Quantifying the Epidemiological Impact of Targeted Indoor Residual Spraying on Aedes-borne Diseases
Phase: NA
Conditions: Aedes-borne Diseases, Dengue, Chikungunya, Zika
Sponsor: Emory University

Study Protocol 
 
TITLE: Quantifying the Epidemiological Impact of Targeted Indoor 
Residual Spraying on Aedes-borne Diseases 
 
NCT NUMBER: NCT04343521 
 
Protocol Date: July 14, 2019 

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Quantifying the Epidemiological Impact of Targeted Indoor Residual Spraying on Aedes-
borne Diseases
Application to:
National Institute of Allergy and Infectious Diseases (NIAID)
DMID Funding Mechanism: 
U01
Principal Investigator:
Gonzalo Vazquez-Prokopec, MsC, PhD
Draft or Version Number:
June 16, 2019

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Statement of Compliance
The study will be carried out in accordance with Good Clinical Practice (GCP) as required by the 
following [use applicable regulations depending on study location and sponsor requirements; 
samples follow]:

U.S. Code of Federal Regulations applicable to clinical studies (45 CFR 46)

ICH GCP E6

Completion of Human Subjects Protection Training

NIH Clinical Terms of Award
Refer to:  http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.htm#46.
http://www.fda.gov/cder/guidance/959fnl.pdf
http://grants.nih.gov/grants/guide/notice-files/NOT-OD-01-061.html
http://cme.cancer.gov/c01/

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SIGNATURE PAGE
The signature below constitutes the approval of this protocol and the attachments, and provides 
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 applicable US federal regulations and ICH guidelines.
Site Investigator:*
Signed:
Date:
01/14/2019
Name: Pablo Manrique-Saide
Title: Professor, Autonomous University of Yucatan. 

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Statement of Compliance ...............................................................................................................i
Signature Page ..............................................................................................................................ii
List of Abbreviations......................................................................................................................iv
Protocol Summary......................................................................................................................…v
1
Key Roles .............................................................................................................................1
1.1
Background Information............................................................................................4
1.2
Scientific Rationale ...................................................................................................6
1.3
Potential Risks and Benefits .....................................................................................7
1.3.1
Potential Risks ..............................................................................................7
1.3.2
Known Potential Benefits ..............................................................................9
2
Objectives...........................................................................................................................10
3
Study Design ......................................................................................................................11
4
Study Population.................................................................................................................16
4.1
Selection of the Study Population...........................................................................16
4.2
Inclusion/Exclusion Criteria.....................................................................................17
5
STUDY PROCEDURES/EVALUATIONS...........................................................................19
5.1
Study Procedures ...................................................................................................19
5.2
Laboratory Evaluations ...........................................................................................20
5.2.1
Laboratory Evaluations/Assays...................................................................20
5.2.2
Specimen Collection, Preparation, Handling and Shipping.........................21
6
Statistical Considerations ...................................................................................................22
6.1
Study Outcome Measures ......................................................................................22
6.2
Sample Size Considerations...................................................................................23
6.3
Participant Enrollment and Follow-Up.....................................................................26
6.4
Analysis Plan ..........................................................................................................26
6.5
Trial Continuation Rules..........................................................................................29
7
Subject Confidentiality ........................................................................................................30
7.1
Future Use of Stored Specimens............................................................................30
8
Informed Consent Process .................................................................................................31
8.1
Informed Consent/Assent Process (in Case of a Minor or Others Unable to 
Consent for Themselves)........................................................................................32
9
DATA MANAGEMENT PLAN.............................................................................................33
10
Literature References .........................................................................................................36
SUPPLEMENTS/APPENDICES

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
Consent and assent Forms, including Future Use Consent

Manual of Procedures

Data Safety and Monitoring Plan (DSMP)

Forms for DSMP

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ABV
Aedes-borne virus (ABVs, Aedes-borne viruses)
AE
Adverse Event
CFR
Code of Federal Regulations
CHIKV
Chikungunya virus
CIOMS
Council for International Organizations of Medical Sciences
CRCT
Clustered randomized controlled trial
CRF
Case Report Form
DENV
Dengue virus
DMID
Division of Microbiology and Infectious Diseases, NIAID, NIH, 
DHHS
DSMB
Data and Safety Monitoring Board
FSD
Familias sin dengue (cohort study)
FWA
Federal-Wide Assurance
GCP
Good Clinical Practice
ICF
Informed Consent Form
ICH
International Conference on Harmonisation
IEC
Independent or Institutional Ethics Committee
IRB
Institutional Review Board
ISM
Independent Safety Monitor
JAMA
Journal of the American Medical Association
MOH
Ministry of Health
MOP
Manual of Procedures
N
Number (typically refers to subjects)
NIAID
National Institute of Allergy and Infectious Diseases, NIH, 
DHHS
NIH
National Institutes of Health
PI
Principal Investigator
SAE
Serious Adverse Event
SMC
Safety Monitoring Committee
SOP
TIRS
Standard Operating Procedure
Targeted Indoor Residual Spraying
WHO
World Health Organization

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Title: Quantifying the Epidemiological Impact of Targeted Indoor Residual Spraying on 
Aedes-borne Diseases
Population:
4,600 children aged 2-15 at enrollment, primarily Hispanic and of mestizo 
origin. All healthy at enrollment. 
Number of Sites:
1
Study Duration:
5 years
Subject Duration:
4.5 years (1 year baseline, up to 3.5 years of trial follow-up)
Objectives:
Our overall study objective is to quantify, via a two-arm, parallel, unblinded, CRCT, the 
efficacy of TIRS in preventing symptomatic disease caused by ABVs in children 2 to 15 
years of age at baseline from the city of Merida, Yucatan State, Mexico. 
We hypothesize that performing preemptive control with TIRS (e.g., applying insecticide 
with high residual activity before the onset of peak transmission season) will 
significantly reduce ABV burden in comparison to reactive vector control activities 
routinely implemented to control ABVs (truck-mounted ULV spraying, larviciding). Our 
proposed TIRS trial will also be highly operationally relevant in that it will quantify the impact of 
an approach with potential for rapid scale-up and implementation within existing MOH 
infrastructures.  
Primary Endpoint: Laboratory-confirmed (virologically [RT-PCR testing of acute samples] or 
serologically [IgM and IgG ELISA testing of paired acute and convalescent samples]) symptomatic 
DENV, CHIKV or ZIKV. 
Secondary endpoints: a) Laboratory-confirmed (serologically, [IgG ELISA and neutralization 
testing of annual surveillance samples]) DENV, CHIKV or ZIKV infection; b) Ae. aegypti mosquito 
infection rates with DENV, CHIKV or ZIKV (assessed by RT-PCR); c) Ae. aegypti indoor 
entomological indices (adult presence and abundance, female presence and abundance, 
bloodfed female presence and abundance). 

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Schematic of Study Design:

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1
KEY ROLES
Individuals:
DMID Representative 
Dr Gregory Deye
BG 5601FL RM 8A39, 5601 FISHERS LN
ROCKVILLE MD 20852
240-292-4199
GREGORY.DEYE@NIH.GOV
Principal Investigator:  
Gonzalo M. Vazquez-Prokopec
Emory University
400 Dowman Drive, Atlanta, GA 30322
4047274217
gmvazqu@emory.edu
Institutions (sub-award PI):
Autonomous University of Yucatan
Pablo Manrique-Saide
Department of Zoology and Director, Collaborative Unit for 
Entomological Bioassays (UCBE) 
Universidad Autonoma de Yucatan, Mexico
pablo_manrique2000@hotmail.com
Fred  Hutchinson Cancer Research Center
M. Elizabeth Halloran
1100 Fairview Ave. N, M2-C200, Seattle, Washington 98109
(206) 667-2722  
betz@fredhutch.org
University of Florida
Ira Longini 
Department of Biostatistics
College of Public Health & Health Professions College of Medicine
2004 Mowry Rd, P.O. Box 117450, Gainesville, FL 32611  
352-294-1938 
 
ilongini@ufl.edu

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Emory will be the project headquarters. Gonzalo Vazquez-Prokopec, MSc;PhD will be the 
project PI and will be in charge of overall project coordination, sub-award management 
communication with NIH and ensuring that milestones are accomplished in a timely manner. 
Lance Waller, PhD, will lead the data infrastructure and management components of the 
proposal, which will include overseeing a full-time database manager in the development of the 
REDcap database schema, setting-up forms for field data collection, overseeing data entry and 
integrity, providing data access and support to all project members, ensure data storage, access 
and sharing is HIPAA compliant. Matthew Collins, MD, PhD, will oversee all diagnostics 
components of the project, both in Merida and at Emory University. Through prior funding from 
Emory University Global Health Institute, Dr. Collins has set-up testing platforms in Merida and 
in Emory University, both capable of performing similar diagnostics (PCR, ELISA IgM, IgG). Dr. 
Collins will be responsible of diagnostics quality control by communicating periodically with 
UADY lab personnel, and by performing blind-tests of a sample of serum samples at Emory 
University to compare validity of field results. Dr. Collins’s lab is fully functional for performing all 
tests (e.g., ELISA and FRNT). Emory University’s Clinical Trials Audit and Compliance (CTAC) 
department (http://www.ctac.emory.edu/) will coordinate with the project monitor (H. Gomez-
Dantes (MD, MPH), National Institute of Public Health) all aspects concerning trial monitoring. 
Dr. Gomez-Dantes has more than 25 years’ experience working with Aedes-borne viruses, 
including the oversight of longitudinal cohort studies, is fluent in English and Spanish, and will 
visit Merida once per year to check patient records, adverse events and participant drop-out 
records and report to the project’s PI any findings. The figure below shows roles of all sub-
awardees (labeled in blue), and here we will report roles of those in leadership positions. 

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The Autonomous University of Yucatan (UADY). Will be responsible for trial implementation. 
Pablo Manrique-Saide, PhD, will coordinate all aspects of the implementation of the trial, which 
will involve communicating with the PI about progress or challenges in the achievement of the 
study milestones, coordinate all regulatory components of the project (e.g., working with local 
IRB, coordinating spraying activities with CENAPRECE, collaboration with Yucatan MOH), 
hiring field personnel, and assure all commodities are procured in time. Given the complexity 
and magnitude of tasks, Dr. Manrique-Saide will be seconded by Norma Pavia-Ruz, MD, who 
will help coordinate all field and laboratory personnel, making sure they perform their activities 
according to plan. Dr. Pavia-Ruz will be responsible for ensuring that the informed consent 
process is performed accordingly, and will evaluate records to make sure all enrolled 
participants have a corresponding consent form. Additionally, Dr. Pavia Ruz will serve as the 
project’s point of contact for any human subjects questions or matters (as an MD, she has the 
credentials to discuss with participants any concerns or issues they may have). Additionally, Dr. 
Pavia-Ruz will be responsible of talking with participants who withdraw from the study, to make 
sure accurate records for participant loss are kept. Dr. Pavia-Ruz will also communicate with Dr. 
Collins at Emory, to ensure that diagnostics are performed at the highest level. Additionally, Dr. 
Manrique-Saide will coordinate shipment of specimens and samples (serum and mosquitoes) to 
Emory University for further testing. Fred Hutchinson Cancer Research Center (FHCRC). Will 
be responsible of trial design and analysis. Elizabeth (Betz) Halloran, MD; PhD; MPH, will lead 
the trial design component of the trial, and have a significant role in the analysis and simulation 
modeling of trial data. Dr. Halloran has extensive experience designing clinical trials and 
analyzing trial data. For this study, Dr. Halloran will oversee the implementation of the variable-
constrained randomization to identify the study clusters that will be included in the trial, and 
advise on the statistical methods needed to quantitatively evaluate the primary and secondary 
endpoints. University of Florida will be responsible for trial analysis and modeling. Ira Longini, 
PhD, will lead all analysis and simulation efforts related to the trial. Dr. Longini will be joined by 
Natalie Dean, PhD, who will have a primary role in data analysis. Both Longini and Dean have 
extensive experience analyzing trial information, including a recent Ebola ring vaccination study 
[REF]. Specifically, Longini and Dean will have access to primary data from our study, and will 
use it to quantify the metrics needed for statistical inference of the primary and secondary 
endpoints. Thomas Hladish, PhD, will use the trial results (i.e., entomological and 
epidemiological efficacy) to project the impact of TIRS at the city and State levels. Dr. Hladish 
will have full access to all trial data, as he will need information on sero-prevalence, mobility and 
TIRS efficacy in order to parameterize the simulation model. Technical support of the US CDC 
(A. Lenhart (PhD, MPH)) for evaluating patterns of insecticide resistance in space and time will 
be very valuable for the study. Mexico’s Federal MOH (CENAPRECE) will contribute with 
personnel for spraying and grant access to project members to their online ABD database. The 
Yucatan State MOH will provide access to samples for laboratory testing in support of the trial’s 
enhanced surveillance procedures, as well as help with communication about TIRS and the 
trial’s goals.

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1.1
Background Information 
Aedes-borne viruses (ABVs; e.g., dengue [DENV], chikungunya [CHIKV], Zika [ZIKV]) pose a 
major public health burden worldwide[1, 2, 3]. Transmitted primarily by the highly anthropophilic 
mosquito, Aedes aegypti, ABVs propagate epidemically, inflicting dramatic healthcare and 
development costs on urban tropical environments. Model projections estimate an average of 390 
million DENV infections occur per year, of which 96 million manifest clinically[4, 5]. Explosive 
DENV outbreaks saturate healthcare systems [6], with worldwide estimates as high as $39 billion 
(2010 US$) per year spent on the costs of medical care, surveillance, vector control, and lost 
productivity[7]. The emergence and rapid epidemic propagation of CHIKV and ZIKV (and 
particularly congenital Zika) have added significant burden and costs to healthcare systems[8, 9]. 
Given the heavy global burden of ABV illness, and in the absence of efficacious vaccines or other 
therapeutic options, the implementation of highly impactful vector control represents the only 
viable approach currently available for effective prevention[10, 11].  
In most of the world, vector control methods such as larval control, source reduction and space 
spraying are widely used against ABVs[12, 13]. Unfortunately, there is limited epidemiological 
evidence that these existing methods are adequate to prevent or contain ABV transmission 
in a sustainable manner[13, 14]. Poorly designed evaluations, a historical lack of focus on 
quantifying intervention impact using epidemiological endpoints, and limited funding for large-
scale randomized controlled trials with epidemiological endpoints have all contributed to the lack 
of rigorous, evidence-based assessments of ABV vector 
control interventions[10, 15]. Furthermore, the classic 
deployment of house-based interventions in response to 
clinical ABV cases has failed to account for the important 
contribution of out-of-home human exposure to Ae. aegypti 
and to onward dengue transmission[16] and the silent 
contribution of asymptomatic infections[17]. Novel vector 
control 
approaches 
and 
intervention 
delivery 
strategies with proven and robust epidemiological 
evidence of their impact on ABVs are urgently needed.
Indoor Residual Spraying (IRS) is the use of long-lasting 
residual insecticides applied to the walls, eaves, and 
ceilings of houses or structures targeting vectors that land 
or rest on these surfaces[18, 19, 20]. The residual 
component of the application means that, for several weeks 
or months, the insecticide will kill mosquitoes and other 
insects that come into contact with treated surfaces. IRS 
does not directly prevent people from being bitten by 
mosquitoes but it reduces overall mosquito density by 
killing mosquitoes contacting treated surfaces. IRS is used 
to control malaria, Chagas and leishmaniasis[19, 20, 21]. 
Historical 
evidence 
has 
shown 
that, 
when 
expeditiously implemented, residual insecticide 
applications can significantly reduce ABVs. The 
elimination of Ae. aegypti from the Mediterranean region 
was attributed to IRS campaigns that were conducted for 
malaria eradication[21], and IRS either alone [22], or in combination with larval control[23], 
Fig 1. TIRS application on walls (above; 
adapted from WHO 2015) and an applicator 
in Merida, Mexico, performing TIRS (below). 

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contributed to the elimination of Ae. aegypti and DENV from (British) Guyana and the Cayman 
Islands, respectively. Despite this evidence, the fact that it is time consuming and dependent on 
specialized human resources has limited IRS widespread adoption by control programs due to 
the perceived challenge of scaling-up the intervention over large urban areas. Work primarily led 
by our team has contributed to a shift in the conceptualization and implementation of IRS to control 
Ae. aegypti in urban areas (reviewed by[14, 24]). Our approach of adapting IRS to the urban 
context of Ae. aegypti control was based on three components: a) modifying insecticide 
application sites to account for Ae. aegypti indoor resting behavior; b) piloting novel 
insecticide formulations that have high residual activity and to which Ae. aegypti are 
susceptible; c) changing the intervention delivery from reactive (after detection of 
symptomatic cases) to preemptive (prior to peak ABV transmission season). 
In urban settings, adult Ae. aegypti typically rest indoors, where they feed frequently and almost 
exclusively on human blood[25]. Studies performed in Panama and our work in Peru and Mexico 
have shown that Ae. aegypti rest predominantly below heights of 1.5 m, mainly inside bedrooms 
and on surfaces made of cement, wood and cloth[26, 27, 28]. Our studies performed in 
experimental houses in Merida, Mexico, show that 
selectively applying residual insecticides below 1.5 m 
and on common mosquito resting surfaces provides an 
entomological impact  similar to spraying entire walls 
(as performed in classic IRS), but in a fraction of the 
time (<18%) and insecticide volume (<30%) compared 
to classic IRS [29]. We call this selective insecticide 
application 
mode 
‘targeted 
indoor 
residual 
spraying’ (TIRS), and it includes the application of 
residual insecticides on exposed low walls [<1.5m], 
under furniture, and on dark surfaces of all house 
quarters with the exception of the kitchen; Fig. 1. As 
such, TIRS can be considered as a rational vector 
control approach that exploits Ae. aegypti resting 
behavior to focalize insecticide applications, thus 
reducing unnecessary exposure to chemicals in 
both 
applicators 
and 
household 
residents and also reducing the time it 
takes to spray a premise with no 
apparent 
loss 
in 
insecticidal 
efficacy[29]. A typical house in Merida, 
Mexico has a floorplan area of ~70 m2 
and can be sprayed with TIRS in an 
average of 9.35 min (± 0.124 min SEM; 
n = 571 houses)[29]. In Cairns, 
Australia, 
we 
performed 
an 
observational study that found that 
TIRS can reduce the probability of 
future DENV transmission by 86-96% 
as 
compared 
to 
unsprayed 
premises[30] (Fig. 2). In that study, 
which analyzed data from enhanced 
surveillance and g eocoded insecticide 
Fig 2. Epidemiological impact of TIRS on dengue in 
Cairns, Australia (Vazquez-Prokopec et al. 2017). 
Fig 3. Entomological impact of TIRS using pirimiphos-methyl 
(Actellic 300CS) applied prior to the transmission season 
(Dunbar et al. in prep.). 

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applications, the remarkable protection provided by TIRS led to the containment of a dengue 
outbreak[30], providing significant cost-savings to the local Ministry of Health (MOH)[11]. 
Furthermore, concurrent trap collections of Ae. aegypti in the heart of the outbreak showed that 
TIRS was associated with a ~70% reduction in female mosquito abundance[31].  
As Ae. aegypti resistance to pyrethroid insecti cides is reported with increasing 
frequency[32, 33], it is crucial to select a chemical that maximizes insecticide efficacy while 
limiting detrimental effects to the environment or human health[34]. In Merida, Mexico, we 
performed a Phase II cluster randomized controlled trial (CRCT) to evaluate the entomological 
impact of IRS with bendiocarb (Ficam®, BAYER, a carbamate insecticide to which local Ae. 
aegypti are fully susceptible) and reported reductions in indoor adult Ae. aegypti abundance up 
to 70% over a 3-month period, compared to no reduction when the pyrethroid deltamethrin was 
used[34]. In further evaluations we showed that bendiocarb can yield up to 5 months of 
control[29]. Our most recent Phase II entomological CRCT (using the same design as in[34]) has 
estimated the impact of TIRS with the organophosphate pirimiphos-methyl (Actellic 300CS®, 
Syngenta) applied 1 month prior to the beginning of the ABV transmission season and evaluated 
over 8 months (Fig. 3). Actellic provided a statistically significant reduction in Ae. aegypti female 
and bloodfed female abundance for up to 7 months post-spraying, covering the entire peak 
transmission season (Fig. 3). Average entomologic reduction in Ae. aegypti female abundance 
(compared to control clusters) across the transmission season was 74%, similar to previous TIRS 
efficacy estimates from Australia[31] and Mexico[34]. Our Phase II CRCTs and experimental 
studies indicate that a ~10-14 min application can reduce Ae. aegypti indoors by ~70% for 
up to 5-7 months, creating the possibility for preemptive vector control (Fig 3).
In Yucatan, as is often observed in tropical settings, ABV transmission is seasonal, with 
~84% of DENV cases reported in 1995-2015 occurring between July 1 and Dec 31 (Fig 3 
and[35]). Using a stochastic simulation model fitted to Yucatan State, Mexico, we found that high 
levels of TIRS coverage (75% houses treated once per year) applied preemptively before the 
typical dengue season (before July) could reduce DENV infections by 89.7% (median of 1,000 
simulations; interquartile range [IQR]:[83.0%, 94.8%]) in year one and 78.2% (IQR: [71.2%, 
88.0%]) cumulatively over the first five years of an annual program [35]. Effectiveness can be 
increased and intervention start date pushed earlier if using an insecticide with longer residual 
power (~150 days, equivalent to Actellic® 300CS) than bendiocarb (~90 days)[35]. As such, our 
theoretical exercise shows that conducting preemptive TIRS will provide high short-term and 
long-term effectiveness in preventing ABVs in areas where transmission is seasonal. 
1.2
Scientific Rationale 
The only epidemiologic evidence of TIRS effectiveness comes from our work in Australia[30, 34], 
which challenges the applicability of such findings to highly endemic tropical settings. 
Furthermore, there is no randomized evidence quantifying the impact of TIRS on clinical 
disease. For TIRS to obtain a full WHO policy recommendation, information about the 
intervention’s public health impact derived from well-designed clinical trials must be available[15]. 
As such, there is a need for rigorously implemented clinical trials evaluating TIRS within 
ABV endemic settings[10, 13]. Such trials will contribute to the body of evidence for the use of 
this intervention to prevent ABVs. 

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1.3
Potential Risks and Benefits
1.3.1 Potential Risks
Overall, the risks to study participants are minimal in all of our study procedures (summarized in 
Table 1). The most sensitive risk is related to potential intoxication with the insecticides used in 
TIRS. 
Both Actellic 300CS® (Syngenta) and Ficam® (Bayer) have been approved by the World Health 
Organization (WHO) for indoor control of mosquitoes18,19. WHO’s hazard assessments 
concluded that, when used for indoor residual spraying as instructed and at the recommended 
doses, both bendiocarb and pirimiphos-methyl 300 CS do not pose undue hazards to the spray 
operators or residents of the treated dwellings[36, 37]. Provided that operational guidelines are 
followed, routine cholinesterase monitoring of spraymen during indoor residual spraying 
programs is not required[36, 37]. 
Both pirimiphos-methyl and bendiocarb insecticides, are reversible inhibitors of cholinesterase, 
an essential nervous system enzyme. Pirimiphos-methyl can cause cholinesterase inhibition in 
humans; that is, it can overstimulate the nervous system causing nausea, dizziness, confusion. 
Very high exposures (e.g., accidents or major spills) to pirimiphos-methyl can produce 
respiratory paralysis & death[38]. Organophosphate vapors rapidly produce mucous membrane 
and upper airway irritation and bronchospasm, followed by systemic muscarinic, nicotinic and 
central effects if exposed to significant concentrations[38]. Symptoms of bendiocarb poisoning 
include weakness, blurred vision, headache, nausea, abdominal cramps, chest discomfort, 
constriction of pupils, sweating, muscle tremors, and decreased pulse[39]. If there is severe 
poisoning (e.g., accidents or major spills), symptoms of twitching, giddiness, confusion, muscle 
incoordination, slurred speech, low blood pressure, heart irregularities, and loss of reflexes may 
also be experienced. Death can result from discontinued breathing, paralysis of muscles of the 
respiratory system, intense constriction of the openings of the lung, or all three [39]. 
Carbamates generally do not accumulate in mammalian tissue and the cholinesterase inhibition 
reverses rapidly once exposure ceases. Complete recovery from an acute poisoning by 
bendiocarb, with no long term health effects, is possible if exposure ceases and the patient has 
time to recover their normal level of cholinesterase before succumbing to symptoms. In non-
fatal cases, the illness usually lasts less than 24 hours. Bendiocarb vapors rapidly produce 
mucous membrane and upper airway irritation and bronchospasm, followed by systemic 
muscarinic, nicotinic, and central effects if exposed to significant concentrations [39].
Our project will carefully monitor potential exposure to chemicals on our study population. In our 
Phase II entomological trial utilizing Actellic 300CS, we performed individual acceptability 
surveys in 160 houses, including 630 individuals (Vazquez-Prokopec et al. unpublished). Our 
survey detected 19 cases (3%) of symptoms compatible with a reaction to the insecticide. The 
most common signs (accounting for 85% of symptoms) were headache, nausea and skin 
irritation. All of the symptoms reversed within 1-2 days without the need for medical intervention. 
Our study will treat any signs of intoxication as adverse events and will report them accordingly 
(see DSMP section). As for insecticide applicators, we will follow WHO recommended practices, 
which include wearing personal protection equipment and making insecticide dilutions using 
pre-filled sachets with the right amount for each machine charge (see manual of procedures for 
detailed steps).  

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Agricultural use of bendiocarb and pirimiphos-methyl have been associated with risk of 
environmental contamination[38, 39], as both insecticides are soluble in water and toxic to birds, 
fish and arthropods. The doses and mode of application in indoor residual spraying are not seen 
as a source of environmental contamination by WHO, due to difficulty in having insecticides 
runoff into surface waters. There is a risk of environmental contamination if water used for 
washing spraying equipment is not properly handled. We will follow Mexico’s MOH strict 
procedures for insecticide preparation, equipment cleaning and insecticide disposal 
(http://www.cenaprece.salud.gob.mx/programas/interior/vectores/descargas/pdf/guia_rociado_r
esidual_intradomiciliar.pdf).  
Risks associated with venipuncture include occasional bruising and a slight risk of infection at 
the site of the venipuncture and, in a small number of participants, dizziness associated with a 
vasovagal response. We do not anticipate any risks associated with our active surveillance. The 
information from this study is not expected to lead to any negative ramifications such as legal or 
employment risks, nor is there is a possibility of physical, psychological, social or legal injury 
from participation in this study and standard measures to protect confidentiality should be 
sufficient. Movement surveys collect detailed information about specific whereabouts of 
individuals prior to infection (Retrospective Movement Survey) or when healthy (Prospective 
Movement Survey), but their sensitivity and risk are also minimal. 
Study Component
Risks
Intoxication due to 
unintended 
exposure to 
insecticides.
Direct (contact) or indirect (inhalation of fumes) intoxications are rare but likely.
Bendiocarb: Symptoms of poisoning include excessive sweating, headache, 
chest tightness, giddiness, nausea, vomiting, stomach pains, salivation, blurred 
vision, slurred speech and muscle twitching.
Pirimiphos-methyl: can cause cholinesterase inhibition in humans; that is, it can 
overstimulate the nervous system causing nausea, dizziness, confusion.
 
Febrile surveillance
Longitudinal cohort
Pain or discomfort, bruising, or infection at venipuncture site or temporary 
dizziness during blood draw.
Use of identifiable information (demographic information, address, febrile status)
DENV+, CHIKV+, 
ZIKV+ participants 
Same as for febrile surveillance and longitudinal cohort
The data gathered in this project will be identifiable and certain data types, such 
as movement interview are sensitive. 
The primary risks lie with identifying the individuals who provided information 
they consider confidential (e.g., movement to private locations).
There is a small risk that the repeated blood collections will cause or exacerbate 
anemia. 
In-depth interviews 
(prospective and 
retrospective 
movement 
interviews)
Risks to study participants are minimal.  Participants may feel that in-depth 
interviews take up too much time – but they have the option of ending their 
participation at any time.  There are no sensitive topics covered, but if any 
participant feels that there is something he/she does not want to talk about, 
he/she does not need to answer all questions.
Table 1. Potential risks associated with specific components of our study. 

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1.3.2 Known Potential Benefits
Our experience with Phase II trials of TIRS has shown that households receiving the 
intervention see a measurable reduction in the number of Ae. aegypti as well as other 
nuisance insects. For instance, Culex quinquefasciatus mosquitoes are highly abundant 
indoors, and householders report being bitten significantly less at night (when this vector is 
active). Other perceived benefits of the intervention include the fact that cockroaches, spiders 
and scorpions are also killed by residual insecticides. Householders report seeing dead insects 
as a sign of the quality and efficacy of the insecticides applied. In fact, a survey performed on 
160 households receiving TIRS with Actellic 300CS reported that 96% of house owners would 
recommend the intervention to others or would receive it again, if provided (Vazquez-Prokopec 
et al. unpublished).  
For viremic participants captured in active surveillance, the clinical laboratory testing and 
medical consultation will allow detecting severe cases that may require hospitalization. A 
common problem in Merida, like other resource-poor environments, is that residents hesitate to 
seek medical help because of economic constraints or fear of hospitals, and allow their disease 
to progress to a stage where clinical intervention is unsuccessful.  Our staff can reassure and 
advise participants to seek care, help navigate the hospital admission process, and provide 
evidence of dengue infection to local physicians. In practice, a definitive diagnosis of dengue 
infection may be useful to aid local physicians in justifying a more aggressive clinical approach 
for dengue-infected patients. Our enhanced surveillance, which will test samples that otherwise 
do not get tested at the state laboratories, will provide benefits to the public health care system, 
as it will enhance quantifications of magnitude of disease throughout the transmission season.
Entomological surveys alert households to Ae. aegypti production sites and our staff can explain 
how production sites can be removed. We also typically remove a significant portion of the adult 
female mosquitoes residing in the household in our routine collections. Although this is a 
temporary measure, it can provide some relief from biting insects. 

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2
OBJECTIVES
Our overall study objective is to quantify, via a two-arm, parallel, unblinded, CRCT, the 
efficacy of TIRS in preventing symptomatic disease caused by ABVs in children 2 to 15 
years of age at baseline from the city of Merida, Yucatan State, Mexico. We hypothesize 
that performing preemptive control with TIRS (e.g., applying insecticide with high 
residual activity before the onset of peak transmission season) will significantly reduce 
ABV burden in comparison to reactive vector control activities routinely implemented to 
control ABVs (truck-mounted ULV spraying, larviciding). Our proposed TIRS trial will also 
be highly operationally relevant in that it will quantify the impact of an approach with potential for 
rapid scale-up and implementation within existing MOH infrastructures.  

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3
STUDY DESIGN
We will pursue a two-arm CRCT with a total of 50 clusters of 5x5 city blocks each, including 
at least 92 enrolled age-eligible participants per cluster, with 25 clusters randomly 
allocated to receive TIRS and 25 clusters not receiving TIRS (Fig 5) (see section 4.4 in the 
Clinical Trial Protocol). A total of 4,600 age-eligible individuals will be recruited for the trial. 
This sample size accounts for a 20% loss to follow-up. Routine MOH vector control actions 
(primarily, truck-mounted ULV spraying of chlorpyriphos, larviciding with altosid® and indoor 
space spraying in premises of suspected ABV cases) performed in response to symptomatic ABV 
cases reported to the healthcare system will not be interrupted and could occur across both study 
arms. Therefore, our trial will compare TIRS to routine ABV prevention and control, 
providing an opportunity to quantify intervention efficacy and potential benefits in 
comparison to existing practice. Clusters will be located within the areas identified as hot-spots 
of ABV transmission[40] (Fig. 5). Placing all clusters within the zone of high ABV incidence 
will increase power by focusing on an area with higher event rates and lead to a more 
homogeneous design (e.g., less 
likely to have imbalance across 
trial arms). We will use a covariate-
constrained 
randomization 
strategy[41] to randomly select 50 
clusters from the set of census 
tracts in the hot-spot region. This 
approach ensures that key cluster-
level variables (e.g., historical ABV 
transmission, population size and 
density, % employed  population) 
are not more than 10% different 
between trial arms and maximizes 
the distance between neighboring 
clusters. 
To 
further 
reduce 
contamination and edge effects, 
while all households in TIRS 
clusters will be offered the 
intervention, epidemiological and 
entomological 
evaluation 
will 
occur in the center of each 
cluster, following a “fried egg” 
design (blue blocks in inset of 
Fig. 5). Enrollment into the trial will 
occur in all clusters before TIRS 
allocation has been determined, in 
order to prevent selection bias.  
The 
steps 
involved 
in 
the 
epidemiological evaluation of TIRS, in order, are (Fig 5): a) random selection of 50 clusters into 
two groups (TIRS allocation not yet determined); b) participant enrollment and consent/assent to 
participate in the trial; c) baseline ABV serological and entomological surveys immediately 
following participant enrollment; d) random allocation of one group to TIRS; e) implementation of 
Fig 5. Proposed trial design. City blocks in yellow show the size of 
clusters within census areas, and blue blocks show the area where 
epidemiological and entomological evaluations will be concentrated 
(“fried egg” design).  

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TIRS intervention; f) active case detection and monthly entomological collections post-
intervention and for a period of 6 months after spraying; g) a post-season serological survey in all 
enrolled children. Steps e), f), and g) will be performed for two transmission seasons, with the 
potential to add a third season should incidence of the primary endpoint be lower than assumed 
(Fig 5). Each step will be explained in the sub-sections below. See table below for a timeline. 
Community Involvement: Engaging communities early in the trial will be essential for maximizing 
participant acceptance and retention[42, 43]. We will continue involving our experienced team of 
10 social scientists and social workers, who interact directly with study participants (through 
informal conversations, games and other educational activities with children) to ensure they 
remain engaged throughout the duration of the study [43]. Other factors previously highlighted as 
important by the communities are: a) the involvement of MOH personnel in spraying, rather than 
personnel specifically contracted for the trial; b) the availability of physicians who will provide 
direct care to enrolled children and their families, including medical diagnoses and results of 
laboratory tests; c) the participation of the local university, the Autonomous University of Yucatan.  
3.2.8. Baseline study: Following participant enrollment, we will conduct a baseline epidemiological 
and entomological assessment (preceding the first TIRS application) to assess household 
characteristics (size, building materials, number of rooms, number of inhabitants), quantify levels 
of ABV baseline seroprevalence in enrolled children, and measure Ae. aegypti infestation and 
susceptibility to insecticides (Fig 6). All enrolled children will provide a blood sample by 
venipuncture, which will be tested for the presence of antibodies against DENV, CHIKV or ZIKV. 
A proportion (10%) of all houses located in the center of the cluster (blue  blocks in Fig 5, equal 
to 1,350 houses across 50 clusters) will be visited monthly to collect mosquitoes indoors and 
outdoors, which will be used to quantify patterns of Ae. aegypti abundance and collect eggs that 
can be reared for assays to detect insecticide resistance. 
Intervention: Personnel from CENAPRECE with experience performing IRS will conduct the 
spraying after being trained using our study procedures. Based on our model predictions[35], 
spraying should start May-June and extend for 1-2 months. Ae. aegypti in Merida and Yucatan 
are susceptible to both carbamates and organophosphates [32]. We will prioritize the use of 
Actellic 300CS®, given its longer residual power in comparison to Ficam® (7-8 months versus 
4-5 months, respectively). Insecticide application will follow strict SOPs developed by our team 
as part of a previous CDC-Emory-UADY collaboration (see manual of procedures). Residents will 
be asked to temporarily leave the house during treatment and wait 0.5-1 h for the product to dry 
before re-entering the house. Staff will wear branded uniforms with identification and use 
appropriate personal protective equipment (Fig 1). 
Intervention evaluation. The epidemiological impact of TIRS on the primary endpoint will be 
evaluated by active (enhanced) surveillance to detect and lab-confirm symptomatic DENV, CHIKV 
or ZIKV from July 1st to December 31st of each season (the period of peak ABV transmission, Fig 
3, and right after TIRS application) (see timeline). As ABV transmission in Merida is highly 
seasonal, and a single TIRS application covers most of the peak transmission season (Fig 3), 
focusing active surveillance during a 6-month period after spraying will be more efficient and cost-
effective to quantify the epidemiological impact of TIRS than spraying twice and maintaining active 
surveillance during the period of low transmission. Epidemiological impact will be further assessed 
in a secondary endpoint using yearly serosurveys performed after the peak transmission season 
(January-April) to serologically detect interval ABV infections. Entomological impact will be 
measured by standardized collections of adult and immature Ae. aegypti in a sample of houses 
from each cluster during July 1st-December 31st, coinciding with active surveillance. 

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Enhanced ABV symptomatic case detection for the primary endpoint will rely on three 
sources: 1) community-based active surveillance based on wellness visits (1x/week) or regular 
phone calls (2x/week) by nurses; 2) self-reported illness by study participants to a toll-free 01-800 
number; 3) State of Yucatan passive surveillance of ABV cases (investigators will mine these data 
to capture symptomatic ABV cases not identified from source 1 or 2 above). Ten field teams 
consisting of a nurse and a social worker or anthropologist each will conduct wellness 
visits to all enrolled children once per week, with the goal of identifying any probable ABV 
case. In addition to wellness visits, nurses will call parent/guardians of enrolled children regularly 
(twice per week) to check for the occurrence of any ABV symptoms. When interacting with 
participant mothers, nurses will also remind them that they can call our toll-free 0-1800 number in 
case of any illness compatible with an ABV infection. Widely used in our FSD cohort, the 01-
800 hotline enhances the detection of symptomatic individuals  by providing study 
participants 24-7 access to a toll-free phone number to consult an ‘on call’ physician about 
any symptom in their families [44]. For instance, between weeks 30-50 of 2015, 373 telephone 
calls were received, with 32.5% of them not related to a febrile episode suggestive of dengue. 
Around 40% (n=149) of febrile patients that contacted the dengue line had previously consulted 
a physician who gave the diagnosis of probable dengue. People then requested that our study 
team draw a blood sample for confirmation of clinical diagnosis. Additionally, our project will 
partner with CENAPRECE and the State Laboratory (see letters of collaboration) to 
enhance the detection and confirmation of symptomatic ABV cases from our cohort 
reporting to the healthcare system. Access to the online ABV database managed by 
CENAPRECE [45] will allow our team to visualize the location of all suspected cases residing 
within our study clusters in real-time. By mapping the geographic location of cases, we will 
be able to identify if enrolled households had cases not detected by our active 
surveillance. CENAPRECE database will also be used to map regular vector control 
actions performed by the MOH. 
A suspected ABV symptomatic case will be a participant with acute onset of fever (axillary 
temperature ≥38°C) OR a non-focal rash PLUS any one supporting symptom such as 
headache, conjunctivitis, arthralgia or myalgia). When a suspected ABV case meeting the 
case definition is identified through active surveillance, they will be visited preferably on the same 
day by one of our four project physicians to perform a physical examination (physical exam, 
temperature, vital signs). The doctor will be joined by one field team, which will obtain 
demographic, behavioral data, and collect blood specimens. Acute and convalescent (preferably 
14-21-days later, but up to 28 days) blood specimens will be collected on each suspected case 
to confirm ABV infection. We will follow detailed protocols for ascertaining each case (see section 
3.3.5 below). Additional information collected from each suspected ABV case at the first visit will 
include: demographic information (age, sex) and history of movement (by a retrospective 
movement survey). Once laboratory confirmation is obtained, participants will meet with our study 
physicians, who will explain the diagnosis and advise on potential steps if symptoms worsen. 
Yearly blood samples from all enrolled participants will be collected after the regular 
transmission season (from January to April) to test for serologic evidence of interval 
infection by DENV, CHIKV or ZIKV. We will follow the same procedures for contacting 
participants and obtaining blood samples as reported in our FSD cohort study [44, 46, 47], with 
the modification that, in addition to blood specimens, our team will perform prospective movement 
surveys to characterize the routine mobility patterns of participants.   
 

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The assessment of the entomological impact of TIRS will rely on monthly household-level 
information characterizing adult Ae aegypti abundance and ABV infection. Briefly, a random 
sample of 10% of the houses located in the center (‘yolk’ of our fried-egg design) of each cluster 
(~1,350 houses in total) will be visited and surveyed for the presence of adult Ae. aegypti 
mosquitoes indoors as described in[34, 48]. Female Ae. aegypti collected indoors will be pooled 
by city block and tested for the presence of DENV, CHIKV or ZIKV infection. Entomological 
surveys will begin right after TIRS spraying (July 1st), and will be performed monthly for six months 
(until Dec 31st). Monthly WHO cone bioassays[34, 49] will be done in a random sample of 20 
treated houses and 5 untreated houses (controls) to characterize the residual efficacy of the 
insecticide used. 
Timeline
Our trial timeline (Table belos) will extend for five years and consist of: a) random selection of 
50 clusters into two groups (TIRS allocation not yet determined); b) participant enrollment and 
consent/assent to participate in the trial; c) baseline ABV serological and entomological surveys 
immediately following participant enrollment; d) random allocation of one group to TIRS; e) 
implementation of TIRS intervention; f) active case detection and monthly entomological 
collections post-intervention and for a period of 6 months after spraying; g) a post-season 
serological survey in all enrolled children. Steps e), f), and g) will be performed for two 
transmission seasons, with the potential to add a third season should incidence of the primary 
endpoint be lower than assumed. Final analysis for quantifying primary and secondary 
endpoints will occur after the second transmission season ends (Q3 of year 4) and continue 
throughout year 5 of the project. 
Activity by Quarter
Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4
Establish sub-awards, IRB approvals, 
Database fully operational.
Finalize Trial Design
Project Annual meeting
Participant Enrollment
Baseline Serology and Entomology
TIRS implementation
Active Surveillance
Annual sero-survey
Trial Monitoring
Trial Analysis
Trial Projection/simulation
Publication
RPPR
Colors in each quarter pinpoints to the period of ABD transmission (blue=low transmission; red = high transmission).                                
Cells in dark gray indicate activities that will be performed if number of cases is low and a new evaluation season is needed. 
2020
2021
2022
2023
2024
Table. Detailed timeline for milestones of the project. Red indicates quarters of high ABD transmission, whereas blue 
indicates quarters of low ABD transmission.
The first two quarters will be dedicated to establish the infrastructure and regulatory 
components of the trial. Emory University will lead all administrative components, which will 
include centralizing IRB protocols and approvals (in Mexico), establish sub-contracts with UFL, 
UADY and FHCRC; purchase insecticides, reagents and purchase travel for project members, 
coordinate with CENAPRECE all components of the spraying. Additionally, Emory University will 
establish the database schema and infrastructure, and provide database access to all project 

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members. Within the first two quarters we will produce our final trial design by refining our 
variable constrained randomization and making sure all clusters are eligible (for instance, they 
do not occur in areas that are unsafe to our personnel). Once clusters are identified, participant 
enrollment will begin. We estimate it will take us approximately 4-6 months to enroll the 4,600 
children in the cohort. Right as enrollment occurs, and after the ABV transmission season (after 
December), we will conduct baseline serological and entomological survey to assess the natural 
immunity to ABVs in the study population. TIRS will be implemented in June-July of years 2 and 
3 of the project (and year 4 if a third season is needed). Epidemiological and entomological 
monitoring of the trial will occur annually. After the regular transmission season we will conduct 
yearly blood draws to quantify seroconversions to ABVs. Once a year (right after the ABV 
transmission season) the trial monitor will travel to Merida to evaluate progress and identify any 
potential issues. Analysis and modeling of trial data will begin after the second transmission 
season (year 4 of the project). We will have in-person project meetings once per year in Merida 
and virtual meetings (using Emory’s site-license for the Zoom videoconferencing system) once 
per month. 

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4
Study Population
4.1
Selection of the Study Population
Our trial will focus on the pediatric population. 
Specifically, children 2-15 years of age at the time of 
enrollment living within the assigned clusters in the 
city of Merida, Yucatan, Mexico. Briefly, the FSD cohort 
indicated that the majority of dengue naïve infections and 
seroconversions were observed in children <15 years old 
47,49,50 (Fig 6) and, while they had lower symptomatic 
illness than adults, the chances of detecting both 
infection and disease is expected to be higher than older 
age groups due to their lower natural immunity to ABVs. 
CHIKV and ZIKV  incidence during the first wave of their 
invasion into the Yucatan was also larger  in the <15 year-
old group (Table 2), supporting our age-group eligibility 
criteria. We will exclude younger children (<2 years) 
because of the inherent difficulties obtaining blood from 
small children, and potential for cross-reactivity of maternal 
antibodies63. 
Table 2. Incidence rates (IR) per 1,000 person-years of arbovirus confirmed symptomatic infections in the cohort in 
Yucatan, Mexico by age, gender. Extracted from Rojas et al. 201852.
There will be no exclusion criteria related to gender, ethnic or racial category. While females 
had slightly higher rates of ABV incidence than males (Table 2), our trial will enroll both male 
and female children. The health status of this population at enrollment will be mostly healthy. 
Based on our FSD cohort studies, we expect to enroll 50% females, and Mexican citizens, 
Spanish speakers, and of mestizo ethnicity (mixed Mayan and white/European). The health 
status of this population at enrollment will be mostly healthy. 
While our study will include participants of both sexes, our analysis plan will not use such 
information to assess the primary or secondary endpoints and quantify the epidemiologic impact 
of TIRS. We will recruit 4,600 children aged 2-15. Our team has vast experience working with 
children as part of our FSD cohort study 47,49,50. Our team of four physicians will include an 
Fig. 6. Age-specific baseline dengue 
seroprevalence in Merida cohort, 
Yucatan, Mexico. From [Rojas et al. 

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infectious disease pediatrician (Adolfo Palmachan, MD), who has extensive experience (~30 
years) diagnosing infections locally circulating in Merida, including ABVs. Table 3 shows the 
sample size and procedures that will be followed for each segment of the population.  
Study Component
Target N 
(expected 
or planned)
Activities
Febrile Surveillance 
4,600
Census, household visits 1x/wk, phone calls 2x/wk. for 
two transmission seasons (and potentially 3).
Probable acute ABV illness. 
(From active surveillance)
1,000
2 blood samples (acute and convalescent, 14-30 d after 
acute) to all cases meeting the ABV case definition. 
Demographic and movement interviews at first visit. 
Serosurvey
4,600
Provide a blood sample 1/yr for two years (and 
potentially 3) to quantify ABV exposure.  
Loss to follow-up interviews
920
Interview participants leaving the study to assess 
potential causes. 
Table 3. Target sample size of children aged 2-15 at the time of enrollment, and activities performed on them. 
 
4.2
Inclusion/Exclusion Criteria
There will be two levels of participation in the trial: 1) at the household level, owners will consent 
to have their house sprayed with TIRS; 2) at the individual level, children 2-15 years of age at the 
time of enrollment will consent/assent to participate in the study, which will require their 
prospective follow-up for signs of ABD illness and yearly blood draws to assess seroconversion. 
The inclusion/exclusion criteria for each level is as follows:
1. Household Level
1.1. Inclusion Criteria:
- Household is located within the bounds of a study cluster (5x5 city-block clusters). 
- City block has at least 60% premises that are residential.
1.2. Exclusion Criteria:
- Households where study personnel identify a security risk (i.e., site where drugs are sold, 
residents are always drunk or hostile)
- Sites where no residents spend time during the day (i.e. work 7d a week outside the 
home).Inability for a resident to provide informed consent.
- Non-residential places (e.g., businesses, schools, markets, etc.).
2. Individual Level
2.1. Inclusion Criteria:
- 2 or more and up to 15 years of age at the time of initial enrollment
- Living in a house that consented to TIRS. 
2.2. Exclusion Criteria:
- Less than 2 years of age or more than 15 years of age at the time of enrollment.
- Not living in a house that consented to TIRS. 

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- Having a medical condition that prevents implementation of study procedures. 
- Temporary visitor to household
- Plans to leave study area within next 12 months

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5
STUDY PROCEDURES/EVALUATIONS
5.1
Study Procedures 
See manual of procedures attachment for detailed procedures and forms for each 
component.
Intervention: Personnel from CENAPRECE with experience performing indoor residual spraying 
and trained to apply TIRS will conduct the spraying which --based on our model predictions-- 
should start between May 1st and June 1st and extend for 1.0-1.5 months. Ae. aegypti in 
Merida and Yucatan are susceptible to both carbamates and organophosphates. Either Ficam® 
(Bayer, a carbamate) or Actellic 300CS® (Syngenta, an organophosphate) will be used on a 
given year to treat all houses. We will prioritize the use of Actellic 300CS®, given its longer 
residual power in comparison to Ficam® (7-8 months versus 4-5 months, respectively). 
However, if insecticide resistance profiles of mosquitoes at baseline or after the first year of 
spraying show decreases in susceptibility to Actellic 300CS®, we will switch to Ficam®. 
Insecticide application will follow strict SOPs developed by our team as part of a CDC-Emory-
UADY collaboration (see Manual of Procedures). Each spraying team will include 2 spraymen 
and one supervisor in charge of communicating with the household owners and evaluating the 
quality of spraying. Team leaders will ask the homeowner if there are areas or rooms they do 
not wish to have treated (and take note in the field spraying form). Residents will be asked to 
temporally leave the room during treatment and wait at least 30 minutes for the product to dry 
before re-entering the home. We will avoid treating near pets, especially near fish tanks, and do 
not spray children’s toys, food, kitchens or exposed cloths and bedding. Staff will wear branded 
uniforms with identification at all times and use appropriate personal protective equipment (Fig. 
1 in Research Strategy document). Relevant treatment data will be captured in the field using 
smartphones connected to the project’s REDCap database. 
Blood Samples: Venipuncture procedures will be performed using standard aseptic techniques. 
An experienced phlebotomist will take the blood sample from an antecubital vein. Blood 
volumes will depend on the participant’s age (5 ml for children under 8 years and 10 ml for 
children 9-18 years). Blood will be collected into Vacutainer® collection tubes or by a needle 
and syringe. A 22-gauge needle will be used for 5-15 yr olds, and a 23-gauge needle for 
children <5 yrs. Serum will be separated and stored at -70°C in labeled polypropylene cryogenic 
vials. When results are available they will be provided to individual study participants with an 
explanation of their significance. We have found in previous studies that participants are curious 
about their results even though results often have no impact on treatment. 
Entomological Surveys: Aedes aegypti surveys will be carried out once per month and for a 
period of 6 months post-TIRS application in a random sample of 10% of the households 
participating in the trial (equaling to ~1,600 houses per sampling period). To maintain quality 
control, an entomological supervisor will accompany the survey team and a second supervisor 
will review data daily. Adult captures:  Adult Ae. aegypti will be collected with Prokopack® 
aspirators 26 and used to calculate six adult indices: prevalence and abundance of adults, 
females and bloodfed females. 

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Movement surveys: Two movement surveys will be implemented (see Manual of Procedures). 
The retrospective movement survey (RMS) obtains information about locations visited in the 
past 15 days. The RMS gathers information on the main activity performed in visited locations 
and the self-reported duration of the visits (frequency and time of visits). The RMS will be used 
to quantify the proportion of time a symptomatic individual spent within their respective cluster 
and identify potential contamination in the trial by pinpointing to participants who live in one 
cluster type (e.g., TIRS) and visit other cluster type (e.g., control). We will apply prospective 
movement surveys (PMS) to all subjects at the time of the yearly serosurveillance blood 
samples. These surveys ask questions about the routine movements, rather than prior 15 days, 
and will be used to quantify the cumulative time under treatment for each child. For children up 
to 8 years, parents are interviewed to ask about time in home and specific places visited, 
whereas children 9 and up are interviewed directly by our skilled team of anthropologists. The 
project PI, GVP, has over 10 years’ experience applying and analyzing RMS and PMS 
instruments. 
5.2
Laboratory Evaluations
5.2.1 Laboratory Evaluations/Assays
Laboratory testing will be shared between UADY and Emory as described below. 
Active (enhanced) surveillance: acute samples from suspected ABV meeting our case definition 
will be tested at UADY using molecular testing by multiplex reverse transcriptase-polymerase 
chain reaction (RT-PCR) [50], NS1 detection and virus-specific IgM testing. Acute and 
convalescent specimens will be shared in parallel with Emory for assessment of IgM and IgG 
seroconversion. Testing of acute specimens in real time on site at UADY is critical as it allows 
immediate feedback to be given to subjects and/or their medical providers which may impact their 
clinical care.
Annual serologic surveillance for total ABV infection will be accomplished by obtaining a single 
serologic specimen from each subject each year of the study, including a baseline specimen prior 
to commencement of the TIRS intervention and following the final transmission season of the 
study (Fig 5). Testing will be performed at Emory University by a stepwise testing approach. Each 
ABV is considered independently – for serosurveillance, each serotype of DENV is considered as 
a distinct virus. Once an infection is detected, no further testing for infection by that virus is 
pursued in subsequent study years. Antigen capture ELISA for human IgG[51] will be the first 
test. Conversion from negative to positive for IgG indicates interval infection. Measurement of 
neutralizing antibody by focus reduction neutralization testing (FRNT) will provide a confirmatory 
and, in some cases, a more specific serologic assay [52, 53, 54].  For flaviviruses, seronegatives 
will be tested each year by IgG ELISA [55]. The first positive IgG ELISA to DENV or ZIKV will 
trigger FRNT testing to determine immunity to specific viruses. For flavivirus IgG positive subjects, 
further assessment for interval infection will rely solely on FRNT testing. CHIKV IgG conversion 
will be confirmed with neutralization testing by FRNT.
Mosquito Infection: The low indoor density of Ae. aegypti [48, 56] would make it too costly to 
quantify ABV infection at the household level. Therefore, we will quantify natural ABV infection 

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rates in Ae. aegypti at the city block level (9 blocks per cluster * 50 clusters = 450 blocks monitored 
monthly). At the UCBE lab, we will pool all female Ae. aegypti collected in a block in vials 
containing 25 mosquitoes (if more than 25 females are collected in a city block, we will make more 
than one vial). Vials will contain RNALater (Quiagen) and will be stored at -80°C for future testing, 
which will occur at the end of each transmission season (January-June). RNA will be extracted 
with an QIAmp Viral RNA kit (QIAGEN, 52904). We will implement RT-PCR procedures [50] for 
ABV detection using a StepOne Plus (Applied Biosystems) thermocycler. 
Insecticide Resistance. Standard CDC bottle bioassays [57] will assess phenotypic resistance of 
adult Ae. aegypti from treatment and control clusters pre-spraying and at 3 and 9 months post-
spraying every year. We will generate 10 Ae. aegypti colonies using eggs collected from 10 
randomly selected clusters (5 intervention and 5 control). F1 or F2 progeny from each colony will 
be screened for susceptibility to the following insecticides: deltamethrin, bendiocarb and 
pirimiphos-methyl using the diagnostic doses (DD) and diagnostic times (DT) previously 
established for Ae. aegypti by the CDC [57]. When resistance is detected, both DNA and RNA 
will be analyzed from a subset of the phenotyped mosquitoes to calculate the frequencies of 
known resistance alleles as well as expression of resistance-associated genes. This will enable 
us to measure changes in resistance mechanisms over time and in response to insecticide 
pressure. All resitance typification activities will occur at the Entomology lab at CDC in Atlanta, 
GA. 
5.2.2 Specimen Collection, Preparation, Handling and Shipping
5.2.2.1
Instructions for Specimen Preparation, Handling, and Storage
Special instructions for the collection, labeling, preparation, handling, and storage of specimens 
are detailed in the Manual of Procedures attachment. 
5.2.2.2
Specimen Shipment
We will be shipping serum samples from UADY to Emory for advanced and confirmatory 
serological assays. We estimate such shipments to occur 2x/year, coinciding with the end of the 
transmission season and the end of the annual sero-survey. Also, we have included specific 
SOPs in the Manual of Procedures for handling blood specimens. Mexico allows human 
specimen sample export to the US as long as a permit is signed by COFEPRIS (the Mexican 
equivalent to FDA). Approvals are protocol-specific, and we will be submitting paperwork 
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6
STATISTICAL CONSIDERATIONS
6.1
Study Outcome Measures
Outcome measures are summarized in the following table:
Type
Name
Time Frame
Brief Description
Primary
Laboratory-confirmPost-treatment
Laboratory-confirmed (virologically [RT-PCR testing of acute samples] 
or serologically [IgM and IgG ELISA testing of paired acute and 
convalescent samples]) symptomatic DENV, CHIKV or ZIKV.
Secondary
Laboratory confirm Post-treatment
Laboratory confirmed (serologically, [IgG ELISA and neutralization 
testing of annual surveillance samples]) DENV, CHIKV or ZIKV 
infection
Secondary
Aedes aegypti infec Post-treatment
Ae. aegypti mosquito infection rates with DENV, CHIKV, ZIKV 
(assessed by RT-PCR)
Secondary
Aedes aegypti infes Post-treatment
Ae. aegypti indoor entomological indices (adult presence and 
abundance, female presence and abundance, bloodfed female and 
abundance)
Secondary
Community acceptaPost-treatment
Percentage of households receiving the intervention that would 
recommend it to others in the community (assessed by household 
surveys)
Secondary
Safety profile
Post-treatment
Percentage of households receiving the intervention that had 
evidence of a reaction to the insecticide (assessed and confirmed by 
study doctors)
The primary endpoint will be binary (infected yes/no) and will be used as is for the Cox 
proportional hazards model. Additional variables will include date of onset of symptoms as well 
as house and cluster ID. No transformations will be made on such variables.
For secondary endpoints, epidemiological data will also be binary (interval seroconversion, 
which is the combined assessment of the serological status of a participant between annual 
serosurveys) and used in logistic models without transformation. 
For infection in mosquitoes, we will collect binary data (a mosquito pool is either infected or not), 
and transform it into minimum infection rates (MIR): (1/# mosquitoes in the pool)*1,000. This 
measure assumes that, when a pool is positive, it was due to at least one mosquito. That is why 
it is called ‘munimum’. 
For mosquito collections, data will be collected as counts (number per house) and used as is for 
negative-binomial analysis or used to calculate infestation (binary at the house level, with a 
house being infested or not). Both measures will be used as described in [34]. 
We will perform household surveys to assess the acceptability of the intervention for those 
households receiving it. We will quantify the % of heads of household that would recommend 
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Upon receipt of a sign of potential health reaction to insecticides used, doctors will visit the 
household and assess whether it could be related to the application. The % of households 
reporting signs of potential intoxication will be quantified for houses receiving the intervention. 
6.2
Sample Size Considerations
The size of clusters for the intervention will 
be 5x5 city blocks. This extent was chosen 
because: a) the area is large enough to contain 
more than the 92 age-eligible children required 
for the epidemiological evaluation; b) Ae. aegypti 
dispersal is limited, rarely exceeding 100m[58], 
thus concentrating our evaluation in the center of 
the cluster will provide a less biased estimate of 
intervention efficacy; c) prospective mobility 
surveys informing how human mobility may affect 
the placement of clusters. Surveys performed on 
800 FSD residents showed that FSD cohort 
participants living in Merida had about 60% their 
mobility occurring within 1km of the house (Fig 7). 
Given the large extent of movement, separating clusters by distance (e.g., larger than 1 km 
separation) would be unfeasible. Instead, our approach for the trial was to focus on a “fried egg” 
design, where entomology and epidemiological follow-up of study participants will occur within 
the central 3x3 city blocks of the 5x5 city-block cluster (Fig 8). 
Randomization:
The study area includes 286 hot-spot census tracts, 192 of 
which have total population size of at least 1,000, per the 
2010 census. Census tracts are categorized into four sectors. 
For each census tract, we have characterized the following 
features that are used as balancing factors:
-
Population size, per 2010 census. 
-
Population density, per 2010 census.
-
Percent employed population, per 2010 census.
-
Cumulative number of ABD cases between 2008 
and 2016 (per passive surveillance).
We simulated 1,000,000+ random allocation patterns (25 clusters in group A, 25 clusters in 
group B – allocation to TIRS/routine vector control determined later), and we use covariate-
constrained randomization[41] to eliminate the allocation patterns with an imbalance of ±10% on 
any of the continuous balancing factors listed above. Specifically, for each balancing factor, if for 
a given allocation pattern, the mean of group A divided by the mean of group B was greater 
Fig. 7. Probability of moving a given distance from 
the home (y-axis) as a function of the distance from 
home (x-axis) for Merida residents, and stratified by 
age group (Vazquez-Prokopec et al. unpublished). 
Fig. 8. Structure of a study cluster. 
Yellow areas will receive TIRS, whereas 
blue areas will receive TIRS and 
participate in the epidemiological and 
entomological evaluation of the 
intervention.

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than 1.1 or less than 1/1.1, that pattern would 
not be considered for potential selection. 
Furthermore, we eliminate any allocation 
pattern with imbalance in the number of 
clusters per arm per sector greater than ±1. 
We will implement checks to satisfy the criteria 
that the design is not overly constrained, as 
proposed by Moulton[41] (e.g. pairs of clusters 
always or never appearing in the same arm).
For each retained allocation patterns, we 
calculate the mean distance from the centroid 
of each cluster to the centroid of its nearest 
neighbor also in the trial. We restrict to the top 
10% of allocation patterns with the largest 
mean distance. Preliminary exercises returned 
over 1,500 allocation patterns satisfying all 
selection criteria. Sample allocation patterns 
are plotted in Fig 9. 
Given the set of allocation patterns that 
meet the above balancing criteria, we will 
use equal probability sampling to randomly 
select one allocation. For participant 
enrollment, the study teams will be provided 
with a list of 50 census tracts for inclusion in 
the study, without record of which census 
tracts are in Group A or B. At this time, households and individuals will be invited to participate 
in the study. No participant or investigator will have knowledge of their allocation. After 
completing the enrollment process, we will then use a random number generator to 
determine which group (A or B) is allocated to TIRS, where each group has a 50% 
probability.
Power and sample size 
To assess power and sample size requirements, we analyzed historical passive surveillance 
data from the 192 hot-spot census tracts with population size of at least 1,000 (from our 
publication characterizing the ABV hot-spot area [40]). We use yearly data from 2008 to 2016 
on the number of dengue, chikungunya, and Zika cases recorded in children 0-14 years each 
year by census tract[40]. Data were combined into pairs of adjacent years to mimic a two-year 
trial period. The table below summarizes the mean incidence (number of cases over two-year 
period/number of children) and intracluster correlation coefficient (ICC) for a given two-year 
period.
Data source
Mean
ICC
2008+2009 Dengue
0.0402
0.0345
2009+2010 Dengue
0.0530
0.0289
2010+2011 Dengue
0.0572
0.0164
2011+2012 Dengue
0.0847
0.0153
2012+2013 Dengue
0.0729
0.0188
Fig 9. Two examples of outputs from the 
constrained randomization into two groups. 

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2013+2014 Dengue
0.0455
0.0256
2014+2015 Dengue/Chik
0.0581
0.0229
2015+2016 Any
0.0385
0.0151
For our sample size calculations, we conservatively assume a low incidence (4% over a 
two-year period) and high ICC (0.035). Sample size is shown below for 50 clusters, 
considering 80% power and 90% power and TIRS efficacy from 70 to 90%. Sample sizes are 
adjusted for the study design effect and expected 20% loss to follow-up (LTFU).
POWER
TIRS 
EFFICACY
NUMBER 
OF 
EVENTS
TOTAL 
EFFECTIVE 
SAMPLE 
SIZE
NUMBER PER 
CLUSTER, 
UNADJUSTED
NUMBER 
PER 
CLUSTER, 
ADJUSTED 
FOR LTFU
TOTAL 
SAMPLE 
SIZE, 
ADJUSTED 
FOR LTFU
90%
70%
38
1390
>500
75%
30
1164
122
152
7600
80%
24
984
62
77
3850
90%
16
714
28
35
1750
80%
70%
28
1038
74
92
4600
75%
22
870
43
54
2700
80%
18
734
30
37
1850
90%
12
534
17
21
1050
Assuming 4% incidence over a two-year period, 70% TIRS efficacy, an ICC of 0.035, and 
20% loss to follow-up, we will require 92 age-eligible children enrolled per cluster for an 
overall sample size of 50 clusters and 4,600 children to have 80% power to detect a 
significant reduction in ABV incidence with TIRS.
Figure 10 below summarizes the distribution of the number of children less than or equal to 14 
years of age in each cluster, based on the 2010 census. The minimum size is 145 children. The 
median is 546 children.

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Figure 10. Distribution of children aged 0-14 per cluster, based on 2010 census. 
6.3
Participant Enrollment and Follow-Up
The duration of individual participation in the trial will vary, but will not be longer than 4.5 
years. Enrollment will occur at the end of Year 1 of the project, and will involve a baseline 
serological characterization of the population. Since then, and for the following 2-3 years, 
participants will be involved in the epidemiological assessment of TIRS (which will rely on active 
surveillance and annual blood draws). On the first two quarters of the 4th year post-enrollment, 
participants will provide the last annual blood sample to assess their serological evidence of 
interval infection. We will emphasize participants the fact that we would like people to 
participate as long as possible within this timeline. Febrile participants are followed clinically 
during the duration of their illness -- which rarely exceeds 7 days-- and we solicit convalescent 
samples 14-30 days after an acute sample. Movement and loss to follow-up interviews will last 
a maximum of 1 hour.
Activity by Quarter
Q1
Q2
Q3
Q4
Q1
Q2
Q3
Q4
Q1
Q2
Q3
Q4
Q1
Q2
Q3
Q4
Q1
Q2
Q3
Q4
Participant Enrollment
Baseline Serology and 
Entomology
Active Surveillance
Annual sero-survey
possible 3season
2020 (Y1)
2021 (Y2)
2022 (Y3)
2023 (Y4)
2024 (Y5)
6.4
Analysis Plan
Primary analysis

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The primary analysis will estimate the overall efficacy of TIRS in reducing the rate of laboratory-
confirmed symptomatic Aedes-borne disease, where the overall efficacy is estimated as one 
minus the hazard ratio from a Cox proportional hazards model. The Cox proportional hazards 
model will be fit using individual-level data for eligible and consenting children. The primary 
endpoint will be time to symptom onset of first laboratory-confirmed ABD. This assumes that 
participants having 2+ disease episodes during the trial period is rare and can be ignored. To 
account for clustering, the model will include a robust variance estimator with two parameters; 
one characterizes the level of correlation in outcomes between children within the same 
household, and one characterizes the level of correlation in outcomes between children in 
different households but within the same cluster.
The primary analysis will be the estimated overall TIRS efficacy against laboratory-confirmed 
Aedes-borne illness (
). The hypothesis test for the primary outcome will be H0:  
 
 𝑇𝐼𝑅𝑆
𝑇𝐼𝑅𝑆=  0
versus Ha:  
. The two-sided score test will be conducted at the 
 level. In 
𝑇𝐼𝑅𝑆 ≠ 0
𝛼= 0.05
addition, a 95% confidence interval will be calculated for 
.
𝑇𝐼𝑅𝑆
The time origin is July 1 prior to the first season. Spraying will be completed prior to the start of 
the first season. The first season will extend for six months, ending on December 31. This is 
henceforth referred to as the peak season. The second season will start on July 1 of the 
following year and extend for six months. The at-risk process for the primary efficacy survival 
model will consider only time during the peak season, which corresponds to time when the 
residual effect of the insecticides used in TIRS is expected to be active.
We will use Schoenfeld residuals to assess departures from proportionality, as would occur if 
the effect of TIRS varies over time. We will use time-dependent (piecewise) models where 
significant non-proportionality occurs.
Secondary analyses 
Epidemiological endpoints
We based our sample size calculations on the primary endpoint. Given the larger number of 
sub-clinical and undetected ABV infections compared to symptomatic manifestations, the study 
will be equally powered to detect a statistical difference in interval-level infections (measured by 
annual serosurveys) 
Planned secondary analyses of clinical and human serological data include:

Cox proportional hazards model with time to first laboratory-confirmed symptomatic ABV 
disease as the endpoint, adjusting for additional cluster- and household-level covariates 
(e.g. population density, household size, socio-economic status).

Cox proportional hazards model with time to first laboratory-confirmed symptomatic ABV 
disease as the endpoint, adjusting for routine human movement as measured by the 
prospective movement survey (measured in all enrolled participants). The proportion of 
time in treated areas will be included as a further covariate. 

Disease-specific versions of the primary analysis (e.g. time to first laboratory-confirmed 
symptomatic dengue disease as the endpoint), if data permit.

Analysis of recent human movements measured by RMS in enrolled participants 
presenting with symptoms for laboratory confirmation. The data will be analyzed using a 
test negative design-type structure, where individuals testing negative for any ABV will 

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serve as a comparator group for individuals testing positive for ABV. The analysis will 
adopt recently developed methods for cluster randomized vector control trials 81.

Binomial generalized linear mixed effects model to assess the efficacy of TIRS for 
reducing laboratory-confirmed DENV, CHIKV, or ZIKV infection, analyzed as cumulative 
incidence over the two (or potentially three) transmission seasons, as measured from 
annual serological samples.
Entomological endpoints:
Planned secondary analyses of mosquito data include:

Ae. aegypti indoor entomological indices (adult presence and abundance, female 
presence and abundance, blood fed female presence and abundance) and ABV 
minimum infection rates (# infected mosquitoes/total mosquitoes tested x 1,000).

Negative binomial GLMMs will test for differences in treatment and control arms for all 
entomologic indices as in our previous work, including Ae. aegypti infection rates with 
DENV, CHIKV, or ZIKV, and Ae. aegypti indoor entomological indices (adult presence and 
abundance, female presence and abundance, blood fed female presence and abundance) 
[34]. 

The following Ae. aegypti adult indices will be calculated for each sampling date and 
compared between treatments and over time: presence (binomial variable) and 
abundance (count variable) of adults, females and bloodfed females per house. 
Generalized linear mixed effects models (GLMM) nested at the cluster (level 1) and city-
block (level 2) levels will be used to compare each entomological index between 
treatment and control arms. Link functions for GLMMs will be binomial for presence 
indices and negative Poisson for abundance indices. The best fit models (after 
comparing AIC values for models including all levels or only Level 1) will be used to 
calculate odds ratios (OR, for mosquito presence/absence) and incidence rate ratios 
(IRR, for mosquito abundance) using control houses as the unit of comparison. We will 
calculate the operational efficacy of the intervention as E=(1-IRR)×100. This measure, 
ranging between 0 and 1, describes the percent reduction of mosquito abundance in 
treated houses with respect to the control. All models will be run with the software 
package lme4 within the software platform R (https://www.r-project.org/).
Transmission modeling
Our existing mathematical model for Yucatan[35, 59] will be used to simulate the effectiveness 
of TIRS for different scenarios of intervention coverage and insecticide residual power, using the 
observed trial data as a critical model input. This agent-based model of individual people and 
mosquitoes incorporates household demography, a spatially heterogeneous population 
structure based on census and remote sensing data, movement of workers and students, and 
seasonal fluctuations in mosquito population and incubation period.
We will model TIRS in treated households as (1) reducing the location's susceptible mosquito 
population and (2) increasing daily mortality probability for infected mosquitoes.  For 
computational efficiency, we represent susceptible mosquitoes probabilistically and infected 
mosquitoes explicitly. For (1) we use the TIRS efficacy to reduce the susceptible population, 
and for (2) we calculate a daily additional mortality probability associated with the overall 
population reduction (e.g, roughly 0.13 for 80% efficacy TIRS). This additional mortality is 
treated separately from the normal daily mortality due to age or other factors. To calculate daily 
mortality, we use the assumed TIRS efficacy combined with our empirically-derived model for 

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mosquito age distribution. The age distribution determines baseline daily mortalities, to which 
we add a constant daily death probability.
We will use our model to evaluate the effectiveness of TIRS for different scenarios of 
intervention coverage and insecticide residual power. Different movement (e.g. mosquito vs. 
human) and transmission (e.g. pathogen introduction and elimination) dynamics become 
relevant at different spatial scales [16], thus we will predict the  impact of scaling-up TIRS to the 
entire state rather than treating single cities. Simulating epidemiological trends of scaled-up 
TIRS for periods longer than the duration of our trial (e.g. a decade) will evaluate the effect of 
changing population-level immunity [35] and generate measures of effectiveness that are more 
informative for programmatic decision making. 
Integrating ABV transmission modeling with the field study will facilitate both interpreting trial 
results [60] and increasing realism of the model.  The explicit spatial structure of our model will 
allow us to separately calculate direct effects (due a person to living in a treated household) and 
indirect effects (due to adjacency to treated households) of TIRS as a function of residence 
position within, or distance from, a treated cluster.  We will also use our model to evaluate 
competing hypotheses for unexpected field results that we observe.
Spatial spread of ABV is driven by both humans and mosquitoes, but because Aedes bites 
during the day when humans are most likely to be away from home, human movement patterns 
are likely particularly important in driving spatial transmission16.  Our model already represents 
both human and mosquito movement, but the trial will produce data that will be highly valuable 
in evaluating and refining both.  Survey data about human movements may be used directly by 
the model, while a probabilistic mosquito movement kernel may be inferred based on spatial 
autocorrelation patterns in ABV case data collected in the trial.
6.5
Trial Continuation Rules
 
The heterogeneous nature of ABV transmission may dictate the need for a third season to 
evaluate the epidemiological impact of TIRS. Decision to continue into a 3rd season will be made 
in conjunction with the program officer assigned to this project. We will follow an event-driven 
decision process.
 
After the second season evaluating TIRS, a project member (Natalie Dean) will quantify the 
number of total primary endpoints. We will pursue the following ranking in order to evaluate 
whether to stop or continue into a third season:
- If 90+ endpoints are detected, stop and analyze data as final.
- If 20-89 endpoints, continue into a third season.
- if <20 endpoints, examine feasibility/futility.
The choice of 90+ endpoints is based on our power calculations, and represents the 
target number of events expected for a power of 80% and a TIRS efficacy of 70%. The choice of 
<20 endpoints represents the target number of events needed for a power of 80% when TIRS 
efficacy is 90%.
 

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7
SUBJECT CONFIDENTIALITY 
The information from this study is not expected to be sensitive or lead to any negative 
ramifications such as legal or employment risks, and standard measures to protect 
confidentiality should be sufficient. All paper data forms will be stored in locked files or cabinets 
at UADY in a specified storage facility with limited access. All data will be stored on secure 
servers managed by Emory University (see below). All files sent to consultants, collaborators 
and co-investigators will have names and address removed. No household identifiers, beyond 
an anonymized household or participant code (see below) will be included within our maps or 
databases or shown on printed maps or in publications of the study area. Such code is 
produced by generating a random number for each city block (without replacement) and a 
random number for each house in the city block (without replacement). Then, the house ID is 
the combined cluster (2 characters) and block (2 characters) code (e.g., code 3355). Once 
house codes are generated, they won’t change throughout the study. Participant codes will add 
two more characters to the house code (e.g., 3355##). The assignment of those last digits will 
be based on the order of enrollment into the study for each member of the house (e.g., 335502 
will be the children enrolled second in that house). Outside of our database these codes will not 
be interpretable, rendering the data effectively unidentifiable without access to our 
servers. Access to the database will be primarily administered through a custom, web-based 
interface with restricted access privileges and encrypted data transfer (REDcap: 
https://it.emory.edu/catalog/data-and-reporting/redcap.html). Access will be limited to certified 
project personnel and certified associates, who will be provided unique login and password 
combinations. Database servers will be protected by multiple layers of security. 
It will be made clear during the consent process that no information can be shared with anyone 
other than designated study personnel, the paper and computer files will be well protected, and 
we will ask that interviews be carried out one-on-one to prevent other family members listening 
in. We will take all necessary measures to ensure confidentiality. It will also be made clear to 
study personnel that any violation of confidentiality would be a fireable offense. Informed 
consent documents will include a specific statement relating to posting of clinical trial 
information at ClinicalTrials.gov, and also link the study to a ClinicalTrials.gov ID. We will permit 
access to all documents and records that may require inspection by the sponsor or its 
authorized representatives.  
7.1
Future Use of Stored Specimens
The consent forms for participation in the epidemiologic evaluation of TIRS will have a separate 
section for requesting permission to store the participant’s blood/serum for future use in studies 
either on dengue or other arboviruses, in view of the fact that: a) it may be necessary to 
revalidate the original laboratory findings at some point in the future; b) new and improved 
serological/diagnostic tests may become available whilst the project is in progress which would 
be beneficial to the project; and c) new viruses may be discovered over the course of the project 
for which it would be advantageous to screen the study participants retrospectively. The 
participants will be able to refuse consent for future use of their blood samples, without any 
repercussions. Should the participants agree to the future use of their specimens, their 
blood/serological samples will be stored indefinitely at secure laboratory facilities at Emory 
University and will not have any participant identifiers, beyond the participant’s code.

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8
INFORMED CONSENT PROCESS
Two levels of consent will be required for inclusion in the trial. First, household owners (adult 
member who owns or is the primary name on a lease of the property) will be presented with 
information materials about the intervention: the implementation of TIRS (and description of 
potential risks and benefits), and the entomological follow-up studies (see Manual of 
Procedures). After being given time to review the information, one household owner will be 
asked for written informed consent to have their houses be part of the trial. This means, 
they will consent to receive the TIRS intervention during at least two consecutive transmission 
seasons (should their house be part of a cluster receiving treatment), and to monthly 
entomological surveys (regardless of receiving or not the intervention). A study performed in 
Cairns, Australia, showed that TIRS coverages higher than 60% of houses were associated with 
a reduction in DENV incidence93. In our trial, city blocks with less than 60% consent to 
participate in the study will be replaced by neighboring blocks (we expect this to be rare, as our 
experience spraying more than 1,000 houses in Merida shows that 100% of blocks had <75% 
acceptance; Vazquez-Prokopec et al. unpublished). 
Second, individuals from houses consenting to participate in the trial and randomly selected to 
be included in the epidemiological evaluation of TIRS (see section 2.4 and section 3.1.1.B) will 
be provided with information about the longitudinal cohort component of the trial. This evaluation 
will involve the prospective monitoring for symptomatic DENV, CHIKV, ZIKV illness and a yearly 
blood-draw for assessing DENV, CHIKV, ZIKV sero-conversion. Parental informed consent 
will be obtained from children aged 2-10 and both assent to participate and a parentally 
signed informed consent document will be obtained for 11-15 year olds. The consent 
process will be conducted by trained UADY social anthropologists already involved in the FSD 
cohort, who have experience working with the communities and addressing potential concerns 
from the population. Consent forms will be written in Spanish at an approximate sixth-grade 
reading level. 
Consent will be obtained in participants’ homes. Study personnel will visit all houses in the 
5x5 city block clusters in a door to door fashion (when seeking household consent) or visit 
houses located in the center 3x3 city blocks of the 5x5 block cluster (when seeking individual 
consent). Study explanations are usually provided to small groups of adults present in the 
household, whereas written consent or assent are obtained from each individual participant. 
Subjects may voluntarily withdraw their consent for study participation at any time 
without penalty or loss of benefits to which they are otherwise entitled. If a subject 
withdraws or is withdrawn prior to completion of the study, the reason for this decision will be 
recorded in a case report form (CRF). The reasons, might include, but are not limited to:
•
Subject no longer meets eligibility criteria
•
Subject becomes noncompliant
•
Medical disease or condition, or new clinical finding(s) for which continued participation, 
in the opinion of the investigator might compromise the safety of the subject, interfere 
with the subject's successful completion of this study, or interfere with the evaluation of 
responses.
•
Subject lost to follow-up

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8.1
Informed Consent/Assent Process (in Case of a Minor or 
Others Unable to Consent for Themselves)
Assent to participate and a parentally signed informed consent document will be 
obtained for 11-15 year olds. The consent document follows Emory IRB policy for 
working with minors, and includes a section separating those individuals assenting from 
those consenting. A copy of the form is attached to this application. 

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9
DATA MANAGEMENT PLAN
DATA MANAGEMENT CORE (DMC): 
Emory University will centralize all aspects related to data storage, management and sharing. 
We proposed a Data Management Core (DMC) to provide timely and efficient curation and 
dissemination of study data from multiple sources (e.g., clinical, laboratory, reagents, passive 
surveillance, entomology, demographic, Ministry of Health interventions), all essential to the 
success of the proposed clinical trial. The DMC will provide secure development of reproducible 
analytic data sets for each publication, presentation, and other research output of the study. 
Members of the DMC will collaborate closely with project leads and the data teams in Mexico 
(field and laboratory) and at Emory (laboratory) to ensure timely, accurate, and reproducible 
data collection, management, and analysis.
Data Management Center Personnel
Lance Waller, Ph.D., Professor of Biostatistics and Bioinformatics, Rollins School of Public 
Health, Emory University will serve as DMC Director.  Dr. Waller has over 27 years’ experience 
in the development, application, and interpretation of biostatistical methods to a wide range of 
complex public health data sets.  Dr. Waller currently directs the DMCs for two large-scale NIH 
projects:  the NIAID Tuberculosis Research Center at Emory (with PI Rengarajan and other 
PREVEN-TB personnel, http://tbru.emory.edu/), and the NHLBI/Gates Household Air Pollution 
Intervention Network trial (http://www.hapintrial.org/). Both projects involve clinical and 
laboratory data in domestic and international settings, and address case report forms, data 
transfer protocols, enrollment reporting, data quality control protocols, and long-term storage of 
study data.  
Additional DMC personnel include: a full-time dedicated Data Manager to provide development, 
customization, adaptation, and testing of the proposed data transfer and storage platforms as 
well as curating our complete data holdings, project-specific research analytic data sets, and 
data archiving to repositories and future use. The database manager will travel regularly to 
Merida to coordinate all aspects of data collection and upload into the database, train database 
managers at UADY in proper data handling, and help with any specific data needs. 
Data and Resource Management
The DMC will implement and oversee the overall plan for data storage, retrieval and curation 
associated with the clinical trial, including, but not limited to, clinical data from medical records, 
specimen tracking of human specimens, mosquitoes, intervention deployment and results of 
multiple types of laboratory analyses.  
Fig. 1 illustrates the flow of data between study sites and provides a map for DMC data 
procurement, transfer, and curation plans. The DMC will collaborate with study sites and the trial 
leadership to refine data sharing, processing, and storage protocols for all elements of the 
projects, building on similar experiences with other large-scale, multi-center studies.
Secure local and cloud storage plans for our study data will be in place. Redundant Emory 
University-owned EMC Isilon arrays provide local storage on dedicated, secure, and HIPPA-
compliant 

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servers. Current policies provide nightly duplication of all data from the on-campus storage array 
to another secure off-site storage array in downtown Atlanta.  An Emory Broad Area Agreement 
with Amazon Web Services provides HIPPA-compliant cloud storage via a Virtual Private 
Network for duplication and storage of study data. We will also post specific study data 
associated with particular publications to appropriate online data repositories (e.g, journal 
archives, dryad.org, etc.). Such data will be de-identified and compliant with all our IRB 
requirements for data sharing and following recent recommendations for clinical trial data 
sharing (Ohmann et al. Sharing and reuse of individual participant data from clinical trials: 
principles and recommendations. BMJ Open. 2017 7(12):e018647. PMID: 29247106).  
Analysis, Integration and Visualization
Fig. 1 also illustrates how the DMC draws data from each field, clinical and laboratory site. Data 
will 
be 
managed 
through REDCap, an 
open-source 
data 
capture 
system 
(developed 
at 
Vanderbilt under the 
auspices of NIH’s 
Clinical 
and 
Translational 
Science 
Awards), 
allowing clinical data 
flow, 
reporting 
of 
completeness, 
and 
secure transfer from 
local 
REDCap 
servers to Emory’s 
REDCap 
server.  
Laboratory data will 
be 
linked 
by 
research 
project 
within the LabKey 
system, 
linking 
heterogeneous 
data sources for the entire project, including REDCap clinical data objects. Data exported from 
the database will be exported into GIS mapping platform (QuantumGIS) for rapid visualization 
and spatial analytics.  
The DMC and lead investigators will establish data quality assurance/quality control (QA/QC) 
procedures, building on established QA/QC protocols for ongoing trials. The DMC will 
coordinate practices across existing collaborations in order to document and implement the 
specific QA/QC guidelines for the trial. These protocols will provide standardized data reports to 
monitor enrollment and progress toward study goals as well as visual analytics for each project.  
The DMC will provide such data reports on a regular basis to project investigators and overall 
study leadership. 
Figure 1: Structure and organization of the Emory DMC.

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As noted above, the DMC will also work to develop archived analytic data sets for each 
publication/presentation conducted using trial data.  These analytic data sets provide a 
snapshot of all relevant data, meta-data, and code for a particular research output (e.g., 
manuscript) to establish reproducible research results.  For example, if a researcher compares 
newer results to an existing publication, they need access to the data and code used in the 
original analysis, and any assumptions, compromises, decisions influencing the results.  For 
primary analyses, the DMC will publish peer-reviewed “data descriptors” in data science 
journals (e.g., Scientific Data) to facilitate future replication and extension of trial results.
Transfer of Data and Reagent/Resource Information
Managing the data transfer infrastructure requires both flexibility and stability in order to respond 
to project needs and to maintain standards across study sites and personnel.  DMC personnel 
are well-versed in this balance and bring an array of technical, analytical, and diplomatic skills to 
the project.  The DMC will participate fully in discussions with the PI, leadership team and 
individual project leaders to establish workable and reliable transfer protocols, building on the 
data management infrastructure.
The DMC will ensure transmission of study data in accordance with HIPPA and other data 
security requirements.  Where possible, the DMC will utilize secure APIs to connect analytic 
components, and where not possible, the DMC will establish data transfer protocols via 
alternative approaches (e.g., secure FTP, secure cloud storage, etc.) in order to enable, 
monitor, and document regular transfers so that data holdings are never more than 1-2 weeks 
out of date.  This practice builds on past success in previous DMC projects. 
Some study projects involve the transfer of specimens from clinical to laboratory sites.   In 
addition to material transfer agreements, the DMC will work with the PI leadership team and 
study investigators to establish and implement specimen tracking and storage across trial-based 
studies.
Data and Research Resources Availability
Finally, the DMC will manage data resources locally within Emory’s secure local and cloud 
storage servers, including the analytic data sets associated with particular research output as 
mentioned above.  These analytic data sets provide working data for ongoing manuscript and 
presentation development, and also provide a record of the trial’s research output.  The DMC 
will work with investigators to provide relevant meta-data for each analytic data set and archive 
the relevant analytic code.  
Making Data Publicly Available
Finally, the DMC will collaborate with trial investigators to upload data to proper databases for 
sharing with the research community in a timely manner. The DMC will develop metadata 
following NIH guidelines to capture protocols for the overall study and individual samples. The 
analytic data sets for each publication, presentation, or other research output will allow transfer 
of data and figures to study and NIH leadership without corruption. This metadata will link each 
analytic data set (to be shared with study leadership and the broader community) with all 
components of each trial-associated component. 

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10
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SUPPLEMENTS/APPENDICES

Consent and assent Forms, including Future Use Consent

Manual of Procedures

Data Safety and Monitoring Plan (DSMP)

Forms for DSMP