Trachoma, caused by Chlamydia trachomatis (Ct), persists as a major cause of preventable blindness despite the global SAFE strategy. Understanding how Ct genovars and genovariants influence infection dynamics and clinical outcomes is crucial for sustaining elimination efforts and informing vaccine development. A four-year longitudinal study was conducted in a trachoma-endemic region of Tanzania across multiple rounds of mass drug administration (MDA) with azithromycin. Ct infections were genotyped by ompA sequencing to identify genovars and genovariants. Associations between genetic variants, bacterial load, and clinical signs of trachoma were assessed. Following MDA, a shift in Ct genovar prevalence occurred from genovar B to genovar A. Genovar B was associated with more severe clinical signs, including follicles, papillae, and scarring, whereas genovar A infections exhibited higher bacterial loads. Among 121 individuals with recurrent infections, 94% were re-infected with the same genovar, indicating limited protective immunity and incomplete clearance despite MDA coverage exceeding 60%. The genovariants B2, B9, and A2 predominated, with an A→T amino acid substitution in B9 potentially modifying antigenic recognition. Post-MDA, normalized genovariant diversity increased, suggesting ongoing transmission or strain reintroduction. Distinct genovar-associated clinical and immunological patterns underscore the need to elucidate genovar-specific virulence and immune evasion mechanisms. These findings provide key insights for optimizing trachoma control and advancing vaccine development.

World Health Organization guidelines for control of soil-transmitted helminths (STHs) rely on coproscopic methods to assess population prevalence. In low-prevalence and light-intensity STH settings, quantitative PCR (qPCR) has higher sensitivity and specificity for detection. For qPCR to accurately identify transmissible infections of public health significance, it is essential to interpret qPCR cycle threshold (Ct) results. As part of the DeWorm3 community-based cluster-randomized trial on interrupting STH transmission, we conducted population-based surveys using high-throughput qPCR and aimed to establish appropriate Ct cutoffs to detect transmissible infections. Experimental approaches, including egg and genome-equivalent spiking experiments, were hindered by inefficient fecal DNA extraction despite optimization efforts. The Ct results for 29,980 samples (pre-intervention, cross-sectional surveys) revealed a bimodal distribution for two of the four species tested, Necator americanus and Ascaris lumbricoides. The first peak was assumed to represent transmissible infections, and the second peak to represent indeterminate or non-transmissible infections. Using a finite mixture model, we defined true qPCR positivity as any Ct result with a ≥5% chance of belonging to the first peak. This approach yielded Ct cutoffs of 34.4398 for N. americanus and 28.57587 for A. lumbricoides. For hookworms, the sensitivity of qPCR was 96.7%, compared to 73.2% for Kato-Katz, and moderate- to heavy-intensity infections (median Ct, 19.1; interquartile range [IQR], 17.9-19.8) were differentiated from both light-intensity infections and Kato-Katz-negative samples (25.3; IQR, 22.5-27.9). Our findings demonstrate the feasibility and utility of evidence-based Ct cutoffs to identify transmissible STH infections in large-scale surveys and categorize infection intensity as programmatically relevant.IMPORTANCECurrently, appropriate cycle threshold (Ct) cutoff values for soil-transmitted helminth quantitative PCR (qPCR) testing do not exist. As a result, it is not possible to differentiate a qPCR "positive" that represents a transmission risk from a signal resulting from non-infectious material such as fragmented eggs or sloughed cells. Leveraging samples collected and tested as part of the DeWorm3 cluster-randomized controlled trials, we used a data-driven statistical approach to establish a qPCR Ct cutoff that distinguishes epidemiologically relevant transmissible infections from non-transmissible DNA. This study highlights the utility of evidence-based qPCR Ct cutoffs to identify soil-transmitted helminth infections when evaluating and informing deworming programs.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT03014167.

BACKGROUND:Soil-transmitted helminths are targeted for elimination as a public health problem. This study assessed whether, with high coverage, community-wide mass drug administration (MDA) could lead to transmission interruption. METHODS:DeWorm3 is an open-label, community cluster-randomised controlled trial in Benin, India, and Malawi. In each country, a single governmental administrative unit (population ≥80 000 individuals) with soil-transmitted helminth endemicity and participation in at least five rounds of community-wide MDA for lymphatic filariasis, was divided into 40 clusters (population ≥1650 individuals), which were randomly assigned (1:1) to community-wide MDA versus school-based deworming. Laboratory personnel were masked to exposure status and all investigators were masked to post-baseline outcome data until unmasking. In all clusters, preschool-aged and school-aged children received school-based deworming as per national guidelines for 3 years. In intervention clusters, door-to-door community-wide MDA (a single oral dose of 400 mg albendazole) was delivered to all eligible individuals biannually by community drug distributors for 3 years. All individuals aged 12 months and older in India and Benin and aged 24 months and older in Malawi were eligible for treatment, except women in the first trimester of pregnancy, those with adverse reactions to benzimidazoles, those who were acutely ill or intoxicated, or those reporting treatment within the previous 2 weeks. The co-primary outcomes were individual-level prevalence and cluster-level transmission interruption (ie, weighted prevalence of predominant species of ≤2%) of the predominant soil-transmitted helminth species, assessed by quantitative PCR (qPCR) 24 months after the last round of MDA. The analysis set contained a subset of randomly selected participants per cluster who enrolled in the endline assessment, provided a stool sample, and had a qPCR result. All individuals who received treatment were eligible for inclusion in the safety population. This trial is registered with ClinicalTrials.gov (NCT03014167), and is active but not recruiting. FINDINGS:Between Oct 10, 2017, and Feb 17, 2023, 120 clusters (40 clusters per country, comprising 357 716 individuals) were randomly assigned, 60 to community-wide MDA and 60 to school-based deworming. 184 030 (51·4%) individuals in the clusters at baseline were female, 173 663 (48·5%) were male, and 23 (<0·1%) were other. The analysis set consisted of 58 827 individuals in the control group and 58 554 in the intervention group 24 months after the cessation of all deworming, Necator americanus prevalence (the predominant species at all sites) in the community-wide MDA group was lower than the school-based deworming group in Benin (adjusted prevalence ratio [aPR] 0·44 [95% CI 0·34-0·58]), India (0·41 [0·32-0·52]), and Malawi (0·40 [0·34-0·46]). Transmission interruption was achieved for N americanus in 11 (55%) of 20 intervention clusters versus six (30%) of 20 control clusters in Benin (p=0·20), in one (5%) intervention cluster versus no control clusters in India (p=1·00), and in no clusters in either group in Malawi (p=1·00). 984 adverse events were reported among 487 participants over the study, of which 32 among 13 participants resulted in hospitalisation and were classified as serious adverse events (three of which were related to study procedures). INTERPRETATION:Soil-transmitted helminth transmission interruption might be possible in focal geographies but does not appear to be programmatically feasible within the evaluated timeframe. Community-wide MDA should be considered as an alternative strategy to school-based deworming programmes to improve equity and outcomes in helminth-endemic areas. FUNDING:The Gates Foundation.

Trachoma is targeted for global elimination as a public health problem by 2030. Measurement of IgG antibodies in children is being considered for surveillance and programmatic decision-making. There are currently no programmatic guidelines based on serology, which represents a generalizable problem in seroepidemiology and disease elimination. Here, we collate Chlamydia trachomatis Pgp3 and CT694 IgG measurements from 48 serosurveys across Africa, Latin America, and the Pacific Islands (41,168 children ages 1–5 years) and propose a novel approach to estimate the probability that population C. trachomatis transmission is below or above levels requiring ongoing programmatic action. We determine that trachoma programs could halt control measures with >90% certainty when seroconversion rates (SCRs) are ≤2.2 per 100 person-years. Conversely, SCRs ≥4.5 per 100 person-years correspond with >90% certainty that further control interventions are needed. More extreme SCR thresholds correspond with higher levels of confidence of elimination (lower SCR) or ongoing action needed (higher SCR). This study demonstrates a robust approach for using trachoma serosurveys to guide elimination program decisions.

Serology is increasingly used to monitor disease transmission and elimination. Embedding dried blood spot collection in trachoma surveys allows transmission intensity inference through population-level seroconversion rates (SCR) estimation, but there is no formal assessment of the required sample size. Using data from 40 prevalence surveys, we estimated intra-cluster correlation coefficient, a key design parameter, and assessed survey design considerations for incorporating serological monitoring. Design scenarios focused on recent proposed operational SCR thresholds (2.2 [no action needed] and 4.5 [action needed] per 100 child years) for interpretation of serological data in low-transmission and post-elimination settings. We evaluated SCR estimation in 42 two-stage designs by calculating precision (confidence interval width around an SCR value) and power (the measure of deviation of suggested thresholds to the SCR value). When the underlying SCR is ≤1.5 and >5.7 per 100 person-years, sample sizes between 300-2000 allowed good precision of SCR estimation. The same sample range would correctly classify areas as above or below the thresholds with >80% power when the underlying SCR is <1.7 or >5 per 100 person-years in lower and higher endemicity settings, respectively. Our results support estimation of serological data via the recommended population-based survey design for trachoma monitoring.

Background: Mass drug administration (MDA) is a key strategy in the response to neglected tropical diseases (NTDs). Operational research indicates that coverage of adult men in MDA is frequently lower as compared to adult women, but this remains poorly understood and obscured by a lack of routine sex-disaggregated reporting. Such differences may represent a potential threat to NTD strategies that require high MDA treatment coverage to achieve control or elimination objectives. Methods: Using detailed treatment data from a cluster-randomised trial of community-based MDA for soil-transmitted helminths (STH) conducted in southern Malawi, this secondary analysis describes sex-specific trends in coverage over six consecutive rounds of MDA delivered biannually at household level. We report community-level coverage by both a per-protocol (trial) definition, and pragmatic operational definition to simulate routine implementation. We used multilevel mixed-effects logistic regression to investigate individual, household and programme factors associated with non-treatment by both coverage definitions amongst adult men. Results: Adult mens participation in MDA was substantially and consistently lower than adult womens. Median difference (female minus male) in community-level protocol coverage was 14.9% (IQR: 9.2, 19.6), increasing to 21.7% (IQR: 15.9, 28.7) by operational coverage. Factors associated with increased odds of non-treatment were similar by coverage definition and included younger age, lower levels of education, and previous reported absenteeism; and were reduced by modifiable programmatic factors such as the day or time of household visit. Conclusions: This analysis highlights the persistence of inequity for adult men in participation in MDA, despite a well-resourced context that achieved high coverage of adult women. More nuanced MDA strategies that respond to the needs of adult men will be required to achieve equitable coverage, accompanied by robust monitoring and evaluation to evaluate their effectiveness at reaching this under-served population. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial NCT03014167 ### Funding Statement The DeWorm3 study was funded through grants to the Natural History Museum of London (OPP1129535) and University of Washington (INV-022149, INV-030049, INV-002114) from the Gates Foundation. The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of this report. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The DeWorm3 parent trial was approved by the College of Medicine Research Ethics Committee at the University of Malawi (P.04/17/2161), the London School of Hygiene and Tropical Medicine Observational/Interventions Research Ethics Committee (12013) and the Human Subjects Division at the University of Washington (STUDY00000180). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced are available online at https://doi.org/10.25934/PR00010754

Trachoma, caused by Chlamydia trachomatis (Ct), remains a leading cause of preventable infection- induced blindness worldwide. We conducted a 4- year longitudinal study in three trachoma- endemic villages in Northern Tanzania, tracking infection dynamics and factors influencing trachomatous scarring progression and persistence pre- and post- mass drug administration (MDA) interventions. We analysed 118 whole genomes of Ct originating from ocular swabs of children. Sample collection was conducted at 3- month intervals over 4 years, encompassing 15 timepoints. We studied Ct phylogeny and patterns of SNP accumulation within sequences in the Ct genotype A (CtA) and Ct genotype B (CtB) phylogenetic clades, with the association of clinical signs of trachoma and scarring progression. Of the samples analysed, 71 (60.2%) were identified as CtA and 47 (39.8%) as CtB. We observed a significant shift in genotype prevalence: CtB predominated in pre- MDA samples (36 out of 40, 90%), whilst CtA became the dominant genotype after the first MDA round (67 out of 78, 85.9%) (P<0.0001). Phylogenetic analysis revealed two distinct CtA clades: clade 1 (29 sequences) was primarily found pre- MDA and shared a common ancestor with Tanzanian CtA reference genomes, whilst clade 2 (42 sequences) emerged post- MDA and exhibited a characteristic -6 kbp reduction in the plasticity zone (PZ). Similarly, CtB sequences formed two distinct clades, both sharing ancestry with a Tanzanian CtB reference genome. Notably, we identified variable genome reduction in the PZ (-4 and -10 kbp) amongst 13 CtB sequences distributed across both clades. We documented a significant shift in Ct genotype distribution following the first round of MDA, characterized by the emergence of CtA strains with distinct genetic profiles compared to pre- MDA strains. The observed reductions in Ct genome size suggest ongoing evolutionary processes shaping these bacterial populations. Additional research is needed to understand the dynamic changes in Ct lineage composition before and after antibiotic interventions and to determine how variations in genome size influence Ct biology and its susceptibility to azithromycin treatment.

Importance:Mass azithromycin distributions may reduce malaria parasitemia in the short term, but longer-term effectiveness is unclear. Objective:To examine whether biannual mass azithromycin distributions are associated with lower rates of malaria parasitemia in preschool children living in Niger. Design, Setting, and Participants:A cluster randomized trial was performed from November 23, 2014, until June 9, 2020, as an ancillary trial to a larger trial studying the effect of mass azithromycin on child mortality. Study communities (ie, government-defined health catchment areas) in Niger were randomized in a 1:1 ratio to biannual (ie, twice-yearly) mass administration of azithromycin or placebo to all children aged 1 to 59 months and followed up for 5 years. Data analyses were performed from June 25, 2023, to April 27, 2025. Intervention:Twice-yearly administration of a single dose of oral azithromycin, 20 mg/kg, or placebo. Main Outcomes and Measures:The prevalence of parasitemia 4 years after the community started treatment, assessed in a random sample of 40 children per community. Results:Among the 30 communities in Niger included in the study at baseline, the 15 communities randomized to azithromycin consisted of 1695 children (mean [SD] age, 30.8 [2.8] months; 858 [51.8%] male) and the 15 communities randomized to placebo consisted of 3031 children (mean [SD] age, 30.6 [2.6] months; 157 [52.0%] male). The mean prevalence of malaria parasitemia at baseline was 8.9% (95% CI, 5.1%-15.7%) in the azithromycin arm and 6.7% (95% CI, 4.0%-12.6%) in the placebo arm. At annual follow-up visits up until month 48, parasitemia was not statistically significantly lower in the azithromycin arm compared with the placebo arm, assuming a 10% prevalence in the placebo arm (-3.3 percentage points [PP]; 95% CI, -5.8 to -0.2 PP; permutation P = .05). The Niger Ministry of Health instituted seasonal malaria chemoprevention (SMC) after the month 36 study visit. Analysis restricted to the period before SMC found significantly less parasitemia in the azithromycin arm compared with the placebo arm (4.8 PP lower; 95% CI, -7.4 to -1.3 PP; permutation P = .02). Conclusions and Relevance:In this placebo-controlled cluster randomized trial, malaria among children aged 1 to 59 months was lower in communities treated with biannual mass azithromycin, but the effect was significant only for the first 3 years of the trial, before SMC. Trial Registration:ClinicalTrials.gov Identifier: NCT02048007.

BACKGROUND:Leptospirosis is a zoonotic bacterial infection occurring worldwide. It is of particular public health concern due to its global distribution, epidemic potential and high mortality without appropriate treatment. The method for the management of leptospirosis, particularly in severe disease, is clouded by methodological inconsistency and a lack of standardized outcome measures. The study this protocol details aims to develop a core outcome set (COS) for leptospirosis research. A COS is a set of outcomes with international consensus as a minimum for reporting in future studies focusing on leptospirosis. Establishing a COS will contribute to harmonizing Leptospirosis treatment research and will be instrumental in constructing a high-quality evidence base to feed into a planned future rigorous international clinical trial on leptospirosis. METHODS:The COS-LEP study will employ a COS development methodology standardized by the COMET initiative framework. This includes (1) a systematic review of available quantitative and qualitative literature reporting therapeutic response and safety outcomes and measures; (2) focused interviews with healthcare professional and people treated for leptospirosis exploring outcomes of interests using qualitative methodology; (3) narrowing the choice of outcomes by international consensus using a Delphi survey process; and (4) undertaking a hybrid consensus meeting with key stakeholders to build the final COS. DISCUSSION:This protocol describes the method to develop the first core outcome set for use in human leptospirosis studies. This will not only be a key feature in the design of a future definitive randomized controlled trial, but also provide a structure for clinicians and researchers collecting treatment cohort data in the various settings where leptospirosis is a public health issue.

Soil-transmitted helminths (STHs) are intestinal parasites that affect over a billion people worldwide. STH control relies on microscopy-based diagnostics to monitor parasite prevalence and enable post-treatment surveillance; however, molecular diagnostics are rapidly being developed due to increased sensitivity, particularly in low-STH-prevalence settings. The genetic diversity of helminths and its potential impact on molecular diagnostics remain unclear. Using low-coverage genome sequencing, we assess the genetics of STHs within worm, faecal, and purified egg samples from 27 countries, identifying differences in the genetic connectivity and diversity of STH-positive samples across regions and cryptic diversity between closely related human- and pig-infective species. We define substantial copy number and sequence variants in current diagnostic target regions and validate the impact of genetic variation on qPCR diagnostics using in vitro assays. Our study provides insights into the diversity and genomic epidemiology of STHs, highlighting both the challenges and opportunities for developing molecular diagnostics needed to support STH control efforts.

Trachoma, a neglected tropical disease caused by Chlamydia trachomatis (Ct) serovars A–C, is the leading infectious cause of blindness worldwide. Africa bears the highest burden, accounting for over 86 % of global trachoma cases. We investigated Ct serovar A (SvA) and B (SvB) whole genome sequences prior to the induction of mass antibiotic drug administration in The Gambia. Here, we explore the factors contributing to Ct strain diversification and the implications for Ct evolution within the context of ocular infection. A cohort study in 2002–2003 collected ocular swabs across nine Gambian villages during a 6 month follow-up study. To explore the genetic diversity of Ct within and between individuals, we conducted whole-genome sequencing (WGS) on a limited number (n=43) of Ct-positive samples with an omcB load ≥10 from four villages. WGS was performed using target enrichment with SureSelect and Illumina paired-end sequencing. Out of 43 WGS samples, 41 provided sufficient quality for further analysis. ompA analysis revealed that 11 samples had highest identity to ompA from strain A/HAR13 (NC_007429) and 30 had highest identity to ompA from strain B/Jali20 (NC_012686). While SvB genome sequences formed two distinct village-driven subclades, the heterogeneity of SvA sequences led to the formation of many individual branches within the Gambian SvA subclade. Comparing the Gambian SvA and SvB sequences with their reference strains, Ct A/HAR13 and Ct B/Jali20, indicated an single nucleotide polymorphism accumulation rate of 2.4×10−5 per site per year for the Gambian SvA and 1.3×10−5 per site per year for SvB variants (P<0.0001). Variant calling resulted in a total of 1371 single nucleotide variants (SNVs) with a frequency >25 % in SvA sequences, and 438 SNVs in SvB sequences. Of note, in SvA variants, highest evolutionary pressure was recorded on genes responsible for host cell modulation and intracellular survival mechanisms, whereas in SvB variants this pressure was mainly on genes essential for DNA replication/repair mechanisms and protein synthesis. A comparison of the sequences between observed separate infection events (4–20 weeks between infections) suggested that the majority of the variations accumulated in genes responsible for host–pathogen interaction such as CTA_0166 (phospholipase D-like protein), CTA_0498 (TarP) and CTA_0948 (deubiquitinase). This comparison of Ct SvA and SvB variants within a trachoma endemic population focused on their local evolutionary adaptation. We found a different variation accumulation pattern in the Gambian SvA chromosomal genes compared with SvB, hinting at the potential of Ct serovar-specific variation in diversification and evolutionary fitness. These findings may have implications for optimizing trachoma control and prevention strategies.

Background Soil-transmitted helminths (STH) affect approximately 1.5 billion people globally. The current STH control strategy is annual or twice-annual preventive chemotherapy, typically school-based deworming targeting children and women of reproductive age. Mathematical modeling suggests that it may be possible to interrupt STH transmission through high-coverage community-wide mass drug administration (cMDA). DeWorm3 is a cluster randomized trial testing cMDA for prevalence reduction and transmission interruption. The purpose of this study is to describe coverage of cMDA in study clusters over time and correlates of coverage at individual and cluster levels.Methods From 2018-2020, DeWorm3 delivered six rounds of cMDA with 400 mg albendazole at sites in Benin, India, and Malawi. We report coverage, treatment uptake, and directly observed therapy across all rounds. Factors associated with coverage at the cluster level were identified using binomial generalized estimating equations, while factors associated with non-treatment at the individual level were identified using binomial mixed-effects models.Results Coverage was high across all clusters and rounds, exceeding the WHO target of 75% in all sites and across all rounds (78% to 95%); cluster-level coverage tended to increase over time. Younger, unmarried, and migratory adults were more likely to be untreated at all sites; adult males were more likely to be untreated in Benin and Malawi. Among children, girls were more likely to be untreated, as were non-school-attending and migratory children. Higher adult education was associated with greater odds of non-treatment among adults, but lower odds among children in the household. Belonging to a less wealthy or minority language-speaking household was associated with non-treatment among both adults and children.Conclusions It is possible to deliver community-wide MDA with high coverage. Unique individual and community-level factors influence treatment across settings, and these may be addressed through targeted programming. Trial Registration : Field Studies on the Feasibility of Interrupting the Transmission of Soil-transmitted Helminths (STH), NCT03014167.

Background School-based approaches are an efficient mechanism for the delivery of basic health services, but may result in the exclusion of children with disabilities if they are less likely to participate in schooling. Community-based ‘door to door’ approaches may provide a more equitable strategy to ensure that children with disabilities are reached, but disability is rarely assessed rigorously in the evaluation of health interventions.Objectives To describe the prevalence and factors associated with disability among children aged 5–17 years and to assess the relative effectiveness of routine school-based deworming (SBD) compared with a novel intervention of community-based deworming (CBD) in treating children with disabilities for soil-transmitted helminths.Setting DeWorm3 Malawi Site (DMS), Mangochi district, Malawi.Participants All 44 574 children aged 5–17 years residing within the DMS.Primary and secondary outcome measures Disability was defined as a functional limitation in one or more domains of the Washington Group/UNICEF Child Functioning Module administered as part of a community-based census. Treatment of all children during SBD and CBD was independently observed and recorded. For both intervention types, we performed bivariate analyses (z-score) of the absolute proportion of children with and without disabilities treated (absolute differences (ADs) in receipt of treatment), and logistic regression to examine whether disability status was associated with the likelihood of treatment (relative differences in receipt of treatment).Results The overall prevalence of disability was 3.3% (n=1467), and the most common domains of disability were hearing, remembering and communication. Boys were consistently more likely to have a disability compared with girls at all age groups, and disability was strongly associated with lower school attendance and worse levels of education. There was no significant difference in the proportion of children with disabilities treated during SBD when assessed by direct observation (−1% AD, p=0.41) or likelihood of treatment (adjusted risk ratio (aRR)=1.07, 95% CI 0.89 to 1.28). Treatment of all children during CBD was substantially higher than SBD, but again showed no significant difference in the proportions treated (−0.5% AD, p=0.59) or likelihood of treatment (aRR=1.04, 95% CI 0.99 to 1.10).Conclusion SBD does not appear to exclude children with disabilities, but the effect of consistently lower levels of educational participation of children with disabilities should be actively considered in the design and monitoring of school health interventions.Trial registration number NCT03014167.

Purpose Population-based prevalence surveys are essential for decision-making on interventions to achieve trachoma elimination as a public health problem. This paper outlines the methodologies of Tropical Data, which supports work to undertake those surveys.Methods Tropical Data is a consortium of partners that supports health ministries worldwide to conduct globally standardised prevalence surveys that conform to World Health Organization recommendations. Founding principles are health ministry ownership, partnership and collaboration, and quality assurance and quality control at every step of the survey process. Support covers survey planning, survey design, training, electronic data collection and fieldwork, and data management, analysis and dissemination. Methods are adapted to meet local context and needs. Customisations, operational research and integration of other diseases into routine trachoma surveys have also been supported.Results Between 29th February 2016 and 24th April 2023, 3373 trachoma surveys across 50 countries have been supported, resulting in 10,818,502 people being examined for trachoma.Conclusion This health ministry-led, standardised approach, with support from the start to the end of the survey process, has helped all trachoma elimination stakeholders to know where interventions are needed, where interventions can be stopped, and when elimination as a public health problem has been achieved. Flexibility to meet specific country contexts, adaptation to changes in global guidance and adjustments in response to user feedback have facilitated innovation in evidence-based methodologies, and supported health ministries to strive for global disease control targets.

Class II HLA loci DRB1, DQB1 and DPB1 were typed for a total of 939 Gambian participants by locus-specific amplicon sequencing. Participants were from multiple regions of The Gambia and drawn from two studies: a family study aiming to identify associations between host genotype and trachomatous scarring (N = 796) and a cohort study aiming to identify correlates of immunity to trachoma (N = 143). All loci deviated from HardyWeinberg equilibrium, likely due to the family-based nature of the study: 608 participants had at least one other family member included in the study population. The most common alleles for HLA-DRB1, DQB1 and DPB1 respectively were DRB1*13:04 (18.8 %), DQB1*03:19 (27.9 %) and DPB1*01:01 (25.4 %). Participants belonged to a variety of ethnicities, including the Mandinka, Fula, Wolof and Jola ethnic groups.

BackgroundOcular infections with Chlamydia trachomatis serovars A-C cause the neglected tropical disease trachoma. As infection does not confer complete immunity, repeated infections are common, leading to long-term sequelae such as scarring and blindness. Here, we apply a systems serology approach to investigate whether systemic antibody features are associated with susceptibility to infection. MethodsSera from children in five trachoma endemic villages in the Gambia were assayed for 23 antibody features: IgG responses towards two C. trachomatis antigens and three serovars [elementary bodies and major outer membrane protein (MOMP), serovars A-C], IgG responses towards five MOMP peptides (serovars A-C), neutralization, and antibody-dependent phagocytosis. Participants were considered resistant if they subsequently developed infection only when over 70% of other children in the same compound were infected. ResultsThe antibody features assayed were not associated with resistance to infection (false discovery rate < 0.05). Anti-MOMP SvA IgG and neutralization titer were higher in susceptible individuals (p < 0.05 before multiple testing adjustment). Classification using partial least squares performed only slightly better than chance in distinguishing between susceptible and resistant participants based on systemic antibody profile (specificity 71%, sensitivity 36%). ConclusionsSystemic infection-induced IgG and functional antibody responses do not appear to be protective against subsequent infection. Ocular responses, IgA, avidity, or cell-mediated responses may play a greater role in protective immunity than systemic IgG.

BackgroundTrachoma is a neglected tropical disease caused by ocular infection with Chlamydia trachomatis, where repeated infections and chronic inflammation can ultimately result in scarring, trichiasis and blindness. While scarring is thought to be mediated by a dysregulated immune response, the kinetics of cytokines and antimicrobial proteins in the tear film have not yet been characterised.MethodologyPooled tears from a Gambian cohort and Tanzanian cohort were semi-quantitatively screened using a Proteome Profiler Array to identify cytokines differentially regulated in disease. Based on this screen and previous literature, ten cytokines (CXCL1, IP-10, IFN-γ, IL-1β, IL-8, IL-10, IL-12 p40, IL-1RA, IL-1α and PDGF), lysozyme and lactoferrin were assayed in the Tanzanian cohort by multiplex cytokine assay and ELISA. Finally, CXCL1, IP-10, IL-8, lysozyme and lactoferrin were longitudinally profiled in the Gambian cohort by multiplex cytokine assay and ELISA.ResultsIn the Tanzanian cohort, IL-8 was significantly increased in those with clinically inapparent infection (p = 0.0086). Lysozyme, IL-10 and chemokines CXCL1 and IL-8 were increased in scarring (p = 0.016, 0.046, 0.016, and 0.037). CXCL1, IP-10, IL-8, lysozyme and lactoferrin were longitudinally profiled over the course of infection in a Gambian cohort study, with evidence of an inflammatory response both before, during and after detectable infection. CXCL1, IL-8 and IP-10 were higher in the second infection episode relative to the first (p = 0.0012, 0.044, and 0.04).ConclusionsThese findings suggest that the ocular immune system responds prior to and continues to respond after detectable C. trachomatis infection, possibly due to a positive feedback loop inducing immune activation. Levels of CXC chemokines in successive infection episodes were increased, which may offer an explanation as to why repeated infections are a risk factor for scarring.

Purpose: Research has shown that primary open-angle glaucoma (POAG) in Zambia has an earlier age of onset and is more clinically severe than in Europe and the United States of America. Mutations of Myocilin have been reported to be associated with POAG in multiple populations. We therefore, investigated the role of myocilin gene mutations in Primary Open Angle Glaucoma in a Zambian population. Methods: The unrelated POAG patients and unaffected controls seen at the University Teaching Hospitals Eye Hospital, Kitwe Teaching Eye Hospital and Lusaka Eye Hospital were recruited for this study. Glaucoma specialists from the participating institutions ascertained all POAG and control patients. Age, sex and ethnicity matched unaffected controls were obtained in patients with an IOP < 22 mm Hg, clear ocular media and normal-appearing optic nerve heads. A complete eye examination, including visual field assessment, was performed in all cases. Genomic DNA was extracted from whole peripheral blood, then subjected to polymerase chain reaction to amplify exons, flanking introns and promoter regions of the myocilin gene. The amplified products were screened for base mutations by autosequence based on the Sanger method. The study used the chi square test and conditional logistic regression to compare the cases and controls. Identified mutations were compared to known myocilin mutations. Results: Unrelated 165 POAG patients and unaffected 173 controls enrolled for the study. The analysis revealed four variants of myocilin mutations in 49 participants which included one synonymous (silent) mutation (Thr474Thr; 45/338) and three missense mutations (Ala446Thr; 16/338), (Leu158Arg; 4/338) and (Arg342Lys; 1/338). The prevalence of myocilin (MYOC) gene mutations in this study was 14.5% (49/338). The study observed two previously reported mutations, Ala446Thr and Arg342Lys, as glaucoma causing mutations. The variant (Lys158Arg) observed in the study was a novel finding. These mutations were detected in age, sex and ethnically matched controls. The missense mutation, Ala446Thr, was found in eight cases and eight controls. Twenty (20) controls and 25 cases had the synonymous or silent (neutral) mutation, (Thr474Thr). Conclusions: The myocilin mutations represent a prevalence of 14.5 in a Zambian population. The Leu158Arg seems to represent novel glaucoma, causing missense mutation. Mutations in myocilin appear to play a big role in the pathogenesis of POAG in a Zambian population.

INTRODUCTION Trichiasis is present when in-turned eyelashes touch the eyeball. It may result in permanent vision loss. Trachomatous trichiasis (TT) is caused by multiple rounds of inflammation associated with conjunctival Chlamydia trachomatis infection. Surveys have been designed to estimate the prevalence of TT in evaluation units (EUs) of trachoma-endemic countries in order to help develop appropriate programme-level plans. In this study, TT-only surveys were conducted in five EUs of The Gambia to determine whether further intensive programmatic action was required. METHODS Two-stage cluster sampling was used to select 27 villages per EU and ~25 households per village. Graders assessed the TT status of individuals aged ≥15 years in each selected household, including the presence or absence of conjunctival scarring in those with TT. RESULTS From February to March 2019, 11595 people aged ≥15 years were examined. A total of 34 cases of TT were identified. All five EUs had an age- and gender-adjusted prevalence of TT unknown to the health system <0.2%. Three of five EUs had a prevalence of 0.0%. CONCLUSION Using these and other previously collected data, in 2021, The Gambia was validated as having achieved national elimination of trachoma as a public health problem. Trachoma is still present in the population, but as its prevalence is low, it is unlikely that today's youth will experience the exposure to C. trachomatis required to precipitate TT. The Gambia demonstrates that with political will and consistent application of human and financial resources, trachoma can be eliminated as a public health problem.