BACKGROUND: Persistent trachoma is increasingly recognized as a serious concern for the global trachoma program. Persistent trachoma is defined as those districts that have had two or more trachoma impact surveys in which the trachomatous inflammation-follicular (TF) prevalence has never been <5%, the elimination threshold for TF. Enhanced tools such as infection and serological monitoring elucidate long-term transmission patterns within persistent districts. This study aimed to clarify trachoma intensity via both traditional indicators and Chlamydia trachomatis (Ct) infection and serologic markers in four districts experiencing persistent trachoma with >10 years of interventions. METHODOLOGY: Population-based surveys were conducted in 2019 in four trachoma persistent districts. Children ages 1-9 years were examined for trachoma clinical signs and children 1-5 years were swabbed for Ct infection. Antibodies to the trachoma antigens Pgp3 and CT694 were measured for all individuals ≥1 year, assessed by multiplex bead assay. Seroconversion rates (SCRs) to both antigens were estimated for children and for individuals of all ages. RESULTS: One district, Ebinat, remained highly endemic, with a TF prevalence and infection prevalence (ages 1-5 years) of 42.5% and 7.1% respectively. Indicators were lower in the other three districts ranging from 10.7%-17.9% TF and 0%-1.7% infection. The Pgp3 SCR among children ages 1-9 years was considerably higher in Ebinat with 10.8 seroconversions per 100 child-years, (95% Confidence Interval [CI]: 8.2, 14.4) compared to the other three districts (SCR range: 0.9-3.9). All-age Pgp3 SCR estimates detected a significant decline in seroprevalence in Machakel district at approximately 12 years prior to 2019. CONCLUSIONS: Infection and serology may be useful tools for clarifying transmission, particularly among persistent districts, and ongoing interventions likely helped push these hyperendemic districts towards the elimination threshold. However, districts such as Ebinat may require more intense interventions to reach elimination within acceptable timelines.

To assess the prevalence of several parasitic infections in Kiribati, dried blood spots collected during trachoma prevalence surveys in the two major population centers in 2015, 2016, and 2019 were tested using multiplex bead-based serologic assays to detect IgG antibodies against four pathogens of public health interest: Toxoplasma gondii (T. gondii), Taenia solium (T. solium), Strongyloides stercoralis (S. stercoralis), and Toxocara canis (T. canis). In Kiritimati Island, the seroprevalences of T. solium recombinant antigen for detection of cysticercosis antibodies (T24H) and recombinant antigen for detection of taeniasis antibodies (ES33) were ≤4% in both surveys, whereas in Tarawa, the T24H seroprevalence was 2% (2016) and 7% (2019) and the ES33 seroprevalence was ≤3% in both surveys. At both sites, the seropositivity of S. stercoralis recombinant antigen for detection of Strongyloides was 0-4%, and for T. canis, the C-type lectin-1 antigen was 0-1% in all surveys. For T. gondii, the surface antigen glycoprotein 2A antigen seroprevalences on Kiritimati Island were 41% (2015) and 36% (2019), and in Tarawa, they were 36% (2016) and 22% (2019), suggesting that T. gondii infections are common in Kiribati, whereas the other pathogens are not.

We assessed severe acute respiratory syndrome coronavirus 2 seroprevalence on residual blood samples for pediatric COVID-19 surveillance: 2263 samples were collected during routine outpatient visits (<18 years, April 2020-August 2021). Seroprevalence increased over time, coinciding with or preceding virus circulation in the community and with or preceding pediatric severe COVID-19 hospitalization peaks. Residual blood sample seroprevalence may be a useful surveillance tool in future outbreaks.

Mozambique is making progress towards elimination of trachoma as a public health problem, but in some districts trachomatous inflammation-follicular (TF) prevalence remains above the 5% elimination threshold despite years of various interventions, including antibiotic mass drug administration. To characterize transmission in four districts, we incorporated testing of ocular infection and serology into routine trachoma impact surveys (TIS) in August 2022. We examined residents aged ≥ 1 year for trachoma and collected information on household water, sanitation, and hygiene. Among children aged 1-9 years, we tested conjunctival swabs for Chlamydia trachomatis nucleic acid and dried blood spots for C. trachomatis antibodies. We modeled age-dependent seroprevalence to estimate seroconversion rate (SCR). We examined 4841 children aged 1-9 years. TF prevalence ranged between 1.1 and 6.0% with three districts below the 5% threshold. PCR-confirmed infection prevalence ranged between 1.1 and 4.8%, and Pgp3 seroprevalence ranged between 8.8 and 24.3%. Pgp3 SCR was 1.9 per 100 children per year in the district with the lowest TF prevalence. Two other districts with TF < 5% had SCR of 5.0 and 4.7. The district with TF ≥ 5% had a SCR of 6.0. This enhanced TIS furthered understanding of transmission in these districts and provides information on additional indicators for monitoring trachoma programs.

Monitoring trachoma transmission with antibody data requires characterization of decay in IgG to Chlamydia trachomatis antigens. In a three-year longitudinal cohort in a high transmission setting, we estimated a median IgG half-life of 3 years and a seroreversion rate of 2.5 (95% CI: 1.6, 3.5) per 100 person-years.

Baseline trachoma surveys in Côte d'Ivoire (2019) identified seven evaluation units (EUs) with a trachomatous inflammation-follicular (TF) prevalence ≥10%, but a trachomatous trichiasis (TT) prevalence in individuals ≥15 y of age below the elimination threshold (0.2%). Two of these EUs, Bondoukou 1 and Bangolo 2, were selected for a follow-up survey to understand the epidemiology of trachoma using additional indicators of Chlamydia trachomatis infection (DNA from conjunctival swabs) and exposure (anti-Pgp3 and Ct694 antibodies from dried blood spots [DBSs]). A two-stage cluster sampling methodology was used to select villages and households. All individuals 1-9 y of age from each selected household were recruited, graded for trachoma and had a conjunctival swab and DBS collected. Conjunctival swabs and DBSs were tested using Cepheid GeneXpert and a multiplex bead assay, respectively. The age-adjusted TF and infection prevalence in 1- to 9-year-olds was <1% and <0.3% in both EUs. Age-adjusted seroprevalence was 5.3% (95% confidence interval [CI] 1.5 to 15.6) in Bondoukou 1 and 8.2% (95% CI 4.3 to 13.7) in Bangolo 2. The seroconversion rate for Pgp3 was low, at 1.23 seroconversions/100 children/year (95% CI 0.78 to 1.75) in Bondoukou 1 and 1.91 (95% CI 1.58 to 2.24) in Bangolo 2. Similar results were seen for CT694. These infection, antibody and clinical data provide strong evidence that trachoma is not a public health problem in either EU.

Trachoma is an infectious disease characterized by repeated exposures to Chlamydia trachomatis (Ct) that may ultimately lead to blindness. Certain areas, particularly in Africa, pose persistent challenges to elimination of trachoma as a public health problem. Efficiently identifying communities with high infection burden could help target more intensive control efforts. We hypothesized that IgG seroprevalence in combination with geospatial layers, machine learning, and model-based geostatistics would be able to accurately predict future community-level ocular Ct infections detected by PCR. We used measurements from 40 communities in the hyperendemic Amhara region of Ethiopia. Median Ct infection prevalence among children 0-5 years old increased from 6% at enrollment to 29% by month 36. At baseline, correlation between seroprevalence and Ct infection was stronger among children 0-5 years old (ρ = 0.77) than children 6-9 years old (ρ = 0.48), and stronger than the correlation between clinical trachoma and Ct infection (0-5y ρ = 0.56; 6-9y ρ = 0.40). Seroprevalence was the strongest concurrent predictor of infection prevalence at month 36 among children 0-5 years old (cross-validated R2 = 0.75, 95% CI: 0.58-0.85), though predictive performance declined substantially with increasing temporal lag between predictor and outcome measurements. Geospatial variables, a spatial Gaussian process, and stacked ensemble machine learning did not meaningfully improve predictions. Serological markers among children 0-5 years old may be an objective, programmatic tool for identifying communities with high levels of active ocular Ct infections, but accurate, future prediction in the context of changing transmission remains an open challenge.SIGNIFICANCE STATEMENT Trachoma is targeted for elimination as a public health problem by 2030. District-level estimates of clinical disease among children 1-9 years old are currently used to guide control programs and assess elimination. However, clinical trachoma is a subjective indicator. Serological markers present an objective, scalable alternative that could be measured in integrated platforms. In a hyperendemic region, community-level seroprevalence aligned more closely with concurrent infection prevalence than clinical trachoma. The correlation between seroprevalence and infection prevalence was stronger among 0–5-year-olds compared to 6–9-year-olds and was consistent over a three-year period of increasing transmission. Serosurveillance among children 0-5 years old may be a promising monitoring strategy to identify communities with the highest burdens of ocular chlamydial infection.Competing Interest StatementThe authors have declared no competing interest.Clinical TrialNCT02754583Funding StatementWe would like to thank the WUHA study participants and field team without whom this research would not be possible. This work was supported by the National Institute of Allergy and Infectious Diseases (R03 AI147128 to BFA) and the National Eye Institute (U10 EY023939 to JDK). This work was also made possible in part by an Unrestricted Grant from Research to Prevent Blindness. We would also like to thank Abbott for its donation of the m2000 RealTime molecular diagnostics system and consumables.Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Approval for the study was obtained from the University of California, San Francisco Institutional Review Board (14-14004).All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI 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).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesDe-identified data and code to replicate this work is available on Github. https://github.com/ctedijanto/swift-spatial-prediction

Background The transmission of trachoma, caused by repeat infections with Chlamydia trachomatis, and many enteropathogens are linked to water quantity. We hypothesized that children living further from a water source would have higher exposure to C. trachomatis and enteric pathogens as determined by antibody responses.Methods We used a multiplex bead assay to measure IgG antibody responses to C. trachomatis, Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae in eluted dried blood spots collected from 2267 children ages 1–9 years in 40 communities in rural Ethiopia in 2016. Linear distance from the child’s house to the nearest water source was calculated. We derived seroprevalence cutoffs using external negative control populations, if available, or by fitting finite mixture models. We used targeted maximum likelihood estimation to estimate differences in seroprevalence according to distance to the nearest water source.Results Seroprevalence among 1–9-year-olds was 43% for C. trachomatis, 28% for S. enterica, 70% for E. histolytica, 54% for G. intestinalis, 96% for C. jejuni, 76% for ETEC and 94% for C. parvum. Seroprevalence increased with age for all pathogens. Median distance to the nearest water source was 473 meters (IQR 268, 719). Children living furthest from a water source had a 12% (95% CI: 2.6, 21.6) higher seroprevalence of S. enterica and a 12.7% (95% CI: 2.9, 22.6) higher seroprevalence of G. intestinalis compared to children living nearest.Conclusion Seroprevalence for C. trachomatis and enteropathogens was high, with marked increases for most enteropathogens in the first two years of life. Children living further from a water source had higher seroprevalence of S. enterica and G. intestinalis indicating that improving access to water in the Ethiopia’s Amhara region may reduce exposure to these enteropathogens in young children.AUTHOR SUMMARY Trachoma, and infection of the eye caused by the bacteria Chlamydia trachomatis, and many diarrhea-causing infections are associated with access to water for washing hands and faces. Measuring these different pathogens in a population is challenging and rarely are multiple infections measured at the same time. Here, we used an integrated approach to simultaneously measure antibody responses to C. trachomatis, Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae among young children residing in rural Ethiopia. We found that the seroprevalence of all pathogens increased with age and that seropositivity to more than one pathogen was common. Children living further from a water source were more likely to be exposed to S. enterica and G. intestinalis. Integrated sero-surveillance is a promising avenue to explore the complexities of multi-pathogen exposure as well as to investigate the relationship water, sanitation and hygiene related exposures disease transmission.Competing Interest StatementThe authors have declared no competing interest.Funding StatementThis study was funded by the National Institutes of Health, National Eye Institute (NEI U10 EY016214)Author DeclarationsAll relevant ethical guidelines have been followed; any necessary IRB and/or ethics committee approvals have been obtained and details of the IRB/oversight body are included in the manuscript.YesAll necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI 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 adva ce).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAssociated data will be available on OSF upon publication

Prevalence of antibodies to Strongyloides stercoralis was measured in 0–12-year-olds using a bead-based immunoassay before and after ivermectin mass drug administration (MDA) for scabies in the Solomon Islands. Seroprevalence was 9.3% before and 5.1% after MDA (p = 0.019), demonstrating collateral benefits of scabies MDA in this setting.Competing Interest StatementThe authors have declared no competing interest.Clinical TrialNCT02775617Funding StatementThe study was funded by a Wellcome Trust Clinical PhD fellowship to MM (102807). The US CDC paid the laboratory costs. The funders did not have any role in the design, conduct or analysis of the study.Author DeclarationsAll relevant ethical guidelines have been followed; any necessary IRB and/or ethics committee approvals have been obtained and details of the IRB/oversight body are included in the manuscript.YesAll necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI 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).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesData available in supplementary material

Monitoring trachoma transmission with antibody data requires characterization of decay in IgG to Chlamydia trachomatis antigens. In a three-year longitudinal cohort in a high transmission setting, we estimated a median IgG half-life of 3 years and a seroreversion rate of 2.5 (95% CI: 1.6, 3.8) per 100 person-years. Copyright The copyright holder for this preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

Trachoma, caused by ocular Chlamydia trachomatis infection, is targeted for global elimination as a public health problem by 2030. To provide evidence for use of antibodies to monitor C. trachomatis transmission, we collated IgG responses to Pgp3 antigen, PCR positivity, and clinical observations from 19,811 children aged 1-9 years in 14 populations. We demonstrate that age-seroprevalence curves consistently shift along a gradient of transmission intensity: rising steeply in populations with high levels of infection and active trachoma and becoming flat in populations near elimination. Seroprevalence (range: 0-54%) and seroconversion rates (range: 0-15 per 100 person-years) correlate with PCR prevalence (r: 0.88, 95% CI: 0.77, 0.93). A seroprevalence threshold of 13.5% (seroconversion rate 2.75 per 100 person-years) identifies clusters with any infection at high sensitivity (>90%) and moderate specificity (69-75%). Antibody responses in young children provide a robust, generalizable approach to monitor population progress toward and beyond trachoma elimination. Copyright The copyright holder for this preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

Baseline mapping in the two major population centers of Kiribati showed that trachoma was a public health problem in need of programmatic interventions. After conducting two annual rounds of antibiotic mass drug administration (MDA), Kiribati undertook trachoma impact surveys in 2019, using standardized two-stage cluster surveys in the evaluation units of Kiritimati Island and Tarawa. In Kiritimati, 516 households were visited and in Tarawa, 772 households were visited. Nearly all households had a drinking water source and access to an improved latrine. The prevalence of trachomatous trichiasis remained above the elimination threshold (0.2% in ≥15-year-olds) and was virtually unchanged from baseline. The prevalence of trachomatous inflammation-follicular (TF) in 1-9-year-olds decreased by approximately 40% from baseline in both evaluation units but remained above the 5% TF prevalence threshold for stopping MDA. TF prevalence at impact survey was 11.5% in Kiritimati and 17.9% in Tarawa. Infection prevalence in 1-9-year-olds by PCR was 0.96% in Kiritimati and 3.3% in Tarawa. Using a multiplex bead assay to measure antibodies to the C. trachomatis antigen Pgp3, seroprevalence in 1-9-year-olds was 30.2% in Kiritimati and 31.4% in Tarawa. The seroconversion rate, in seroconversion events/100 children/year, was 9.0 in Kiritimati and 9.2 in Tarawa. Seroprevalence and seroconversion rates were both assessed by four different assays, with strong agreement between tests. These results show that, despite decreases in indicators associated with infection at impact survey, trachoma remains a public health problem in Kiribati, and provide additional information about changes in serological indicators after MDA.

Trachoma, caused by ocular Chlamydia trachomatis infection, is targeted for global elimination as a public health problem by 2030. To provide evidence for use of antibodies to monitor C. trachomatis transmission, we collated IgG responses to Pgp3 antigen, PCR positivity, and clinical observations from 19,811 children aged 1-9 years in 14 populations. We demonstrate that age-seroprevalence curves consistently shift along a gradient of transmission intensity: rising steeply in populations with high levels of infection and active trachoma and becoming flat in populations near elimination. Seroprevalence (range: 0-54%) and seroconversion rates (range: 0-15 per 100 person-years) correlate with PCR prevalence (r: 0.87, 95% CI: 0.57, 0.97). A seroprevalence threshold of 13.5% (seroconversion rate 2.75 per 100 person-years) identifies clusters with any PCR-identified infection at high sensitivity ( >90%) and moderate specificity (69-75%). Antibody responses in young children provide a robust, generalizable approach to monitor population progress toward and beyond trachoma elimination.

Seroepidemiology, or measuring antibodies to pathogens to estimate population-level exposure, can provide useful public health data. The tests used, however, often lack sufficient validation data due to absence of a gold standard. For many pathogens, serum antibodies can be detected long after resolution of infection, but infection status is often used as a gold standard for antibody positivity. To ensure that recently developed antibody tests for seroepidemiology of Chlamydia trachomatis (Ct), the causative agent of urogenital chlamydia and the blinding eye disease trachoma, have high performance, we generated a chimeric antibody to the immunodominant Ct antigen Pgp3. Two clones were selected to evaluate the test performance of three assays to measure antibodies to Pgp3: multiplex bead assay (MBA), enzyme-linked immunosorbent assay (ELISA), and lateral flow assay (LFA). Overall, each assay demonstrated high accuracy and precision when tested using either clone, and the clones were stable when stored at - 20 °C and 4 °C for almost 2 years. The limit of detection was similar for MBA and LFA, but almost a log-fold higher (i.e. less sensitive) using ELISA. Overall, the chimeric antibodies represent stable control reagents for tests with robust performance and will facilitate deployment of these tests to other laboratories.

OBJECTIVES: Determining an accurate estimate of SARS-CoV-2 seroprevalence has been challenging in African countries where malaria and other pathogens are endemic. We compared the performance of one single-antigen assay and three multi-antigen SARS-CoV-2 IgG assays in a Nigerian population endemic for malaria. METHODS: De-identified plasma specimens from SARS-CoV-2 RT-PCR positive, dried blood spot (DBS) SARS-CoV-2 RT-PCR positive, and pre-pandemic negatives were used to evaluate the performance of the four SARS-CoV-2 assays (Tetracore, SARS2MBA, RightSign, xMAP). RESULTS: Results showed higher sensitivity with the multi-antigen (81% (Tetracore), 96% (SARS2MBA), 85% (xMAP)) versus the single-antigen (RightSign (64%)) SARS-CoV-2 assay. The overall specificities were 98% (Tetracore), 100% (SARS2MBA and RightSign), and 99% (xMAP). When stratified based on <15 days to ≥15 days post-RT-PCR confirmation, the sensitivities increased from 75% to 88.2% for Tetracore; from 93% to 100% for the SARS2MBA; from 58% to 73% for RightSign; and from 83% to 88% for xMAP. With DBS, there was no positive increase after 15-28 days for the three assays (Tetracore, SARS2MBA, and xMAP). CONCLUSION: Multi-antigen assays performed well in Nigeria, even with samples with known malaria reactivity, and might provide more accurate measures of COVID-19 seroprevalence and vaccine efficacy.

Serosurveillance can provide estimates of population-level exposure to infectious pathogens and has been used extensively during the COVID-19 pandemic. Simultaneous, serological testing for multiple pathogens can be done using bead-based immunoassays to add value to disease-specific serosurveys. We conducted a validation of four SARS-CoV-2 antigens-full-length spike protein, two receptor binding domain proteins, and the nucleocapsid protein-on our existing multiplex bead assay (MBA) for enteric diseases, malaria, and vaccine preventable diseases. After determining the optimal conditions for coupling the antigens to microsphere beads, the sensitivity and specificity of the assay were determined on two instruments (Luminex-200 and MAGPIX) when testing singly (monoplex) versus combined (multiplex). Sensitivity was assessed using plasma from 87 real-time reverse transcription polymerase chain reaction (rRT-PCR) positive persons collected in March-May of 2020 and ranged from 94.3% to 96.6% for the different testing conditions. Specificity was assessed using 98 plasma specimens collected prior to December 2019 and plasma from 19 rRT-PCR negative persons and ranged from 97.4% to 100%. The positive percent agreement was 93.8% to 97.9% using 48 specimens collected > 21 days post-symptom onset, while the negative percent agreement was ≥ 99% for all antigens. Test performance was similar using monoplex or multiplex testing. Integrating SARS-CoV-2 serology with other diseases of public health interest could add significant value to public health programs that have suffered severe programmatic setbacks during the COVID-19 pandemic.

BACKGROUND: The epidemiology of trachoma in several Pacific Islands differs from other endemic settings, in that there is a high prevalence of clinical signs of trachoma, particularly trachomatous inflammation-follicular (TF), but few cases of trichiasis and limited evidence of ocular chlamydial infection. This so-called "Pacific enigma" has led to uncertainty regarding the appropriate public health response. In 2019 alongside Nauru's national trachoma population survey, we performed bacteriological and serological assessments of children to better understand the typology of trachoma and to determine whether there is a need for trachoma interventions. METHODS: We used two-stage cluster sampling, examining residents aged ≥1 year and collecting household-level water, sanitation, and hygiene (WASH) variables. Children aged 1-9 years provided conjunctival swabs and finger-prick dried blood spots to investigate the presence of Chlamydia trachomatis nucleic acid and anti-Pgp3 antibodies, respectively. PRINCIPAL FINDINGS: In 818 participants aged 1-9 years, the age-adjusted TF prevalence was 21.8% (95% CI 15.2-26.2%); ocular C. trachomatis prevalence was 34.5% (95% CI 30.6-38.9), and anti-Pgp3 antibody prevalence was 32.1% (95% CI 28.4%-36.3%). The age- and gender-adjusted prevalence of trichiasis in ≥15-year-olds was 0.3% (95% CI 0.00-0.85), but no individual with trichiasis had trachomatous scarring (TS). Multivariable analysis showed an association between age and both TF (OR per year of age 1.3 [95% CI 1.2-1.4]) and anti-Pgp3 positivity (OR 1.2 [95% CI 1.2-1.3]). There were high rates of access to water and sanitation and no WASH variable was associated with the presence of TF. CONCLUSIONS: TF, nucleic acid, and age-specific antibody prevalence collectively indicate that high levels of C. trachomatis transmission among children present a high risk of ocular damage due to trachoma. The absence of trichiasis with trachomatous scarring suggest a relatively recent increase in transmission intensity.

Trachoma is an infectious disease characterized by repeated exposures to Chlamydia trachomatis (Ct) that may ultimately lead to blindness. Efficient identification of communities with high infection burden could help target more intensive control efforts. We hypothesized that IgG seroprevalence in combination with geospatial layers, machine learning, and model-based geostatistics would be able to accurately predict future community-level ocular Ct infections detected by PCR. We used measurements from 40 communities in the hyperendemic Amhara region of Ethiopia to assess this hypothesis. Median Ct infection prevalence among children 0-5 years old increased from 6% at enrollment, in the context of recent mass drug administration (MDA), to 29% by month 36, following three years without MDA. At baseline, correlation between seroprevalence and Ct infection was stronger among children 0-5 years old (ρ = 0.77) than children 6-9 years old (ρ = 0.48), and stronger than the correlation between active trachoma and Ct infection (0-5y ρ = 0.56; 6-9y ρ = 0.40). Seroprevalence was the strongest concurrent predictor of infection prevalence at month 36 among children 0-5 years old (cross-validated R2 = 0.75, 95% CI: 0.58-0.85), though predictive performance declined substantially with increasing temporal lag between predictor and outcome measurements. Geospatial variables, a spatial Gaussian process, and stacked ensemble machine learning did not meaningfully improve predictions. Serological markers among children 0-5 years old may be an objective tool for identifying communities with high levels of ocular Ct infections, but accurate, future prediction in the context of changing transmission remains an open challenge.

Trachoma is the leading infectious cause of blindness. In 2016, Morocco was validated by WHO as having eliminated trachoma as a public health problem. We evaluated two previously endemic districts in Morocco for trachomatous inflammation-follicular (TF), trachomatous trichiasis (TT), and antibodies against Chlamydia trachomatis, the causative agent of trachoma. Community-based cross-sectional surveys in the districts of Boumalene Dades and Agdez included 4,445 participants for whom both questionnaire and serology data were available; 58% were aged 1-9 years. Participants had eyes examined for TF and blood collected for analysis of antibodies to the C. trachomatis antigen Pgp3 by both a multiplex bead assay (MBA) and lateral flow assay (LFA). Seroconversion rates (SCR) per 100 people per year were used to estimate changes in the force of infection using Bayesian serocatalytic models. In Agdez, TF prevalence in 1-9-year-olds was 0.3%, seroprevalence ranged from 9.4% to 11.4%, and SCR estimates ranged from 2.4 to 3.0. In Boumalene Dades, TF prevalence in 1-9-year-olds was 0.07%, and modeling data from the different assays indicated a decrease in transmission between 20 and 24 years ago. The TF data support an absence of active trachoma in the two districts examined. However, seroprevalence and SCR in younger people were higher in Agdez than Boumalene Dades, showing that there can be differences in serology metrics in areas with similar TF prevalence. Data will be included in multicountry analyses to better understand potential thresholds for serological surveillance in trachoma.

BACKGROUND: As prevalence decreases in pre-elimination settings, identifying the spatial distribution of remaining infections to target control measures becomes increasingly challenging. By measuring multiple antibody responses indicative of past exposure to different pathogens, integrated serological surveys enable simultaneous characterisation of residual transmission of multiple pathogens. METHODOLOGY/PRINCIPAL FINDINGS: Here, we combine integrated serological surveys with geostatistical modelling and remote sensing-derived environmental data to estimate the spatial distribution of exposure to multiple diseases in children in Northern Ghana. The study utilised the trachoma surveillance survey platform (cross-sectional two-stage cluster-sampled surveys) to collect information on additional identified diseases at different stages of elimination with minimal additional cost. Geostatistical modelling of serological data allowed identification of areas with high probabilities of recent exposure to diseases of interest, including areas previously unknown to control programmes. We additionally demonstrate how serological surveys can be used to identify areas with exposure to multiple diseases and to prioritise areas with high uncertainty for future surveys. Modelled estimates of cluster-level prevalence were strongly correlated with more operationally feasible metrics of antibody responses. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the potential of integrated serological surveillance to characterise spatial distributions of exposure to multiple pathogens in low transmission and elimination settings when the probability of detecting infections is low.

BACKGROUND: WHO promotes the SAFE strategy for the elimination of trachoma as a public health programme, which promotes surgery for trichiasis (ie, the S component), antibiotics to clear the ocular strains of chlamydia that cause trachoma (the A component), facial cleanliness to prevent transmission of secretions (the F component), and environmental improvements to provide water for washing and sanitation facilities (the E component). However, little evidence is available from randomised trials to support the efficacy of interventions targeting the F and E components of the strategy. We aimed to determine whether an integrated water, sanitation, and hygiene (WASH) intervention prevents the transmission of trachoma. METHODS: The WASH Upgrades for Health in Amhara (WUHA) was a two-arm, parallel-group, cluster-randomised trial in 40 rural communities in Wag Hemra Zone (Amhara Region, Ethiopia) that had been treated with 7 years of annual mass azithromycin distributions. The randomisation unit was the school catchment area. All households within a 1·5 km radius of a potential water point within the catchment area (as determined by the investigators) were eligible for inclusion. Clusters were randomly assigned (at a 1:1 ratio) to receive a WASH intervention either immediately (intervention) or delayed until the conclusion of the trial (control), in the absence of concurrent antibiotic distributions. Given the nature of the intervention, participants and field workers could not be masked, but laboratory personnel were masked to treatment allocation. The WASH intervention consisted of both hygiene infrastructure improvements (namely, construction of a community water point) and hygiene promotion by government, school, and community leaders, which were implemented at the household, school, and community levels. Hygiene promotion focused on two simple messages: to use soap and water to wash your or your child's face, and to always use a latrine for defecation. The primary outcome was the cluster-level prevalence of ocular chlamydia, measured annually using conjunctival swabs in a random sample of children aged 0-5 years from each cluster at 12, 24, and 36 month timepoints. Analyses were done in an intention-to-treat manner. This trial is ongoing and is registered at ClinicalTrials.gov, NCT02754583. FINDINGS: Between Nov 9, 2015, and March 5, 2019, 40 of 44 clusters assessed for eligibility were enrolled and randomly allocated to the trial groups (20 clusters each, with 7636 people from 1751 households in the intervention group and 9821 people from 2211 households in the control group at baseline). At baseline, ocular chlamydia prevalence among children aged 0-5 years was 11% (95% CI 6 to 16) in the WASH group and 11% (5 to 18) in the control group. At month 36, ocular chlamydia prevalence had increased in both groups, to 32% (24 to 41) in the WASH group and 31% (21 to 41) in the control group (risk difference across three annual monitoring visits, after adjustment for prevalence at baseline: 3·7 percentage points; 95% CI -4·9 to 12·4; p=0·40). No adverse events were reported in either group. INTERPRETATION: An integrated WASH intervention addressing the F and E components of the SAFE strategy did not prevent an increase in prevalence of ocular chlamydia following cessation of antibiotics in an area with hyperendemic trachoma. The impact of WASH in the presence of annual mass azithromycin distributions is currently being studied in a follow-up trial of the 40 study clusters. Continued antibiotic distributions will probably be important in areas with persistent trachoma. FUNDING: National Institutes of Health-National Eye Institute. TRANSLATION: For the Amharic translation of the abstract see Supplementary Materials section.

INTRODUCTION: To date, eleven countries have been validated as having eliminated trachoma as a public health problem, including Ghana in 2018. Surveillance for recrudescence is needed both pre- and post-validation but evidence-based guidance on appropriate strategies is lacking. We explored two potential surveillance strategies in Ghana. METHODOLOGY/PRINCIPAL FINDINGS: Amongst randomly-selected communities enrolled in pre-validation on-going surveillance between 2011 and 2015, eight were identified as having had trachomatous-inflammation follicular (TF) prevalence ≥5% in children aged 1-9 years between 2012 and 2014. These eight were re-visited in 2015 and 2016 and neighbouring communities were also added ("TF trigger" investigations). Resident children aged 1-9 years were then examined for trachoma and had a conjunctival swab to test for Chlamydia trachomatis (Ct) and a dried blood spot (DBS) taken to test for anti-Pgp3 antibodies. These investigations identified at least one community with evidence of probable recent Ct ocular transmission. However, the approach likely lacks sufficient spatio-temporal power to be reliable. A post-validation surveillance strategy was also evaluated, this reviewed the ocular Ct infection and anti-Pgp3 seroprevalence data from the TF trigger investigations and from the pre-validation surveillance surveys in 2015 and 2016. Three communities identified as having ocular Ct infection >0% and anti-Pgp3 seroprevalence ≥15.0% were identified, and along with three linked communities, were followed-up as part of the surveillance strategy. An additional three communities with a seroprevalence ≥25.0% but no Ct infection were also followed up ("antibody and infection trigger" investigations). DBS were taken from all residents aged ≥1 year and ocular swabs from all children aged 1-9 years. There was evidence of transmission in the group of communities visited in one district (Zabzugu-Tatale). There was no or little evidence of continued transmission in other districts, suggesting previous infection identified was transient or potentially not true ocular Ct infection. CONCLUSIONS/SIGNIFICANCE: There is evidence of heterogeneity in Ct transmission dynamics in northern Ghana, even 10 years after wide-scale MDA has stopped. There is added value in monitoring Ct infection and anti-Ct antibodies, using these indicators to interrogate past or present surveillance strategies. This can result in a deeper understanding of transmission dynamics and inform new post-validation surveillance strategies. Opportunities should be explored for integrating PCR and serological-based markers into surveys conducted in trachoma elimination settings.

Programs to eliminate trachoma as a public health problem use prevalence of the clinical sign trachomatous inflammation-follicular (TF) in 1- to 9-year-olds in endemic districts to make decisions to begin or end mass drug administration with azithromycin. Trachomatous inflammation-follicular is used as a proxy for transmission of ocular Chlamydia trachomatis infection. Long-term monitoring of previously endemic districts for recrudescence of ocular C. trachomatis infection would benefit from a simple blood test that could be integrated with other public health programs. In this study, we evaluated multiple tests to measure antibodies against the C. trachomatis antigen Pgp3-a multiplex bead assay (MBA), an ELISA, and two versions of a lateral flow assay (LFA)-in four districts of the Amhara region of Ethiopia with varying levels of TF. Seroprevalence and seroconversion rate (SCR) results were proportional to TF prevalence by district for most tests, with the notable exception of the LFA using colloidal gold as the developing reagent. Changing the test developing reagent to black latex improved agreement between serological measures and TF prevalence and in inter-rater agreement. Seroconversion rate estimates using data derived from the LFA-gold assay were inconsistent with the shape of the age-seroprevalence curve, which did not increase in older ages. These data revealed potential complications with using SCR that will need further evaluation. Data from MBA, ELISA, and LFA with the black test line showed good agreement with each other and proportionality to TF estimates, providing further data that serology has potential utility for trachoma surveillance.

Two plasmid gene protein (Pgp3)-based serological assays, the Pgp3-ELISA and multiplex bead assay (Pgp3-MBA), were compared and used to estimate seropositivity of Chlamydia trachomatis (CT) among females 14 to 39 years old participating in the National Health and Nutrition Examination Survey between 2013-2016. Of the 2,201 specimens tested, 502 (29.5%, 95% CI 27.6-31.5) were positive using Pgp3-ELISA and 624 (28.4%, 95% CI 26.5-30.3) were positive using Pgp3-MBA. The overall agreement between the assays was 87.7%. Corresponding nucleic acid amplification test (NAAT) results were available for 1,725 specimens (from women 18-39 years old); of these, 42 (2.4%, 95% CI 1.8-3.3) were CT NAAT-positive. Most of the CT NAAT-positive specimens had corresponding positive serological assay results; 33 (78.6%, 95% CI 62.8-89.2) were Pgp3-ELISA-positive and 36 (85.7%, 95% CI 70.8-94.1) were Pgp3-MBA-positive. Although Pgp3-ELISA and Pgp3-MBA demonstrated equivalent performance in this study, an advantage of the Pgp3-MBA over Pgp3-ELISA is that it is well suited for high sample throughput applications.

Multiplex bead assays (MBAs) for serologic testing have become more prevalent in public health surveys, but few studies have assessed their test performance. As part of a trachoma study conducted in a rural part of Ethiopia in 2016, dried blood spots (DBS) were collected from a random sample of 393 children aged 0 to 9 years, with at least two separate 6-mm DBS collected on a filter card. Samples eluted from DBS were processed using an MBA on the Luminex platform for antibodies against 13 antigens of nine infectious organisms: Chlamydia trachomatis, Vibrio cholera, enterotoxigenic Escherichia coli, Cryptosporidium parvum, Entamoeba histolytica, Camplyobacter jejuni, Salmonella typhimurium Group B, Salmonella enteritidis Group D, and Giardia lamblia. Two separate DBS from each child were processed. The first DBS was run a single time, with the MBA set to read 100 beads per well. The second DBS was run twice, first at 100 beads per well and then at 50 beads per well. Results were expressed as the median fluorescence intensity minus background (MFI-BG), and classified as seropositive or seronegative according to external standards. Agreement between the three runs was high, with intraclass correlation coefficients of > 0.85 for the two Salmonella antibody responses and > 0.95 for the other 11 antibody responses. Agreement was also high for the dichotomous seropositivity indicators, with Cohen's kappa statistics exceeding 0.87 for each antibody assay. These results suggest that serologic testing on the Luminex platform had strong test performance characteristics for analyzing antibodies using DBS.

BACKGROUND: Chlamydia trachomatis causes pelvic inflammatory disease (PID) and tubal infertility. Pgp3 antibody (Pgp3Ab) detects prior chlamydial infections. We evaluated for an association of high chlamydial seropositivity with sequelae using a Pgp3Ab multiplex bead array (Pgp3AbMBA). METHODS: We performed chlamydia Pgp3AbMBA on sera from women 18-39 years old participating in the 2013-2016 National Health and Nutrition Examination Survey (NHANES) with urine chlamydia nucleic acid amplification test results. High chlamydial seropositivity was defined as a median fluorescence intensity (MFI ≥ 50,000; low-positive was MFI > 551-<50,000. Weighted US population high-positive, low-positive, and negative Pgp3Ab chlamydia seroprevalence and 95% confidence intervals (95% CI) were compared for women with chlamydial infection, self-reported PID, and infertility. RESULTS: Of 2,339 women aged 18-39 years, 1,725 (73.7%) had sera and 1,425 were sexually experienced. Overall, 104 women had high positive Pgp3Ab (5.4% [95% CI 4.0-7.0] of US women); 407 had low positive Pgp3Ab (25.1% [95% CI 21.5-29.0]), and 914 had negative Pgp3Ab (69.5% [95% CI 65.5-73.4]).Among women with high Pgp3Ab, infertility prevalence was 2.0 (95% CI 1.1-3.7) times higher than among Pgp3Ab-negative women (19.6% [95% CI 10.5-31.7] versus 9.9% [95% CI 7.7-12.4]). For women with low Pgp3Ab, PID prevalence was 7.9% (95% CI 4.6-12.6) compared to 2.3% (95% CI 1.4-3.6) in negative Pgp3Ab. CONCLUSIONS: High chlamydial Pgp3Ab seropositivity was associated with infertility although small sample size limited evaluation of an association of high seropositivity with PID. In infertile women, Pgp3Ab may be a marker of prior chlamydial infection.

Trachoma, caused by repeated ocular infection with Chlamydia trachomatis (Ct), is targeted for elimination as a public health problem. Serological testing for antibodies is promising for surveillance; determining useful thresholds will require collection of serological data from settings with different prevalence of the indicator trachomatous inflammation-follicular (TF). Dried blood spots were collected during trachoma mapping in two districts each of Togo and Democratic Republic of the Congo. Anti-Ct antibodies were detected by multiplex bead assay (MBA) and three different lateral flow assays (LFA) and seroprevalence and seroconversion rate (SCR) were determined. By most tests, the district with > 5% TF (the elimination threshold) had five-sixfold higher seroprevalence and tenfold higher SCR than districts with < 5% TF. The agreement between LFA and MBA was improved using a black latex developing reagent. These data show optimization of antibody tests against Ct to better differentiate districts above or below trachoma elimination thresholds.

Programs seeking to eliminate the eye disease trachoma use prevalence of the clinical sign trachomatous inflammation–follicular (TF) in 1- to 9-year-olds as a proxy for population-level transmission of ocular Chlamydia trachomatis (Ct). TF prevalence determines the need for the A, F, and E components of the “SAFE” (surgery, antibiotics, facial cleanliness, environmental improvement) strategy. Ocular Ct infection, like its associated signs of conjunctival inflammation, is most common and most intense in young children [1, 2] who are repeatedly infected in areas of active transmission [3]: a model suggests that people can be infected more than 150 times in their lifetime [4]. Repeated infection leads to multiple episodes of TF plus more intense conjunctival inflammation and eventually conjunctival scarring (trachomatous scarring [TS])[5, 6]. Contraction of conjunctival scar can, over a period of years or decades, cause the upper eyelid to turn in and the eyelashes to rub against the eyeball (trachomatous trichiasis [TT]), which can lead to corneal opacity (CO) and blindness.

The transmission of trachoma, caused by repeat infections with Chlamydia trachomatis, and many enteropathogens are linked to water quantity. We hypothesized that children living further from a water source would have higher exposure to C. trachomatis and enteric pathogens as determined by antibody responses. We used a multiplex bead assay to measure IgG antibody responses to C. trachomatis, Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae in eluted dried blood spots collected from 2267 children ages 0-9 years in 40 communities in rural Ethiopia in 2016. Linear distance from the child's house to the nearest water source was calculated. We derived seroprevalence cutoffs using external negative control populations, if available, or by fitting finite mixture models. We used targeted maximum likelihood estimation to estimate differences in seroprevalence according to distance to the nearest water source. Seroprevalence among 1-9-year-olds was 43% for C. trachomatis, 28% for S. enterica, 70% for E. histolytica, 54% for G. intestinalis, 96% for C. jejuni, 76% for ETEC and 94% for C. parvum. Seroprevalence increased with age for all pathogens. Median distance to the nearest water source was 473 meters (IQR 268, 719). Children living furthest from a water source had a 12% (95% CI: 2.6, 21.6) higher seroprevalence of S. enterica and a 12.7% (95% CI: 2.9, 22.6) higher seroprevalence of G. intestinalis compared to children living nearest. Seroprevalence for C. trachomatis and enteropathogens was high, with marked increases for most enteropathogens in the first two years of life. Children living further from a water source had higher seroprevalence of S. enterica and G. intestinalis indicating that improving access to water in the Ethiopia's Amhara region may reduce exposure to these enteropathogens in young children.

The Western Pacific Region (WPR) established a goal to decrease chronic hepatitis B virus (HBV) infection among children to <1% and to achieve >/=95% hepatitis B vaccine birth dose (HepB-BD) and >/=95% three-dose (HepB3) coverage by 2017. In 2016, we conducted a national serosurvey in the Solomon Islands among 6-7-year-old school children to assess progress towards the control goal and immunity to measles, rubella, tetanus and diphtheria. Eighty schools were selected systematically proportional to their 6-7-year-old population; all 6-7-year-olds were enrolled. We collected basic demographic information and vaccination history. Children were tested for HBV surface antigen (HBsAg) using a rapid test, and for immunity to measles, rubella, tetanus, and diphtheria using a multiplex bead assay. In total, 1,249 out of 1,492 children (84%) were enrolled, among whom 1,169 (94%) underwent HBsAg testing and 1,156 (93%) provided dried blood spots. Almost 80% (n = 982) of enrolled children had vaccination cards, among whom 59% (n = 584) received a timely HepB-BD (within 24 hours of birth), 95% (n = 932) received HepB3, and >90% received vaccines for diphtheria, tetanus, and measles (rubella vaccine was not available at the time). HBsAg prevalence was 3.1% (95% confidence interval (CI): 2.0%-4.9%), with 55% of identified cases from one province. Among 982 children with vaccination cards, HBsAg prevalence was higher among children who had not received a timely HepB-BD and at least two HepB doses compared to those who had (4% vs. 2%). Of 1,156 tested children, immunoprotection estimates were 99% (95% CI: 98%-99%) for measles, 99% (95% CI: 97%-100%) for rubella, 85% (95% CI: 83%-87%) for tetanus, and 51% (95% CI: 47%-55%) for diphtheria. Improving timely HepB-BD coverage and maintaining high HepB3 coverage could help Solomon Islands reach the regional HBV control goal. Low immunity to tetanus and diphtheria suggests the need to introduce booster doses to ensure long-term protection.