. The antigen tests detect antigen released from living adult 
filarial worms potentially capable of resuming MF production in the future. The filarial antigen tests are 
much more sensitive for infection in treated subjects than tests for microfilaremia. However, antigen 
tests may remain positive for years after treatment has cleared microfilaremia. The antigen tests will be 
performed on blood from individual residents 5 years of age and older. This will be done by sampling 
populations in sentinel sites (villages) to produce N of 400-500 per site. There will be several sentinel 
sites for each treatment zone (annual vs. twice annual MDA). The sample size number  per sentinel site 
is close to the overall minimum target sample size of 335 needed to show that the filarial antigen rates 
are less than 2% (with 95% confidence and 80% power, assuming an expected rate of 0.5%, which is an 
LF elimination target). While all subjects will be tested with the Binax Now Filariasis card test, not all 
blood samples will be tested with the Alere Filariasis Test Strip and the TropBio antigen test. The 
purpose of parallel antigen testing of a subset of blood samples is to compare the performance of these 
three filarial antigen tests.  
Subjects with positive filarial antigen tests will be tested for microfilaremia with 60 µl night thick blood 
smears collected by finger prick at night. Night blood samples are needed for microfilaria detection, 
because the parasites only circulate in peripheral blood at night.  
Onchocerca volvulus infection- nodule palpation, skin snips and oncho serology: We are using these 
tests as measures of onchocerciasis endemicity. Due to the presence of deep nodules which cannot be 
assessed by superficial palpation, nodule data provide only an inaccurate estimate of endemicity. 
However, this method is used for rapid mapping of onchocerciasis and will be included. The assessment 
of MF in the skin is a more reliable measure of endemicity in areas with no community directed 
treatment using ivermectin. After treatment the presence of MF gives valuable information about the 
MF available for transmission. Two skin snips from skin over the iliac crest will be taken from subjects 
the same subjects who are tested for filariasis as described above. Blood collected for W. bancrofti 
antigen detection (see above) will be also used for onchocerciasis serology.  
Stool examination for infection with STH and schistosomiasis. We will examine stool samples of the 
study population mentioned above for the presence and the density of STH eggs and S. mansoni eggs. 
We will use the FLOTAC and Kato-Katz (KK) methods to examine a single stool sample from each subject. 
These methods have excellent sensitivity and will provide accurate data on both the prevalence and 
intensity of infection. We will use community egg loads as a main indicator which combines both 
prevalence and density of infection and we target 95% reduction for each species. The Liberian MOH has 
agreed to provide Praziquantel to communities shown by our study to have high rates of 
schistosomiasis.  
Urine examination for infection with Schistosoma hematobium We will examine urine samples by 
microscopy for the presence and the density of S. hematobium eggs. The Liberian MOH has agreed to 
provide Praziquantel to communities shown by our study to have high rates of schistosomiasis.  
 
 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
15 
 
Continuation Activities (2019) 
With 4 years of annual cross-sectional surveys conducted by DOLF researcher and completed in both 
Maryland (last survey in 2016) and Lofa (last survey in 2017), we plan to conduct an additional one-
time cross-sectional survey in the Harper site in Maryland district in 2019 and in Lofa (Foya & Kolahun 
Districts) in 2020 to measure the long-term impact of MDA on W. bancrofti (lymphatic filariasis), O. 
volvulus (onchocerciasis) and on STH infection parameters following these cumulative 7-9 rounds of 
MDA. Since the last DOLF surveys, there have been a total of 3 annual rounds of MDA in both areas.  
The study will recruit 2,500 participants in the Maryland area villages and 3,200 in the Lofa area 
villages. The proposed activities will comprise of identical survey methods as previously conducted 
surveys in both study areas. The survey includes: 
• Stool sample collection with Kato Katz analysis for STH infections,  
• Blood collection by finger prick during the day with analysis by FTS and thick blood smear for 
detection of W. bancrofti antigen and microfilaria (respectively) in the blood (TropBio antigen 
test and Binax Now card tests will not be used as they were found to be inferior to the Alere 
Test Strip (FTS)) 
• Skin snips for detection of O. volvulus microfilaria in the skin  
As a final note, we will not provide MDA as part of this research, but we will share our findings to the 
MOH and others to support their decision-making for MDA continuation or cessation. We will publish 
the findings to support global policy decision making.  
 
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 have been endemic at the 
beginning of the study for lymphatic filariasis (LF) and onchocerciasis (oncho).  The LF endemicity rate 
(MF rate) should at the study begin should be about 10% and there should have been no MDA in area 
immediately prior to study begin. The prevalence of oncho is secondary, but should be at least 5% 
nodule rate or 10% skin MF. Eligibility criteria will remain the same in the continuation surveys in both 
areas.  
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 
from population-based studies. There is also no reason to exclude children from the studies, and they 
are included. Exclusion of children less than 5 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.  

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
16 
 
 
14. 6 
STUDY PROCEDURES / LABORATORY EVALUATIONS 
Study procedures include collection of blood/skin biopsies and testing for microfilaremia, filarial 
antigenemia, filarial antibodies, microfiladermia and onchocercal nodules. We will perform stool 
examination to detect STH infections. We will examine urine samples to detect S. hematobium 
infections.  
All parasitology tests will be performed in Liberia (W. bancrofti MF smears, antigen tests, O. volvulus 
MF detection, antibody tests, stool and urine examinations). The TropBio antigen test (ELISA) will be 
performed at Washington University (St. Louis, USA) in a laboratory that has facilities and experience 
needed for this assay.  
15. 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), filarial antigenemia and antibodies (300 microliters),  performance of skin snips (two superficial 
biopsies of about 2 mg skin each) for onchocerciasis testing,  collection of stool (at least 1g of stool) for 
detection of parasite eggs. , and collection of urine (10 ml) for detecting S. hematobium eggs.  Not all 
tests will  be performed on all samples (blood, stool and/or urine). 
Test methods: 
Filarial antigen testing: The ICT card test (Binax Filariasis Now test  [Alere, Portland, ME) 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 100 
µl capillary tube and placed on a sample pad on the card. The card is closed, and the result is read at 10 
minutes.  
The Alere Filariasis Test Strip will be performed according to the manufacturer’s instructions. Finger 
prick blood is placed on the strip and the test is read at 10 minutes. This test uses the same reagents as 
the Binax Filariasis Now card test, and laboratory evaluations have shown that it may be superior to the 
card test, which is widely used for filariasis testing. This evaluation will determine whether the new strip 
test should replace the card test. The TropBio antigen test (a commercial ELISA assay) will be performed 
according to the manufacturer’s instructions.  
W. bancrofti microfilaria (MF) detection: MF tests will be performed for subjects with positive filarial 
antigen tests. Three-line thick smears are prepared with a measured 60 µl quantity of finger prick blood 
collected between 8 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. 
O. volvulus microfilaria (MF) detection: Two skin snips will be taken from the buttocks using Holth 
corneoscleral punches. Biopsies will be individually place in a well of a microtiter plate and incubated in 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
17 
 
100 µl phosphate buffer at ambient temperature. Wells will be check for emerging MF after 30 min and 
after overnight incubation. After the incubation skin snips will be weighted [10]. 
Onchocerciasis serology:  Small amounts of blood (<100 µl) from a subset of individuals may be tested 
for onchocerciasis infection markers (antibodies or antigens).  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, mini-FLOTAC, and KK methods [11]. These tests are widely 
used for detecting and quantifying helminth eggs.  
Testing urine for Schistosoma hematobium eggs. Urine will be collected in 40 ml containers and 
preserved in formalin (final concentration 5%). Ten ml of urinary sediment will be examined by 
microscopy to detect parasite eggs.  
16. 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. ICT cards will be read in day light 10 
minutes after finishing the procedure as required for the current test. Persons with positive antigen tests 
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 not in the direct sunlight. 
Skin snips will be placed in separate wells of labeled microtiter plates immediately after collection, 
phosphate buffer will be added and wells will be covered with tape. Plates will be stored at ambient 
temperature (25-32°C) protected from direct sunlight. After incubation skin snips and emerged MF may 
be preserved by adding a drop of 10% formalin. 
Stool samples will be preserved with formalin (final concentration 5%) and stored at RT until examination 
for helminth eggs. Samples will be labeled with barcode numbers that can be linked to unique identifiers 
(ID numbers).  
Urine samples will also be preserved with 5% formalin for later microscopic examination.  
Safety: Project personnel will treat all human blood, skin, urine, 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, skin biopsies, urine, or stool samples.  
17. 6.3 
Specimen Shipment 
 Some preserved stool samples and questionable blood smears or skin biopsies (with buffer) will be 
tested in parallel in the reference laboratory at Washington University in St. Louis for quality control. 
In addition, serum samples will be tested with the TropBio ELISA at Washington University, as 
mentioned above. Shipment will comply with Liberia rules and with IATA regulations.  
 
18. 7 
STATISTICAL CONSIDERATIONS 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
18 
 
19. 7.1 
Study Outcome Measures 
The following variables will be treated as binary variables: Microfilaremia, antigenemia, 
microfiladermia, onchocercal nodules, stool parasites (Ascaris, Hookworm, and Trichuris) and 
Schistosoma mansoni and S. haematobium where applicable microfilaremia counts will also be 
recorded as MF/60 µl smear, microfiladermia counts as MF/mg skin (and MF/skin snip), nodules as 
nodules per person. Urine eggs will be recorded as eggs/ml of urine, and stool egg counts will be 
recorded as eggs/gram of feces.  
  
20. 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, microfiladermia, schistosomiasis, and 
STH infections are provided in Section 4, above, and this is not repeated here. 
  
21. 7.3 
Participant Enrollment and Follow-Up 
This project aims to enroll at least 3,200 subjects per year in Foya District and 2,000 subjects per year in 
Maryland County. Study areas will have cluster surveys of approximately 400-500 people per 4 sentinel 
site (4 sites each in the annual and semiannual MDA areas). The community/household surveys are cross-
sectional with no follow-up planned. 
  
22. 7.4 
Analysis Plan 
The following outcome variables will be collected and treated as binary variables: Microfilaremia, 
microfiladermia, filarial antigenemia and antibody, and stool/urine parasites (separate analyses for 
Ascaris, Trichuris, schistosomes, and Hookworm). Microfilaria counts (recorded as MF/60 µl blood), 
microfiladermia counts as MF/mg skin, nodules as nodules per person, and stool egg counts for 
each species (recorded as eggs/gram of feces or eggs/ml urine) will be taken as count variables.  
Data will also be collected for demographic characteristics and covariates including age, gender, 
and 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 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
19 
 
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 the 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. 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 will be expressed as the filarial antigenemia rate (proportion of examined 
individuals with filarial parasite antigenemia detected with the Binax Now Filariasis card test. If Alere 
stops selling the card test, we may need to switch to their antigen strip test as the primary antigen test 
in later years of the project. 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 (e.g., 
microfilaremia and microfiladermia) will be analyzed with the same technique.  
 
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 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
20 
 
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, community O. volvulus Mf load, and for community egg loads for each of 
three intestinal worm species.  
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 for controlling STH 
and onchocerciasis in the Foya district. The primary endpoint for assessing the impact of MDA on 
STH infections is community egg load (average stool egg count per species).The primary endpoint 
for onchocerciasis will be Mf prevalence by skin snip. We will also compare community Mf loads 
for onchocerciasis. 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.  
 
23. 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 
Principal Investigator. The study monitor or sponsor representatives may inspect all documents and 
records required to be maintained by the Investigator. 
 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
21 
 
Data privacy control: 
1- Every individual included in the study will have an ID number (unique identifier) that can be 
used to identify Locality ID, House ID, and Person ID. 
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 an Android Smartphone, 
and the information will be backed up daily from Smartphones to a laptop. The data from the 
Smartphones will be uploaded as it is collected to a central database on a secure Microsoft 
Azure cloud server under an account that is covered by a business service agreement between 
the Washington University in St. Louis School of Medicine and Microsoft to be HIPAA compliant. 
Information that includes names will be kept in a separate database that will be password 
protected and accessed only by the Liberian Project Data Manager (PDM) the DOLF PI (Dr. Weil), 
the Objective 1 PI (Dr. Fischer), country PI (Dr. Bolay), or DOLF Project Manager. This dataset will 
only be used during the data cleaning process to answer any discrepancies between 
serial/sample numbers and ID numbers. Names will be removed from the standard project 
database as the identifying data is not necessary for analysis. 
5- The Data Management Team will be responsible for ensuring that no one in the project can 
link the sample IDs with the person IDs and that no one can use the names database 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. 
7- Each computer, Smartphone, or smart telephone with project data will have a separate 
password. The PDM is responsible for controlling these permissions, and she/he is the only 
one that can change this. 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 DOLF 
Project Manager will be responsible for managing access to the Microsoft Azure database.  
24. 8.1 
Future Use of Stored Specimens 
Residual specimens (serum, blood smears) will be maintained after the study is completed for 
future research on infectious diseases. 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 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
22 
 
be filtered out prior to bioinformatic analysis and no genetic testing of humans will be 
performed. 
 
25. 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 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 throughout 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. 
26. 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.  
Human studies for this project are pending at FWA registered institutional review boards at Washington 
University (Dr. Fischer) and at IRB of the University of Liberia, Monrovia (Dr. Bolay); no human studies will 
be conducted prior to approval by both IRBs. 
Participants will be re-consented with a revised informed consent document that includes a summary 
of progress on disease elimination (data findings) to date as well as a description of the survey 
procedures (similar to the previously used informed consent).  
  
27. 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. 
 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
23 
 
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. 
Fischer P, Kipp W, Bamuhiga J, Binta-Kahwa J, Kiefer A, Büttner DW. Parasitological and 
clinical characterization of Simulium neavei-transmitted onchocerciasis in western 
Uganda. Trop Med Parasitol. 1993. 44:311-21. 
11. 
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. 
 
 
 
 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
24 
 
28. Annex 1: Protocol for the Survey on resumption of mass treatment for parasitic infections in 
Foya District following the recent Ebola outbreak 
 
Background 
In March 2014 all DOLF study related work was suspended after cases of Ebola were reported in and 
around our research site at Foya District. Since then, no government MDA has taken place in this area, 
which turned out to be the early epicenter of the Ebola outbreak in Liberia. Now the Ebola outbreak is 
under control in Liberia. There have been no new reported cases in the country since the end of 
February according to Mar 11, 2015 WHO situation report. This means that important public health 
campaigns like MDA can resume. However, given these communities’ difficult experience with the 
outbreak, their trust in the national health system may be strained or damaged in way that could 
negatively impact MDA programs for parasitic infections.  
 
Objective 
Before the government restarts its MDA program, it is important for us to know how communities will 
respond to these drug distribution programs. We need to understand communities’ feelings and 
opinions toward these programs following the recent Ebola outbreak. 
 
Study Design 
This is a cross sectional, questionnaire study.  The questionnaire will be administred to community 
leaders in 21 of the 32 DOLF MDA community survey study villages. Due to time constraints, we are 
using a purposive sample of at least two adult influential community leaders (ex. village chief, religious 
leader, teacher) and at least one community directed distributor per village. The questionnaire includes 
questions on demographics, community feelings toward MDA, and about Ebola’s effect on personal and 
community life. Several open ended questions have been included to elicit rich qualitative data. 
Reponses to the survey will be audio recorded  
 
Analysis Plan 
The main outcomes of interest from the questionnaire of community leaders in Foya are whether or not 
village leaders think their communities are ready to participate in MDA and/or continue participation in 
the community surveys. Frequency of categorical responses will be aggregated for each village and as a 
whole for all of the villages and used together with qualitative data to describe overall feelings in these 
communities toward mass treatment programs for parasitic infections in Foya. 
 
Subject Confidentiality 
Participant’s names will not be recorded in the survey. Audio recordings and other information will be 
kept confidential and only made available to project researchers. Group results from this survey will be 
reported without the names or identifying information of participants. Data stored on password 
protected laptops or paper copies will be kept with researchers and stored in locked rooms, until they 
can be saved on secure Washington University servers.  Data will not be shared with third parties 
without permission from the PI (Gary Weil).  
 
Informed Consent 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
25 
 
Informed consent 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. Researchers will read the consent 
script (with potential risks and benefits) in a locally understood language to subjects and leave a copy of 
this with each participant. The consent document will be reviewed and approved by project IRB’s prior 
to initiation of the study. Researchers will explain the purpose of the study to subjects and answer any 
questions that may arise. The subjects may withdraw consent at any time throughout the course of the 
study. Only adults 18 years or older will be included in this study. 
(See the Informed Consent Script to Be Read for Questionnaire of Community Leaders in Foya District 
included in Supplements/Appendices) 
 
 
 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
26 
 
29. SUPPLEMENTS / APPENDICES 
A. 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.  
Microfilaria testing (W. bancrofti): 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.  
Microfilaria testing (O. volvulus): Two skin biopsies will be incubated in an isotonic buffer and emerging 
MF will be detected using a microscope. 
Filarial antigen tests: Binax Now Filariasis card tests using finger prick blood (100 µL) will be performed 
in the laboratory according to the kit protocol and read at 10 minutes for W. bancrofti.  The Alere 
Filariasis Test Strip, and TropBio ELISA tests will be performed according to the manufacturer’s 
instructions for W. bancrofti .  Tests for onchocerciasis are to be determined.  
Stool examinations for STH: 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 and KK methods [10]. This 
method provides a sensitive means of detecting and quantifying helminth and Schistosoma eggs.  
Urine examinations for Schistosoma hematobium: Urine will be collected in 40 ml containers 
and preserved in formalin (final concentration 5%). The sediment from 10 ml of urine will be 
examined by microscopy.   
Specimen archive: The Laboratory will be responsible for maintenance of archived samples 
(serum, preserved stool and urine samples, etc.) collected during the DOLF project.  
QC activities: DOLF investigators will visit study sites to review procedures and results. We will also 
keep in close contact by email, telephone, and during visits by collaborating scientists from disease-
endemic countries 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.   
 
 
 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
27 
 
B. Standard Operating Procedures Summary (SOP) for Data Collection and Field Work 
Field work will be done 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, preparing blood smears, performing card serological tests, skin snips, and 
labeling specimens (barcode stickers). 
3. Field data will be entered in the field using questionnaires on Smartphones. Finger-prick blood 
and skin biopsies will be collected by physicians or trained technicians for laboratory studies. All 
samples will be labeled in the field with barcode stickers. 
4. Technicians will be responsible for transferring all blood and skin samples to the Laboratory 
immediately after field trips (same night of collection). 
5. Stool and urine samples will be collected by the field epidemiological personnel during the 
survey or on the next day. 
 
 
 
 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
28 
 
C. 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. F. Bolay (country Principal Investigator) 
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 (river blindness and lymphatic filariasis and 
intestinal worm infections) that are common in this area.  
2.  Purpose:  The purpose of this project is to assess the continued effects of mass drug administration 
on filariasis and other worm infections in Liberia. This project is being conducted by the Liberia Institute 
for Biomedical Research (LIBR) in cooperation with scientists at Washington University in St. Louis USA.    
We came to this area several years ago to do a similar study. We found that four years of the drug 
administration decreased the amount of river blindness and lymphatic filariasis in your area and we 
found out that once a year treatment is enough to reduce the infections. Now villages in your area have 
had three more years of the mass drug administration, we want to test you for the infections and find 
out if the number of infections in your area has gone down even more.  
3.  Procedures:  Your finger will be cleaned with alcohol, and a few drops of blood will be collected by 
finger prick. We will keep blood samples collected now to do other research studies of infectious 
diseases in the future. We will also palpate your pelvic region for worm nodules and take two small skin 
samples (skin snips). 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 
and skin samples. We will also collect stool and urine samples to test for other worm infections 
4.  Risks:  There are no significant risks.  Collection of blood (a few drops by finger prick) and skin (less 
than the size of a grain of rice) samples 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 and the small piece of skin being collected will not harm your health.   
5.  Number of participants and duration of the project:  Approximately 5,700 people will participate in 
the project (3,200 people from Foya district and 2,500 people from the Maryland area) per year; the 
tests will be given once in each area. 
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 your local health center for treatment. The 
project will also help the Ministry of Health by providing information on the effects of their mass drug 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
29 
 
administration program on filariasis and other worm infections in your community. The test we are 
going to do will find out if you have the infections, but we are not providing additional treatment or a 
cure. 
7.  Confidentiality:  Results of any abnormal blood, stool or urine 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. Bolay, who can be reached by telephone Tel. +2316516302 / +2316516803/ 
+231886513040 or Email:  director.libr@gmail.com  
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.  
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.  
 
 

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
30 
 
C. Informed Consent Script to Be Read for Questionnaire of Community Leaders in Foya District 
For the survey on resumption of mass treatment for parasitic infections in Foya district following the 
recent Ebola outbreak  
Invitation: Because you are considered to be a leader in this community, you are being invited to participate in a 
survey to help understand the best way to resume mass treatment for parasitic infections following the recent 
Ebola epidemic. This program was started in 2012 to measure how these treatments improve the health of people 
in your village.   
Purpose: The purpose of this questionnaire is to learn how your community feels about the mass treatment 
program that started in 2012 and their willingness to take the medicines when this public health program resumes 
in the near future. We would like to understand whether any actions or special education is needed to reassure 
your community about the purpose and value of this free drug distribution program. The project is being 
conducted by the Liberia Institute for Biomedical Research (LIBR) together with scientists at Washington University 
in Saint Louis, USA. 
Procedures: During the questionnaire you will be asked questions about yourself, your community, mass drug 
administration programs, and Ebola. Your responses will be audio recorded. The questionnaire will take between 
10-15 minutes. You may choose not to answer any question at any time.  Your name will not be collected in the 
questionnaire. 
Risks: There are no significant risks. Some of the questions may be uncomfortable or difficult for you to answer. 
You are not required to answer any question and may choose not to answer or stop the questionnaire at any time. 
Number of participants and duration of the project: Approximately 100 adults will participate in the project. We 
will talk to 4 to 5 people per village in about twenty villages in Foya district over the course of 7 days. 
Benefits: This information will benefit the Ministry of Health by providing information about how communities will 
respond to disease control programs that were interrupted by the Ebola epidemic. There is no direct benefit to 
you for participating in this survey. However by sharing your opinion you can help improve how mass drug 
administration programs operate in your community. 
Confidentiality: Your name will not be recorded as part of the survey. Audio recordings and other information will 
be kept confidential and only made available to project researchers. Group results from this survey will be 
reported and (and perhaps published) without the names or identifying information of participants 
Participation is voluntary:  Your participation in this survey is entirely voluntary. Refusal to participate will not 
penalize you in any way, and you are free to withdraw at any time.  
Consent: Your participation in this survey (answering our questions) will indicate that you have consented to join 
the survey.   
Questions:  You are free to ask any question regarding this questionnaire now. If questions arise later, please 
contact Dr. Bolay, who can be reached by telephone Tel. +2316516302 / +2316516803/ +231886513040 or Email: 
mailto: director.libr@gmail.com 
  

DOLF project protocol –Liberia 
Version 6.2 
 
09 May, 2019 
 
31 
 
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 with each participant.