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. 2026 Aug 20;6(8):e0006518. doi: 10.1371/journal.pgph.0006518

A single dose of azithromycin versus placebo for malaria in infants: A cluster-randomized trial

Rikhard Ihamuotila 1,*, Jane Juma 2, Fadima Haidara 2, Oumar Samaké 2, Juho Luoma 1, Laura Adubra 1, Yin Bun Cheung 1, Dagmar Alber 3, Awa Traoré 2, Camilla Ducker 4, Ulla Ashorn 1, Per Ashorn 1, Samba Sow 2, Yue-Mei Fan 1
Editor: Alassane Dicko5
PMCID: PMC13492813  PMID: 42623398

Abstract

Azithromycin mass drug administration (MDA) has been shown to promote child survival in high mortality settings. Azithromycin has antimalarial effects, and one possible mechanism behind mortality reduction with azithromycin may be reduction in malaria. We aimed to test the effect of a single dose of azithromycin on malaria prevalence and parasite load in infants. This malaria substudy was part of a large double-blinded cluster-randomized clinical trial in Mali studying the effect of azithromycin MDA on infant mortality (ClinicalTrials.gov NCT04424511). Villages were randomized into either placebo or azithromycin MDA groups. We collected dry blood spot samples among 4–11-month-old infants right before and two weeks after an azithromycin MDA dose (20mg/kg). Malaria positivity and concentration of Plasmodium falciparum DNA (parasite load) were determined by quantitative PCR. Hemoglobin concentration was measured with a rapid test. A total of 1158 infants at baseline were treated, with 324 infants in 20 villages in the control group, and 834 infants in 38 villages in the azithromycin group. At baseline, prevalence of PCR-positive malaria was 9.4% in the control group and 9.9% in the azithromycin group. On day 14 after receiving the study drug, prevalence was 8.9% among controls and 8.0% in the azithromycin group. Adjusting for baseline and clusters, malaria prevalence risk ratio was 0.96 (95% CI: 0.63 to 1.48) favoring azithromycin. The prevalence risk ratio for baseline malaria PCR-positive infants was 0.81 (0.55 to 1.19). The ratio of means for parasite load was 0.91 (0.66 to 1.25) favoring azithromycin. Hemoglobin was 0.1 g/L (-3.9 to 4.1) higher with azithromycin. The findings do not conclusively support, but are consistent with, a hypothesis that a single dose of azithromycin reduces malaria prevalence and parasite load among infants in a malaria-endemic West African setting.

Introduction

Mass drug administration (MDA) of azithromycin to under 5-year-old children is a promising intervention to reduce child mortality in sub-Saharan Africa [1–4]. While the intervention has been shown to reduce pneumonia, diarrhea, malaria in under 5-year-old children [5–8], the effect on these conditions has been marginal and the mechanism behind mortality reduction remains unclear. To mitigate the development of antimicrobial resistance, the 2020 WHO guideline on azithromycin MDA for child survival recommended giving the intervention only to 1–11-month-old infants [9], prompting large trials to test the efficacy when given only to this subgroup [3,10–12].

Malaria remains one of the main causes of child mortality, and reduction of malaria burden is a plausible mechanism behind mortality reduction with azithromycin [13]. Although weak, the clinical antimalarial effect of azithromycin is well established [14–16]. Azithromycin has a proven effect on the malaria parasite apicoplast organelle [17], but may also inhibit red blood cell invasion, and mosquito stage development [18,19]. Some azithromycin MDA trials have seen well over 20% reduction in malaria prevalence, parasite density, malaria mortality and clinic visits [6,20–23]. Conversely, many trials have reported no effect on malaria [24–26]. Discerning the effect of azithromycin MDA on malaria in areas with a seasonal malaria chemoprevention (SMC) program has also proven difficult [24,25,27–29].

Evidence is scant on the effect of azithromycin MDA on malaria prevalence and parasite load in the WHO-recommended infant subgroup. We aimed to test the short-term effect of one dose of azithromycin on malaria prevalence and parasite load among infants in a malaria-endemic West African setting.

Methods

Ethics statement

Ethical approval for the LAKANA trial was received from the Mali Institutional Review Board (IRB), the Comité d’Éthique de l’Université des Sciences, des Techniques et des Technologies de Bamako (USTBB), (IRB approval number: N2019/181/CE/FMPOS/FAPH, initially approved 27 December 2019, with the most recent reapproval granted on 13 October 2025). Additionally, the Ethics Committee of Pirkanmaa Hospital District gave a favorable opinion for Tampere University researchers to participate in conducting the LAKANA trial. The trial followed the Declaration of Helsinki and Good Clinical Practice guidelines.

Informed consent was received on three levels: village level, household level, and caregiver level. Verbal consent by household heads or proxies was documented with a digital signature. The caregiver gave final consent for treatment. Substudy-specific information was given to those participating in this substudy and written or fingerprint signed guardian consent was sought.

Trial design

This cluster-randomized placebo-controlled trial was conducted as a prespecified substudy in a subset of villages within a large-scale trial in Western Mali testing the effect of azithromycin MDA on infant mortality: the LAKANA trial (ClinicalTrials.gov NCT04424511, registered 11 June 2020) [30]. The study protocol, statistical analysis plan, and all statistical operating procedures for the main trial and this substudy have been published prospectively [31–33].

The LAKANA trial randomized villages (clusters) into three trial arms: placebo, azithromycin MDA two times yearly, or azithromycin MDA four times yearly (quarterly). The villages were randomized into these three arms in a 3:4:2 ratio respectively. The data collection for this study (February 17 to May 30, 2022, dry season) was conducted during a single MDA round. The villages were visited every three months in the LAKANA trial, and the data collection for this substudy happened on the fourth MDA round, nine months after village enrollment. We consolidated the twice yearly and quarterly azithromycin arms into one treatment group, because villages in both azithromycin arms were receiving azithromycin at the time of this substudy MDA round.

Randomization and blinding

The LAKANA study villages were randomized into the three intervention arms in a public allocation event. Each village representative pulled a lottery ticket from a container with a letter code. The letter code defined the trial arm for the village. The participants, their guardians, village leaders, study site and laboratory personnel, and investigators were all masked. Only external partners facilitating the randomization were unmasked. The placebo and azithromycin substances and their packaging were indistinguishable. Further details of the randomization and masking have been published in the main trial report [30].

Eligibility

The 59 LAKANA substudy villages were in Kita region in Western Mali and were chosen due to their proximity to the Kita study headquarters, facilitating the timely transportation and storage of biological samples. All infants aged 4–11 months (120–364 days of age) in these villages were eligible for this substudy. Infants with severe illness, known macrolide allergy, or weighing less than 3.0 kg were excluded. Severely ill infants were excluded and referred to the nearest health facility.

Intervention and sample collection

Data collectors visited all households included in the parent trial and identified eligible infants. The infants and caregivers were accompanied to a temporary village facility, where the study drug was administered and baseline blood samples were obtained. Infants were weighed with an electronic scale (ADE Model M111600-01, Germany) and given reconstituted suspension of azithromycin 20mg/kg or placebo orally. Caregivers were asked to inform about any adverse events within 14 days of receiving the drug.

The study nurse performed a heel prick with a small lancet to obtain blood drops. At least two blood spots were applied to a dry blood spot (DBS) collection card (PerkinElmer PKI RUO spot Saver Card, USA). Blood hemoglobin (Hb) concentration was determined from a heel prick blood drop using a bedside kit (Orion QuickRead go, Finland), which gave immediate results. The DBS cards were air-dried for 3 hours on a drying rack and then placed in individually sealed plastic bags with a desiccant bag (QIAGEN WB100003, Germany) to prevent humidification. The sample bags were transported to Bamako laboratory without cold storage within 1–2 days from sample collection. If Hb was lower than 80 g/L, the child was referred to the nearest health facility.

The data collectors returned to the village after 14 days, identified the infants and accompanied them to the temporary village facility, where blood samples were obtained again. Caregivers were interviewed on day 14 about adverse events. LAKANA adverse events were reported in the main trial report [30].

Outcomes

The outcomes were malaria prevalence (PCR-detected parasitemia), mean parasite load (DNA concentration in ng/μL as a proxy for disease severity and parasites per blood volume), and mean hemoglobin concentration (g/L) [34,35]. Variables tested as possible effect modifiers were infant age, sex, weight-for-age z-score (WAZ), household asset index, and water, sanitation, and hygiene (WASH) index. The asset and WASH indices are described in more detail in the trial statistical analysis plan [31].

Laboratory analyses

The DNA extraction of Plasmodium falciparum was carried out using the Extracta DBS reagent (Quantabio BioSciences), a ready-to-use protocol optimized for PCR-ready DNA recovery from DBS. We performed quantitative real-time polymerase chain reaction (PCR) to amplify the lactate dehydrogenase (LDH) gene, a marker of Plasmodium falciparum, in the isolated DNA. Reaction volumes were 25 µl each, consisting of 12.5 µl Master Mix, 5 µl of gDNA, forward and reverse primers, probe, and molecular-grade water. Genomic DNA (10 µg/mL) from Plasmodium falciparum strain 3D7 (MRA-102G, BEI Resource, USA) was used to generate serial dilutions for the qPCR standard curve. All reactions were run in duplicate. Samples were considered positive if both had a cycle threshold (Ct) value of < 38 [36,37]. If the two results had discrepancies, we repeated the PCR. The blood DNA concentration of Plasmodium falciparum as a proxy for parasite density and disease severity was derived from real-time PCR standard curves, as validated by others [34,35,38].

Sample size

We prospectively estimated the 59 substudy villages to have 333–450 infants in each study arm and 1000–1350 infants aged 1–11 months in total. The twice-yearly and quarterly yearly azithromycin arms were pooled. This offered an expected statistical power of 80% to detect an effect size of 0.2-0.3 standard deviations comparing azithromycin and placebo at 5% two-sided type 1 error rate for each outcome, given the inter-cluster correlation coefficient was not larger than 0.03 [31]. One substudy village served as a pilot village for testing substudy data and sample collection and was therefore excluded from analyses.

Statistical analysis

We used mixed-effects regression models with random intercepts for villages to estimate the effect of the intervention on malaria prevalence, Hb concentration, and malaria parasite load (logistic model for prevalence and linear models for continuous variables). Marginal risk ratios were predicted from the obtained model coefficients. The independent variables were baseline outcome measures (PCR-positivity, parasite load, or Hb) and intervention group. The day 14 outcome measures were the dependent variables. For the effect modifier analyses we accounted for interactions between the compared subgroups and tested the statistical significance of interactions (predefined p-for-interactions <0.1). All statistical analyses were done with R programming language version 4.5.2.

Because parasite load data distributions were highly skewed, we reported geometric means for parasite load and used log-transformed parasite load values in the regression model. Because log-transformation cannot accommodate zero values, malaria negative samples were assigned a value equal to half of the lowest serial dilution (0.000005 ng/µl of Plasmodium falciparum DNA). This approach, commonly used in antibody titer studies, was applied to include participants who were malaria negative on day 0 or 14 [39].

Results

A total of 1158 infants from 58 LAKANA villages were eligible for this malaria substudy (Fig 1). Malaria status at baseline (day 0) was available for 1038 infants, of whom 101 were malaria PCR-positive. At baseline, malaria prevalence was 9.4% in the control group and 9.9% in the azithromycin group (Table 1). Mean parasite load at baseline was 0.0056 ng/μL (geometric mean 0.0000089 ng/μL) in the control group and 0.0036 ng/μL (geometric mean 0.0000101 ng/μL) in the azithromycin group, with values ranging from 0.000054 to 0.5881 ng/μL among malaria PCR-positives. Mean Hb concentrations at baseline were 103 g/L in the control group and 99 g/L in the azithromycin group, with values ranging from 50 to 224 g/L. Severe anemia (Hb < 70 g/L) was observed in 8.6% of infants at baseline.

Fig 1. Participant flow chart.

Fig 1

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Table 1. Baseline characteristics (day 0) of infants by treatment group.

Variable Control Azithromycin
Number of infants 324 834
Number of villages 20 38
Average number of infants per village (SD) 16.2 (8.9) 21.9 (22.5)
Proportion of girls, % 51.5 48.3
Mean (SD) age, months 7.8 (2.4) 7.7 (2.3)
Prevalence of PCR-positive malaria, % 9.4 9.9
Mean (SD) Hb, g/L 103 (26) 99 (24)
Mean (SD) parasite load, 10–3 ng/μL 5.6 (44.7) 3.6 (26.8)
Proportion with self-reported fever in preceding 2 weeks, % 0.0 1.1
Proportion of infants who received SMC in the past 6 months, % 0.6 1.4
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SD = standard deviation, SMC = seasonal malaria chemoprevention, Hb= hemoglobin.

A total of 1036 infants were revisited on day 14, with 890 malaria results available. Infants were revisited on average 16.0 (3.1SD) days after receiving the study drug (range: 8–30 days). Loss to follow-up rates for malaria results were 21.9% in the control group and 15.3% in the azithromycin group. One azithromycin group village with only one eligible infant was lost to follow-up for malaria outcomes.

Primary outcomes

On day 14, malaria prevalence was 8.9% in the control group and 8.0% in the azithromycin group, with mean parasite loads of 0.0052 ng/μL (geometric mean 0.0000094 ng/μL) and 0.0038 ng/μL (geometric mean 0.0000087 ng/μL) respectively (Table 2). There was no statistically significant difference in malaria prevalence with azithromycin compared to placebo (Table 2). Adjusted for clusters and baseline values, malaria parasite load was 9% lower with azithromycin (ratio of geometric means 0.91, 95% CI: 0.66 to 1.25), but the result was not statistically significant (p = 0.56). There was no statistically significant difference in Hb concentration with azithromycin.

Table 2. Malaria prevalence, parasite load, and Hb concentration at day 14 by treatment group.

Variable Control Azithromycin Risk ratioa

estimate (95% CI)		 p-value
Prevalence of PCR-positive malaria, % 8.9 8.0 0.96 (0.63 to 1.48) 0.86
Prevalence of PCR-positive malaria among baseline malaria positives, % 66.7 55.7 0.81 (0.55 to 1.19) 0.28
Ratio of geometric

meansb estimate (95% CI)
Mean (SD) parasite load, 10–3 ng/μL 5.2 (32.2) 3.8 (43.5) 0.91 (0.66 to 1.25) 0.56
Difference in means estimate (95% CI)
Mean (SD) Hb concentration, g/L 99 (24) 99 (21) 0.1 g/L (-3.9 to 4.1) 0.97
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SD = standard deviation, Hb= hemoglobin

aRatio estimates account for baseline values and are regression coefficients calculated with a mixed-effects regression model with random intercepts for clusters.

bRatio of means estimate uses geometric means due to log-transformation.

Effect modifiers and post-hoc analyses

The test for interactions was not significant for any of the tested potential effect modifier variables for malaria prevalence. No effect modifier subgroup was statistically significantly favoring either azithromycin or placebo for malaria prevalence or parasite load (Fig 2 and 3). Subgroup sample sizes were small, resulting in wide confidence intervals.

Fig 2. Prevalence of PCR-positive malaria (%) at day 14 for each effect modifier by treatment group.

Fig 2

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WAZ = weight-for-age Z-score. Predefined statistical significance at p-for-interactions = 0.1. Risk ratio estimates account for baseline values and are regression coefficients calculated with a mixed-effects regression model with random intercepts for clusters and accounting for interactions.

Fig 3. Parasite load (10-3 ng/μL) at day 14 for each effect modifier by treatment group.

Fig 3

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WAZ = weight-for-age Z-score. aPredefined statistical significance at p-for-interactions = 0.1. bRatios of means are estimated ratios of geometric means due to the log-transformation of parasite load values. They account for baseline valuesand are regression coefficients calculated with a mixed-effects regression model with random intercepts for clusters and accounting for interactions.

In a post-hoc analysis including only infants who were PCR-positive at baseline, mean parasite load at baseline was 0.0642 ng/μL (geometric mean 0.0035 ng/μL) in the control group and 0.0366 ng/μL (geometric mean 0.0066 ng/μL) in the azithromycin group. On day 14, mean parasite loads were 0.0510 ng/μL (geometric mean 0.0014 ng/μL) in the placebo group and 0.0363 ng/μL (geometric mean 0.0003 ng/μL) in the azithromycin group. Log-transformed, and accounting for baseline values and clusters, the ratio of means was 0.15 (95% CI: 0.06 to 0.37, p < 0.001) favoring azithromycin, indicating an 85% reduction in parasite load on average among malaria positive participants.

Discussion

We aimed to test the effect of a single dose of azithromycin on malaria prevalence and parasite load among Malian 4–11-month-old infants. In a sample of 58 villages with 1158 infants, we did not find a statistically significant differences between azithromycin and control groups regarding prevalence of PCR-positive malaria, mean parasite load or hemoglobin levels.

These null results are likely because the study was underpowered, due to a lower-than-expected malaria prevalence and subsequently a smaller than expected number of malaria cases. At the time of the design of this LAKANA substudy, the 2018 Demographic Health Survey of Mali reported a 13% prevalence of malaria in Kayes region and 22% in neighboring Koulikoro region among 6–59 months old children [40]. In our sample, the baseline prevalence was clearly lower, at around 9%. Notably, all the point estimates in our study consistently favored azithromycin, including a 4% overall risk reduction, a 19% risk reduction among baseline malaria positive infants, a 9% reduction in overall parasite load, and an 85% reduction in parasite load among baseline PCR-positive infants. However, we were unable to reject the null hypotheses due to the low number of malaria cases. Common to many other studies not seeing a convincing effect on malaria is a smaller sample size (below 300 in follow-up) in both treatment groups [41–43].

Results from trials looking at the effects of azithromycin MDA on malaria vary widely. Many azithromycin trials have observed effects on malaria prevalence, parasite density, antibody response (force of infection), malaria clinic visits, and malaria mortality [6,20–23,44]. Some of the starkest reductions in malaria have been in a trachoma azithromycin MDA trial in Tanzania (73% reduction in malaria cases), the MORDOR trial in Niger (malaria parasitemia OR 0.54), and the CHAT trial in Burkina Faso (malaria mortality OR 0.67) [6,21,23]. Conversely, there are large-scale trials reporting no effect on malaria. A trial conducted in Mali and Burkina Faso testing SMC given together with placebo or azithromycin, observed more than 20 000 malaria cases, but reported no significant reduction in parasitologically confirmed malaria with azithromycin [24]. The MORDOR Tanzania and Malawi trials observed no significant reduction in malaria outcomes [26,43]. A recent large-scale trial in Burkina Faso (MIRAMA trial), with a similar design as LAKANA, treating only 1–11-month-olds only, reported no statistically significant reduction in malaria mortality in verbal autopsy analyses, but did not report parasitemia prevalence or parasite density [11]. Two trials in Burkina Faso, with similar timing design as our study (two weeks), tested malaria right before and two weeks after azithromycin distribution and reported no reduction in malaria [41,42].

Growing evidence points to a herd protection effect as a possible mechanism for azithromycin MDA mortality reduction [3,45,46]. Reduction in mortality has only been observed when all under 5-year-olds in the community have been treated [1–3,11,12]. One azithromycin trial in Niger demonstrated that treating only 1–4 year-olds resulted in a mortality reduction in untreated infants, suggesting a spillover protective effect [46]. Another azithromycin MDA study also reported possible herd protective spillover effects in untreated individuals [45]. Malaria is transmitted mostly from the immediate community reservoir, and if azithromycin has only a weak antimalarial effect, then treating only a small subset of the community should not result in notable herd protection against malaria. Because we treated only 4–11-month-old infants, any possible herd effects from azithromycin may have been missed. Similarly, the lack of a herd protection effect in the MIRAMA trial (also treating only infants), may explain the non-significant reduction in malaria deaths in their study [11].

Our study had several limitations. We only tested for Plasmodium falciparum, but this is unlikely to cause any major bias since Plasmodium malariae, vivax and ovale are very rare in Kayes region [47,48]. In the analysis of parasite load, malaria negative samples were assigned a value equal to half of the lowest serial dilution to avoid exclusion during log-transformation. While this approach is generally accepted in antibody titer studies, it may introduce a minor bias [49]. Although prespecified, the study was not powered for subgroup analyses; these were conducted to provide preliminary evidence on possible effect modifiers, requiring confirmation in larger studies. Our results cannot be generalized to settings with an ongoing rainy season malaria outbreak, extensive SMC coverage, or when older children are also treated.

In conclusion, this trial introduced new evidence on the effect of azithromycin on malaria in the WHO 2020 guidelines recommended infant subgroup. We observed a slightly lower malaria prevalence and parasite load with a single dose of azithromycin, but the results were not statistically significant, possibly due to a low number of malaria cases. The findings do not confirm, but are consistent with, the hypothesis that a single dose of azithromycin reduces malaria prevalence or parasite load among infants in this malaria-endemic West African setting.

Supporting information

S1 CONSORT Checklist. CONSORT (Consolidated Standards of Reporting Trials) checklist.

(PDF)

pgph.0006518.s001.pdf (114.8KB, pdf)
S1 LAKANA Original Protocol. LAKANA trial original protocol, 2019-11-27.

(PDF)

pgph.0006518.s002.pdf (2.1MB, pdf)
S1 LAKANA Latest Protocol. LAKANA trial protocol with latest amendments, 2024-11-13.

(PDF)

pgph.0006518.s003.pdf (1.8MB, pdf)

Acknowledgments

We thank the communities and families in Kita region for participating in the study. Great thanks go to the field team, nurses and data collectors for their efforts in a challenging setting and terrain. We thank the CVD-Mali laboratory team for conducting the PCR analyses. We are grateful to Nigel Klein for his contributions to the study design and trial administration.

Data Availability

Data is available in Zenodo in a public repository with restricted access (https://doi.org/10.5281/zenodo.19470319) at request from the LAKANA trial project steering group by contacting the LAKANA team at lakana@tuni.fi. R codes for the statistical analyses are available in Zenodo (https://doi.org/10.5281/zenodo.19501230).

Funding Statement

This work was funded by the Gates Foundation (OPP1210821, INV-003354, and INV-005877). RI received personal grants for this work from Finska Läkaresällskapet (The Medical Society of Finland). The study drugs were provided by Pfizer Inc. (New York City, NY, USA). The funders had no role in the conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

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PLOS Glob Public Health. 2026 Aug 20;6(8):e0006518. doi: 10.1371/journal.pgph.0006518.r001

Author response to Decision Letter 0

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5 May 2026

PLOS Glob Public Health. doi: 10.1371/journal.pgph.0006518.r002

Decision Letter 0

Alassane Dicko

30 Jun 2026

PGPH-D-26-01585

A single dose of azithromycin versus placebo for malaria in infants: A cluster-randomized trial

PLOS Global Public Health

Dear Dr. Ihamuotila,

Thank you for submitting your manuscript to PLOS Global Public Health. After careful consideration, we feel that it has merit but does not fully meet PLOS Global Public Health’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Alassane Dicko

Academic Editor

PLOS Global Public Health

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Additional Editor Comments (if provided):

This manuscript reports the findings of a well-conducted cluster-randomized trial evaluating the effect of a single dose of azithromycin on malaria prevalence and parasite density among infants in Mali. The study addresses an important research question and adds to the growing body of evidence on the potential impact of azithromycin on malaria. Overall, this is a high-quality manuscript that is suitable for publication with minor revisions are required to further improve the manuscript. The authors are encouraged to carefully address the specific comments provided by the reviewers to strengthen the manuscript prior to final acceptance.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does this manuscript meet PLOS Global Public Health’s publication criteria?>

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?-->?>

Reviewer #1: No

Reviewer #2: I don't know

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: This study examines azithromycin treatment in a CRT substudy, hypothesizing that azithromycin reduces malaria prevalence, parasite load, and improves hemoglobin concentration. However, the analyses are not presented clearly enough to indicate the expected statistical power with the current sample size. Ultimately, no results reach statistical significance, and the manuscript appropriately acknowledges the insufficient power.

Additional details are needed regarding the regression analyses in the statistical methods. The tables and figures are well designed.

More specific comments below.

Abstract: The conclusion of the abstract should be revised. I do not agree with the statement that the results are consistent with the hypothesis.

Methods

Lines 96-98: The statement that data were collected during a single MDA should be clarified as it was the fourth round of a set of MDA. This part requires clarification.

Effect size 0.2-0.3 SD. Since three different outcomes are reported, specify which effect size refers to each outcome.

This also requires clarification.

Did the pilot study provide estimates for baseline and mean parasite load?

As this is a substudy, the Methods section should report the cluster size for each arm.

Also, it may be best to evaluate the expected power using the known cluster sizes, number of clusters, baseline prevalence, and other relevant measures. Data from the pilot study could be used for these estimates.

Statistical analysis: Logistic regression was used to model outcome prevalence, but the analytic approach for parasite load and hemoglobin concentration should also be specified clearly in the Methods section.

Results

Graphics and tables are well prepared. The results indeed do not indicate significant effects.

Discussion

Lines 277-279: This statement is speculative and should be removed from the discussion.

Reviewer #2: This manuscript describes findings from a sub-study nested within a cluster randomized controlled trial in Mali, where the sub-study aimed to assess if azithromycin mass treatment for infants has any effect on malaria prevalence and mean parasitemia in infants. The study was intended to assess if changes in malaria burden are an intermediate factor which is attributable for changes in all cause mortality observed in other studies after azithromycin mass treatment. However, since no effect of azithromycin on ACCM was observed in the main trial, it is perhaps not surprising that no significant effect of azithromycin on malaria was observed. Nevertheless, this research merits publication, is generally well-designed and presented, and contributes to the evolving evidence base on this theme.

1. Line 50. I recommend modifying here and throughout the rest of the paper to clarify between “malaria” (generally used as an indication of clinically-evident or symptomatic disease) and Plasmodium infection (a more specific description of the outcomes actually presented here).

2. Line 81. This sentence seemed to directly contradict the previous paragraph, where you have described multiple other trials of azithromycin with malaria-related outcomes.

3. Line 89. It may be informative for readers who are not familiar with the LAKANA trial to state here or in the next paragraph if this substudy was completed in all trial clusters, or only a subset. Line 128 is also slightly ambiguous and hints that this study was done in a subset of the most accessible clusters.

4. Line 184. It is not clear which outcome was used for the sample size calculation. What was the anticipated prevalence of P. falciparum by PCR in this population?

5. Lines 190-192. Presumably the mixed-effects model used individual level data, with the outcome being positive/negative status by PCR? I would argue that if this is the case, it may be clearer to describe the outcome as “P. falciparum infection by PCR” rather than prevalence which is an aggregate measure at the cluster level. Furthermore, I assume the model uses the PCR result at the 14-day follow-up as the outcome, and the PCR result at baseline as a covariate? This detail would be helpful to include.

6. Table 1. I believe that the notation for the Mean parasite load should be 10^-3 ng/ul, not 10^3. Please check this throughout the paper.

7. Methods/results. Please include a statement somewhere in these sections describing the dates of data collection, and how these dates relate to the usual seasonal transmission period in Mali. The low SMC coverage suggests that data collection was done during the dry season?

8. Line 236. Did you collect data on how many of the children with severe anemia that were referred to the health center actually completed this referral, and if this referral rate differed between arms? This is a potential bias in your interpretation of azithromycin effects on Hb.

9. Table 2. I’m not sure what is meant by “general” prevalence of malaria. Suggest changing this to “baseline prevalence of P. falciparum by PCR”. Similarly, I recommend modifying “prevalence of malaria among baseline malaria positives” to something like “Day 14 prevalence of P. falciparum by PCR among those PCR-positive at baseline”.

10. Lines 255-256. This model result is very surprising considering the descriptive results reported earlier in this paragraph, which state that the mean parasite density among PCR positives is essentially unchanged in the azithromycin group (0.0366 at baseline, 0.0363 at day 14) while the parasite densities reduced in the placebo arm among malaria positives (0.0642 at baseline, 0.0510 at day 14). I may have misunderstood the data which is being used in the model, but this indicates some further explanation is required to ensure to either explain how the results in the adjusted model are so different from the descriptive analysis, or explain if there is a different population assessed in the model.

11. Line 266. It is difficult to interpret or agree with this statement since the assumed prevalence is not reported in the sample size section, suggesting that prevalence was not a contributing factor in the calculation.

12.Lines 274. As with my earlier comment, the 85% reduction in parasite load among baseline positive infants does not seem to agree with description of the results, suggesting substantial confounding or imbalance that is being accounted for in the adjusted model.

13. Lines 298-306. This may be a suitable place to discuss whether exclusion of children identified as severely ill from trial was a potential limitation. My expectation is that for studies with ACCM as the outcome, these most vulnerable children are the ones that would benefit most from this kind of intervention, whether the effect is mediated through malaria parasitemia reduction or some other mechanism. Did these trials all exclude severely ill children, similar to the LAKANA trial?

14. Lines 318. The statement “ongoing seasonal malaria outbreak” isn’t clear. Does this mean a usual seasonal malaria transmission setting, but during the seasonal peak (implying this study was done in the low transmission season)? Or does this relate to an epidemic, where transmission accelerates beyond the usual expected level?

15. Line 323. I don’t agree that you have definitively shown that the reason for the null result was the low prevalence of malaria. It seems that azithromycin did not have a substantial effect on clearing parasites among those who were infected, which aligns with the Cochrane review showing that azithromycin has only a weak antimalarial effect.

Reviewer #3: 1. Question: was a sensitivity analysis done regarding the missing village?

2. What are the restrictions on data availability for the repository? I do not find anything unreasonable about the way the data are being provided.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLOS Glob Public Health. doi: 10.1371/journal.pgph.0006518.r004

Decision Letter 1

Alassane Dicko

5 Aug 2026

A single dose of azithromycin versus placebo for malaria in infants: A cluster-randomized trial

PGPH-D-26-01585R1

Dear Dr. Ihamuotila,

We are pleased to inform you that your manuscript 'A single dose of azithromycin versus placebo for malaria in infants: A cluster-randomized trial' has been provisionally accepted for publication in PLOS Global Public Health.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Global Public Health.

Best regards,

Alassane Dicko

Academic Editor

PLOS Global Public Health

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 CONSORT Checklist. CONSORT (Consolidated Standards of Reporting Trials) checklist.

    (PDF)

    pgph.0006518.s001.pdf (114.8KB, pdf)
    S1 LAKANA Original Protocol. LAKANA trial original protocol, 2019-11-27.

    (PDF)

    pgph.0006518.s002.pdf (2.1MB, pdf)
    S1 LAKANA Latest Protocol. LAKANA trial protocol with latest amendments, 2024-11-13.

    (PDF)

    pgph.0006518.s003.pdf (1.8MB, pdf)
    Attachment

    Submitted filename: Response to Reviewers.docx

    pgph.0006518.s005.docx (32.5KB, docx)

    Data Availability Statement

    Data is available in Zenodo in a public repository with restricted access (https://doi.org/10.5281/zenodo.19470319) at request from the LAKANA trial project steering group by contacting the LAKANA team at lakana@tuni.fi. R codes for the statistical analyses are available in Zenodo (https://doi.org/10.5281/zenodo.19501230).

    Articles from PLOS Global Public Health are provided here courtesy of PLOS

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