Study Title, Statistical Analysis Plan

Version 2, 2020-04-23

Biannual mass azithromycin

distributions and serologic measures of
malaria and enteric pathogen infection
among pre-school children in Niger
A cluster-randomized, placebo-controlled trial

Statistical Analysis Plan

1. Administrative Information

Trial registration: NCT02048007

Bill & Melinda Gates Foundation (OPP1032340)
Funder:
SAP Version: Version 2, Updated 2020-04-23
A revision history for this document is included at the end.
Protocol Version: Refers to Protocol Version 1.6
This is a detailed analysis protocol for the pre-specified secondary
outcomes of antibody response, to supplement the original trial’s SAP
(current version 28).
Contributors: Statistician responsible:
Benjamin F. Arnold 1
Principal Investigator:
Thomas M. Lietman 1

1
Francis I. Proctor Foundation for Research in Ophthalmology,
University of California, San Francisco, USA

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SAP Table of Contents

1. ADMINISTRATIVE INFORMATION ............................................................................................................. 1
2. INTRODUCTION ............................................................................................................................................ 3
2.1. BACKGROUND AND RATIONALE ................................................................................................................... 3
2.2. OBJECTIVES AND HYPOTHESES .................................................................................................................. 3
3. STUDY METHODS ........................................................................................................................................ 4
3.1. TRIAL DESIGN ............................................................................................................................................. 4
3.2. RANDOMIZATION ......................................................................................................................................... 4
3.3. SAMPLE SIZE .............................................................................................................................................. 4
3.4. STATISTICAL FRAMEWORK .......................................................................................................................... 4
3.5. STATISTICAL INTERIM ANALYSES AND STOPPING GUIDANCE ........................................................................ 5
3.6. TIMING OF THE FINAL ANALYSIS ................................................................................................................... 5
3.7. TIMING OF OUTCOME ASSESSMENTS ........................................................................................................... 5
4. STATISTICAL PRINCIPLES ........................................................................................................................ 5
4.1. CONFIDENCE INTERVALS AND P-VALUES ..................................................................................................... 5
4.2. PROTOCOL DEVIATIONS .............................................................................................................................. 5
5. TRIAL POPULATION .................................................................................................................................... 5
5.1. SCREENING DATA ....................................................................................................................................... 5
5.2. ELIGIBILITY ................................................................................................................................................. 5
5.3. RECRUITMENT ............................................................................................................................................ 6
5.4. WITHDRAWAL/FOLLOW-UP .......................................................................................................................... 6
5.5. BASELINE PATIENT CHARACTERISTICS ........................................................................................................ 6
6. ANALYSIS...................................................................................................................................................... 6
6.1. OUTCOME DEFINITIONS ............................................................................................................................... 6
6.2. ANALYSIS METHODS ................................................................................................................................... 7
6.2.1. Geometric mean antibody levels ..................................................................................................... 7
6.2.2. Seroprevalence................................................................................................................................. 7
6.2.3. Seroconversion rate (force of infection) .......................................................................................... 8
6.2.4. Subgroup analyses ........................................................................................................................... 8
6.3. MISSING DATA ............................................................................................................................................ 9
6.4. MASKING .................................................................................................................................................... 9
6.5. ADDITIONAL ANALYSES ............................................................................................................................... 9
6.6. HARMS ..................................................................................................................................................... 10
6.7. STATISTICAL SOFTWARE AND REPLICATION FILES ..................................................................................... 10
7. REFERENCES ............................................................................................................................................. 11
8. REVISION HISTORY ................................................................................................................................... 12

This SAP was organized following guidelines proposed in: Gamble C, Krishan A, Stocken D,
Lewis S, Juszczak E, Doré C, et al. Guidelines for the Content of Statistical Analysis Plans in
Clinical Trials. JAMA. 2017;318: 2337–2343. PMID: 29260229

The companion computational notebook with underlying detectable effect calculations

presented herein is entitled: MORDOR-Ab-detectable-effect-calculation.Rmd/.html; it is
saved in the same directory as this document.

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2. Introduction
2.1. Background and rationale
MORDOR (Macrolides Oraux pour Réduire les Décès avec un Oeil sur la Résistance) was a
cluster-randomized trial conducted in Malawi, Tanzania, and Niger to measure the effect of
providing biannual azithromycin distributions on all cause mortality over a 24 month period.1 In
Niger, longer term follow-up found sustained reductions in mortality after 36 months.2

Azithromycin is a broad-spectrum antibiotic with antimicrobial activity against multiple

pathogens, so a key question following the trial was the mechanism of impact. Verbal autopsy
results from the Niger trial verbal suggested broad impact across many diseases, including:
diarrhea, malaria, pneumonia, and meningitis.3 The MORDOR Niger study included a separate
morbidity trial in 30 communities selected from the original trial population and randomized with
equal allocation (15 azithromycin, 15 placebo) to further elucidate intervention impacts on
endpoints beyond mortality such as antimicrobial resistance. The intensive monitoring trial has
thus far documented increases in macrolide resistance,4 changes to the microbiome including
reductions in campylobacter,5 and reductions in malaria parasitemia.6

Multiplex bead assays enable the simultaneous detection of serologic antibody response to
multiple pathogens that could potentially mediate the effects of azithromycin on mortality, such
as enteric pathogens and malaria.7 Children ages 13-59 months in the 30-community intensive
monitoring substudy of the Niger trial contributed dried blood spot specimens at baseline (month
0) and repeatedly at months 12, 24, and 36.

2.2. Objectives and Hypotheses

The objective of this study is to measure the effect of biannual azithromycin distribution on
serologic measures of enteric pathogen infection and malaria infection among children ages 13-
59 months in rural Niger.

We hypothesize that biannual azithromycin administered to children 6-59 months will reduce the
number or duration of infection for multiple enteric pathogens and multiple species of malaria,
and that reduced infection will result in lower IgG antibody levels, lower seroprevalence, and
lower force of infection measured through the seroconversion rate.

Rationale, enteric pathogens: Azithromycin has broad-spectrum activity against gram-positive

and atypical bacteria and has been shown to be highly effective for treatment of bacterial enteric
pathogen infections, including Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC),
Vibrio cholerae, and non-typhoidal Salmonella enterica, which are a focus of this study.8–10
Azithromycin has been shown to have modest anti-parasitic activity against Cryptosporidium
parvum.11 Less is known about its effect on Giardia intestinalis infections in humans, but in vitro
and in vivo animal studies have shown azithromycin had comparable antimicrobial activity
against G. intestinalis to metronizadole (the first line treatment)12 and small scale (uncontrolled)
human studies suggest cure rates in excess of 90%.13

Rationale, malaria: Azithromycin has modest activity against Plasmodium falciparum through
action against the parasite’s apicoplast, and high efficacy against P. vivax both as a prophylactic
to prevent infection14 and as a treatment therapy.15 To our knowledge, studies have not
examined the effect of azithromycin on P. malariae or P. ovale. Among preschool aged children

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in Niger, mass distribution of azithromycin has reduced malaria parasitemia in two trials, 6,16 has
reduced malaria-attributed mortality in the MORDOR trial,3 and has been associated with
reduced P. falciparum MSP-119 antibody levels and seroprevalence.17

3. Study Methods
3.1. Trial design
The trial was a parallel, community-based, cluster-randomized controlled trial with equal
allocation. From 30 communities reserved for morbidity monitoring, 15 were allocated to active
azithromycin and 15 were allocated to placebo. At months 0, 6, 12, 18, 24, 30, 36, 48, and 60
children were identified through a census, specimens collected among those age-eligible, and
all children identified ages 1-59 months received either azithromycin or placebo, according to
their community-level assignment. Dried blood spots were collected at months 0, 6, 12, 24, 36,
48, and 60 months.

3.2. Randomization
30 communities were selected from the original study frame of the MORDOR trial and
designated as morbidity substudy communities. The substudy communities were randomized in
equal proportion (1:1) to azithromycin or placebo using a random number generator at the trial’s
data coordinating center at the University of California, San Francisco.

3.3. Sample size

We informed the sample size calculation with measurements from the PRET trial, where 991
children ages 1-5 years old were measured for antibody response to Plasmodium falciparum
MSP-119 across 24 communities.17 In that study, mean seroprevalence to MSP-1 was 65% and
the intra-class correlation coefficient for seropositivity was 0.004. We used a standard sample
size equation for cluster randomized trials in binary outcomes (equation 7.11 of Hayes and
Moulton).18

Under these assumptions, we estimated that with 15 communities per arm, 140 measurements
per community (four phases) and 80% power, the minimum detectable relative reduction is 8%
(prevalence difference = –5.4 percentage points). At 90% power, the detectable relative
reduction is 10% (prevalence difference = –6.3 percentage points).

We estimated under the same assumptions that with 35 children per community (single phase),
at 80% power the minimum detectable relative reduction is 14% (prevalence difference = –9.3
percentage points). At 90% power the detectable relative reduction is 16% (prevalence
difference = –10.7 percentage points).

Detailed calculations are included in the companion computational notebook saved in the same
directory as this SAP entitled: MORDOR-Ab-detectable-effect-calculation.Rmd/.html

3.4. Statistical framework

MORDOR was designed as a superiority trial. We will compare azithromycin versus placebo for
each antibody outcome.

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3.5. Statistical interim analyses and stopping guidance

There were no planned interim analyses or stopping rules for the morbidity substudy trial.

3.6. Timing of the final analysis

The primary analysis for antibody outcomes is planned for after the 36 month visit and analysis
of all specimens collected. If additional specimens from the 48 and 60 month visits are analyzed
for antibody response then we will amend the analysis with the additional data.

3.7. Timing of outcome assessments

Dried blood spots were collected at baseline (month 0) and at 6, 12, 24, 36, 48, and 60 months.
All outcome measurements through 36 months will be included in the primary analysis.

4. Statistical Principles
4.1. Confidence intervals and P-values
We plan to estimate 95% confidence intervals. We will report p-values unadjusted for multiple
comparisons and adjusted across antibody outcomes using a Benjamini-Hochberg procedure.19

4.2. Protocol deviations

Children will be analyzed according to their assignment in an intention to treat (ITT) analysis.
We will report the proportion of children treated in each biannual phase of the trial but we will
not use adherence in the outcome analysis.

5. Trial Population
5.1. Screening data
MORDOR Niger was designed as a large simple trial that enrolled a majority of rural villages in
the Boboye and Loga departments of Niger. The 30 communities in the morbidity trial were
sampled from the overall 646 community clusters that were eligible for the study (the remaining
616 clusters were enrolled in the main mortality endpoint monitoring trial).

5.2. Eligibility
Communities with a population between 200 and 2,000 on the most recent government census
were eligible.

Children were eligible for dried blood spot collection if they were between 0 and 59 months of
age. A random sample of 40 children ages 0-59 months from each community were selected to
contribute to blood specimen collection.

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5.3. Recruitment
The trial will track and report the following populations in the CONSORT flow for the trial:
• Number of communities screened and enrolled
• Number of children screened, enrolled, and treated in each measurement round
• Number of children tested in each measurement round
• Number of children and communities analyzed for each outcome

5.4. Withdrawal/follow-up
The trial will report withdrawal of any enrolled and randomized study communities and the
reason for withdrawal. Since measurement of children within communities relies on repeated
cross-sectional surveys, children will not be considered lost to follow-up.

5.5. Baseline patient characteristics

We will report community size (estimated total population, number of children ages 0-9 years),
age and sex distributions, baseline prevalence of malaria parasitemia, and baseline
seroprevalence to all antigens planned for this analysis.

6. Analysis
6.1. Outcome definitions
Antibody responses were measured at the US Centers for Disease Control (CDC) in a multiplex
bead assay on the Luminex platform that included the following antigens (Table). The primary
analysis will include antibody responses to malaria and enteric pathogens. The NTD antigens
were included as ancillary (opportunistic) measurements: there was no anticipated trachoma
transmission in the study population based on programmatic monitoring of clinical symptoms,
and azithromycin would not be expected to have any impact on Strongyloides stercoralis.
infection.

Pathogen Antigen
Malaria
Plasmodium falciparum PfCSP
Plasmodium falciparum LSA1
Plasmodium falciparum GLURP-Ro
Plasmodium falciparum HRP2
Plasmodium falciparum PfAMA
Plasmodium falciparum PfMSP1
Plasmodium ovale PoMSP1
Plasmodium vivax PvMSP1
Plasmodium malariae PmMSP1
Enteric pathogens
Giardia duodenalis VSP-3
Giardia duodenalis VSP-5
Cryptosporidium parvum Cp17
Cryptosporidium parvum Cp23

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Campylobacter jejuni p18

Campylobacter jejuni p39
Salmonella enterica typhimuriam group B LPS
Salmonella enterica enteritidis group D LPS
Enterotoxigenic E. coli LTB subunit
Vibrio cholerae CTB
Other Neglected Tropical Diseases
Chlamydia trachomatis Pgp3
Chlamydia trachomatis CT694
Strongyloides stercoralis NIE

We will analyze responses using both antibody levels and seroprevalence. Antibody responses
are reported in median fluorescence units minus background (MFI-bg), and will be log10
transformed before analysis due to their skewed distribution.20 The CDC team has developed
seropositivity cutoffs using receiver operating characteristic (ROC) curves from known positive
and negative samples for all antigens except those for Campylobacter, Salmonella, ETEC, and
cholera. For pathogens without ROC-based seropositivity cutoffs, following our previous work
we will identify seropositivity cutoffs with either: (i) finite Gaussian mixture models among
children <1 years old with 2 components to identify a seronegative distribution, and will estimate
a seropositivity cutoff as the mean plus 3 standard deviations of the seronegative distribution;
or, if a mixture model fails to adequately fit the data, (ii) using children measured longitudinally
to identify a subset of responses among presumed unexposed to define the cutoff as their mean
plus 3 standard deviations. Our previous work has shown that all approaches had high
agreement across multiple cohorts, and the choice of approach used should be largely
pragmatic.21

6.2. Analysis methods

6.2.1. Geometric mean antibody levels
Translating quantitative antibody levels to seroprevalence can mask variation in response above
and below the seropositivity cutoff. For high transmission pathogens, as we expect for malaria
and many of the enteric pathogens in this study, changes in mean log10 antibody response
could be detectable above the seropositivity cutoff.20 We will compare geometric mean antibody
levels between arms using a linear model to estimate the mean difference in log10 antibody
response, with standard errors estimated using a non-parametric bootstrap that resamples
communities with replacement to preserve outcome correlation in the data. We will estimate
differences between arms for each antigen. We will additionally estimate a P-value based on a
randomization test that permutes community treatment assignments and uses the difference in
means as a test statistic.

6.2.2. Seroprevalence
Mean antibody levels are in arbitrary units, and for antibody responses that are censored at the
high end of the assay’s dynamic range we could under-estimate true mean antibody levels. We
will additionally estimate a difference in seroprevalence, using cutoffs described in the outcome
definitions (Section 6.1). We will use a logistic regression model of individual level outcomes to
model the probability of infection conditional on arm and community-level baseline
seroprevalence. We will use marginal standardization to estimate differences in prevalence
between arms,22 with standard errors estimated using a non-parametric bootstrap that

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resamples communities with replacement. We will estimate P-values for the differences using a
randomization test that permutes community treatment assignments and uses the difference in
prevalence as a test statistic.

We will estimate differences between arms separately for each pathogen. Our main analysis for
seroprevalence will be based on composite measures for each pathogen whereby an individual
will be considered seropositive if they are positive to any antigen measured for each pathogen.
We will additionally report differences in seroprevalence for each antigen individually.

6.2.3. Seroconversion rate (force of infection)

Seroconversion rates are often used to measure a pathogen’s force of infection.23 will estimate
the seroconversion rate as measure of force of infection using age-structured seroprevalence
using a generalized linear model with a complementary log-log link, age as an offset and
treatment arm as a covariate.23,24 The exponentiated coefficient on treatment arm will estimate
the hazard ratio of seroconversion. We will estimate 95% confidence intervals and P-values
similarly to seroprevalence and geometric mean outcomes: with a non-parametric bootstrap that
resamples communities with replacement (confidence intervals) and with a permutation test
based on the hazard ratio (permutation P-value).

In this analysis, we will use a single outcome per enteric pathogen for parsimony; an individual
will be considered seropositive to each pathogen if they are seropositive based on any antibody
response measured for that pathogen (e.g., for Campylobacter jejuni, an individual will be
considered seropositive if their response to p18 or p39 is above the seropositivity cutoff).

6.2.4. Subgroup analyses

Subgroup analysis by trial phase: A pre-specified subgroup analysis will include a comparison of
antibody response between arms stratified by phase (6, 12, 24, 36 months). We will repeat the
analysis separately by phase to determine whether the intervention had differential impacts on
antibody response at 6, 12, 24, and 36 months of intervention. We will test for interaction on the
additive scale using a linear model that includes an interaction term between arm and phase,25
with confidence intervals for the additive interaction parameter estimated through a
nonparametric bootstrap as described above.

Subgroup analysis by season: Rainfall is highly seasonal in Niger. We hypothesize that enteric
pathogen and malaria antibody responses could vary seasonally, as we have previously shown
in the Gambia, and that intervention effects could differ by season. We will conduct a subgroup
analysis where we use weekly rainfall to determine the rainy season in each study year. The
analysis approach for measures of additive interaction by rainy versus dry season will be the
same as describe above for trial phase.

Subgroup analysis by age: A pre-specified subgroup analysis will examine the effect among
children who were 6 months or younger at the start of the trial. IgG response tends to increase
with age and transmission could be intense for many of the pathogens. Thus, it is possible that
older children will already have high IgG levels to specific pathogens after multiple infections
before the study that will not wane with treatment. In contrast, for children born since the start of
the trial, treatment could reduce the severity or duration of infections, which in turn could result
in a less robust IgG response. We define the subgroup as children 6 months or younger
(including not yet born) at the start of the trial because age 6 months is approximately when we

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would expect children to begin to produce their own IgG response.26 The analysis approach for
measures of additive interaction will be the same as describe above for trial phase.

Age restrictions: The primary analysis will exclude children <6 months old to prevent maternal
IgG contributions from influencing the analysis.26 If age-dependent IgG levels show evidence of
substantial maternal contributions to IgG levels to ages older than 6 months for any antigens
then we will consider limiting the analyses of those antigens to ages 12 months and older. We
will include the younger children in exploratory and descriptive analyses. We have found in
previous studies that for highest transmission enteric pathogens there is very little heterogeneity
in antibody response after age 24 months because most children have been infected (possibly
repeatedly) with little antibody waning.21 If we find an absence of heterogeneity in response
above ages 24 months in the MORDOR Niger trial in masked summaries of age-dependent
antibody response, we will limit comparisons between randomized arms to only include children
in the narrower age range (6-24 months), and exclude responses among children >24 months;
we acknowledge that the exclusion of ~75% of measurements could reduce the statistical power
of the analysis significantly.

6.3. Missing data

Complete case analysis. We propose to analyze only the existing data. In the case of data
missing from an entire community, we will repeat the permutation test including the community
or communities with missing outcomes. Communities with missing outcomes are still able to
receive randomization allocations during the test.

6.4. Masking
The analysis will remain partially masked until the completion of the results outlined in section
6.2. That is, we will complete all analyses using re-randomized and obscured treatment
assignments until final tables and figures are populated.27,28 We will then repeat the analysis
with the true, treatment labels. The reason the approach is “partially masked” is because the
trial’s primary endpoints have been reported, and the broader analysis team has access to the
true treatment labels. Adherence to the analysis masking will be documented through GitHub
commits, but is on the honor system.

6.5. Additional analyses

In addition to comparing arms based on a single summary measure of mean antibody levels,
seroprevalence, and the seroconversion rate, we will estimate age-dependent curves of
antibody levels, seroprevalence, and force of infection. We will estimate age-specific curves
using a semiparametric, cubic spline in a generalized additive model we have used previously
and which captures the variation in age-dependent patterns as well as more flexible machine
learning approaches.20,21 We hypothesize that age-dependent mean and seroprevalence
curves will rise more slowly among children in azithromycin communities compared with those
who receive placebo,20 which will in turn be reflected in lower age-specific force of infection.

The primary analysis approach for the seroconversion rate will estimate rates from age-
structured seroprevalence, treating the data as repeated cross-sectional measurements. As a
sensitivity analysis, we will estimate prospective seroconversion rates and seroreversion rates
among the subset of children for whom the study collects longitudinal measurements.

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To help inform future studies, we will estimate community level intra-class correlation
coefficients (ICCs) for each outcome using a mixed effects model that includes random effects
for the community. We will estimate the ICC as the between-cluster variance (𝜎" ) of the total
variance (between plus within):29
'(
𝐼𝐶𝐶 = ' )' .
( *

6.6. Harms
The trial was overseen by a Data and Safety Monitoring Committee. Monitoring for adverse
outcomes due to treatment has been completed as part of the overall trial and will not be a part
of the present analysis of serological outcomes.

6.7. Statistical software and replication files

We will use R statistical software for all analyses (version 3.6.1 or later).

All data and computational notebooks (.Rmd/.html) required to reproduce the analysis will be
made publicly available through GitHub and a permanent repository such as the Open Science
Framework, Dryad, or Zenodo.

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7. References
1. Keenan, J. D. et al. Azithromycin to Reduce Childhood Mortality in Sub-Saharan Africa. N.
Engl. J. Med. 378, 1583–1592 (2018).
2. Keenan, J. D. et al. Longer-Term Assessment of Azithromycin for Reducing Childhood
Mortality in Africa. N. Engl. J. Med. 380, 2207–2214 (2019).
3. Keenan, J. D. et al. Cause-specific mortality of children younger than 5 years in communities
receiving biannual mass azithromycin treatment in Niger: verbal autopsy results from a
cluster-randomised controlled trial. Lancet Glob. Health 8, e288–e295 (2020).
4. Doan, T. et al. Macrolide Resistance in MORDOR I - A Cluster-Randomized Trial in Niger.
N. Engl. J. Med. 380, 2271–2273 (2019).
5. Doan, T. et al. Gut microbiome alteration in MORDOR I: a community-randomized trial of
mass azithromycin distribution. Nat. Med. 25, 1370–1376 (2019).
6. Arzika, A. M. et al. Biannual mass azithromycin distributions and malaria parasitemia in pre-
school children in Niger: A cluster-randomized, placebo-controlled trial. PLoS Med. 16,
e1002835 (2019).
7. Arnold, B. F., Scobie, H. M., Priest, J. W. & Lammie, P. J. Integrated Serologic Surveillance
of Population Immunity and Disease Transmission. Emerg Infect Dis 24, 1188–1194 (2018).
8. Kuschner, R. A. et al. Use of azithromycin for the treatment of Campylobacter enteritis in
travelers to Thailand, an area where ciprofloxacin resistance is prevalent. Clin. Infect. Dis.
Off. Publ. Infect. Dis. Soc. Am. 21, 536–541 (1995).
9. Tribble, D. R. Antibiotic Therapy for Acute Watery Diarrhea and Dysentery. Mil. Med. 182,
17–25 (2017).
10. Saha, D. et al. Single-dose azithromycin for the treatment of cholera in adults. N. Engl. J.
Med. 354, 2452–2462 (2006).
11. Checkley, W. et al. A review of the global burden, novel diagnostics, therapeutics, and
vaccine targets for cryptosporidium. Lancet Infect Dis 15, 85–94 (2015).
12. Gardner, T. B. & Hill, D. R. Treatment of giardiasis. Clin. Microbiol. Rev. 14, 114–128
(2001).
13. Jeske, J. et al. Effectiveness of azithromycin in the treatment of giardiasis. Med. Sci. Monit.
4, 547–550 (1998).
14. Taylor, W. R. et al. Malaria prophylaxis using azithromycin: a double-blind, placebo-
controlled trial in Irian Jaya, Indonesia. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 28,
74–81 (1999).
15. Dunne, M. W. et al. A double-blind, randomized study of azithromycin compared to
chloroquine for the treatment of Plasmodium vivax malaria in India. Am. J. Trop. Med. Hyg.
73, 1108–1111 (2005).
16. Gaynor, B. D. et al. Impact of mass azithromycin distribution on malaria parasitemia during
the low-transmission season in Niger: a cluster-randomized trial. Am. J. Trop. Med. Hyg. 90,
846–851 (2014).
17. Oldenburg, C. E. et al. Biannual versus annual mass azithromycin distribution and malaria
seroepidemiology among preschool children in Niger: a sub-study of a cluster randomized
trial. Malar. J. 18, 389 (2019).
18. Hayes, R. J. & Moulton, L. H. Cluster randomised trials. (Taylor & Francis Group, 2017).
19. Benjamini, Y. & Hochberg, Y. Controlling the False Discovery Rate: A Practical and Powerful
Approach to Multiple Testing. J. R. Stat. Soc. Ser. B Methodol. 57, 289–300 (1995).
20. Arnold, B. F. et al. Measuring changes in transmission of neglected tropical diseases,
malaria, and enteric pathogens from quantitative antibody levels. PLoS Negl Trop Dis 11,
e0005616 (2017).

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21. Arnold, B. F. et al. Enteropathogen antibody dynamics and force of infection among children
in low-resource settings. eLife 8, e45594 (2019).
22. Muller, C. J. & MacLehose, R. F. Estimating predicted probabilities from logistic regression:
different methods correspond to different target populations. Int J Epidemiol 43, 962–970
(2014).
23. Hens, N., Shkedy, Z., Aerts, M., Damme, C. F. P. V. & Beutels, P. Modeling Infectious
Disease Parameters Based on Serological and Social Contact Data. (Springer, 2012).
24. Jewell, N. P. & van der Laan, M. Generalizations of current status data with applications.
Lifetime Data Anal 1, 101–109 (1995).
25. VanderWeele, T. J. & Knol, M. J. A Tutorial on Interaction. Epidemiol Method 3, 33–72
(2014).
26. Zinkernagel, R. M. Maternal antibodies, childhood infections, and autoimmune diseases. N
Engl J Med 345, 1331–1335 (2001).
27. Nuzzo, R. How scientists fool themselves - and how they can stop. Nature 526, 182–185
(2015).
28. Munafò, M. R. et al. A manifesto for reproducible science. Nat. Hum. Behav. 1, 0021 (2017).
29. Stoffel, M. A., Nakagawa, S. & Schielzeth, H. rptR: repeatability estimation and variance
decomposition by generalized linear mixed-effects models. Methods Ecol. Evol. 8, 1639–
1644 (2017).

8. Revision history

Version Date Summary of Changes, Justification, and Timing vis-à-vis key

trial events (enrollment completion, interim analyses,
unmasking, etc)
1 2020-04-01 • First draft of the statistical analysis plan

2 2020-04-23 • Added pre-specified subgroup analyses by season and by

age. Outcome data have not been examined at this time.
Analysis is still fully masked.

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