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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., Competing Interests: Declaration of interests BA reports financial support to attend global health meetings from the Bill & Melinda Gates Foundation. All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

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., (Published by Elsevier Inc.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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.

Background: In Melanesia, the prevalence of trachomatous inflammation-follicular (TF) suggests that public health-level interventions against active trachoma are needed. However, the prevalence of trachomatous trichiasis is below the threshold for elimination as a public health problem and evidence of conjunctival infection with trachoma's causative organism (Chlamydia trachomatis [CT]) is rare. Here, we examine the prevalence of ocular infection with CT and previous exposure to CT in three evaluation units (EUs) of Papua New Guinea., Methods: All individuals aged 1-9 years who were examined for clinical signs of trachoma in 3 Global Trachoma Mapping Project EUs were eligible to take part in this study (N = 3181). Conjunctival swabs were collected from 349 children with TF and tested by polymerase chain reaction to assess for ocular CT infection. Dried blood spots were collected from 2572 children and tested for anti-Pgp3 antibodies using a multiplex assay., Results: The proportion of children with TF who had CT infection was low across all 3 EUs (overall 2%). Anti-Pgp3 seroprevalence was 5.2% overall and there was no association between anti-Pgp3 antibody level and presence of TF. In 2 EUs, age-specific seroprevalence did not increase significantly with increasing age in the 1- to 9-year-old population. In the third EU, there was a statistically significant change with age but the overall seroprevalence and peak age-specific seroprevalence was very low., Conclusions: Based on these results, together with similar findings from the Solomon Islands and Vanuatu, the use of TF to guide antibiotic mass drug administration decisions in Melanesia should be reviewed., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

The 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 = .019), demonstrating collateral benefits of ivermectin MDA in this setting., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Serological surveillance for trachoma could allow monitoring of transmission levels in areas that have achieved elimination targets. Platforms that allow testing in basic laboratories or testing of easy-to-manage samples such as dried blood spots would contribute to the feasibility of serologic testing. Blood from 506 1-12-year-olds in 2 villages in Kongwa district, Tanzania, was tested for antibodies against the antigen Pgp3. Whole blood, plasma, and dried blood spots (DBS) were tested in lab and field settings using a cassette-enclosed Pgp3 lateral flow assay (LFA-cassette) and a pared-back "dipstick" assay (LFA-dipstick). DBS were also tested with a bead-based multiplex assay (MBA). There was no significant difference in antibody positivity between the MBA and either LFA format (ranging from 42.5% to 48.4%). Interrater agreement between an expert rater and 3 different raters in field and lab settings was uniformly good, with Cohen's kappa >0.81 in all cases., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Validation of elimination of trachoma as a public health problem is based on clinical indicators, using the WHO simplified grading system. Chlamydia trachomatis (Ct) infection and anti-Ct antibody responses (anti-Pgp3) have both been evaluated as alternative indicators in settings with varying levels of trachoma. There is a need to evaluate the feasibility of using tests for Ct infection and anti-Pgp3 antibodies at scale in a trachoma-endemic country and to establish the added value of the data generated for understanding transmission dynamics in the peri-elimination setting., Methodology/principal Findings: Dried blood spots for serological testing and ocular swabs for Ct infection testing (taken from children aged 1-9 years) were integrated into the pre-validation trachoma surveys conducted in the Northern and Upper West regions of Ghana in 2015 and 2016. Ct infection was detected using the GeneXpert PCR platform and the presence of anti-Pgp3 antibodies was detected using both the ELISA assay and multiplex bead array (MBA). The overall mean cluster-summarised TF prevalence (the clinical indicator) was 0.8% (95% CI: 0.6-1.0) and Ct infection prevalence was 0.04% (95%CI: 0.00-0.12). Anti-Pgp3 seroprevalence using the ELISA was 5.5% (95% CI: 4.8-6.3) compared to 4.3% (95%CI: 3.7-4.9) using the MBA. There was strong evidence from both assays that seropositivity increased with age (p<0.001), although the seroconversion rate was estimated to be very low (between 1.2 to 1.3 yearly events per 100 children)., Conclusions/significance: Infection and serological data provide useful information to aid in understanding Ct transmission dynamics. Elimination of trachoma as a public health problem does not equate to the absence of ocular Ct infection nor cessation in acquisition of anti-Ct antibodies., Competing Interests: The authors have declared that no competing interests exist.

Antibody-based epidemiologic surveillance to determine population-level exposure to sexually transmitted infections could help inform public health fertility preservation strategies. We compared the performance of three platforms to detect antibodies against the Chlamydia trachomatis (CT) antigen Pgp3 - multiplex bead array (MBA), enzyme-linked immunosorbent assay (ELISA), and lateral flow assay (LFA) - on sera from adolescents and young adults (AYAs) with pelvic inflammatory disease (PID). Ninety-five of 118 AYAs diagnosed with PID (80.5%) had positive antibody response to Pgp3 antigen by at least one test, and 78 (66.1%) tested positive by all three tests. Among 27 individuals with infection detected using nucleic acid amplification testing, 92.6% were positive by MBA (25/27), 77.8% (21/27) were positive by ELISA, and 74.1% (20/27) were positive by LFA. These data suggest that the MBA was the most sensitive of the three tests and could be useful in seroepidemiologic studies designed to assess population-level exposure to CT.

Latent class modeling can be used to combine the results of multiple tests to compare the sensitivity and specificity of those tests in the absence of a gold standard. Seroepidemiology for chlamydia infection may be useful for determining the cumulative risk of infection within a population. Initial studies using the Chlamydia trachomatis immunodominant antigen Pgp3 have shown utility for seroepidemiology of sexually transmitted chlamydia and the eye disease trachoma. We present our latent class modeling results for comparison of antibody data obtained from three different Pgp3-based platforms - multiplex bead array, ELISA, and lateral flow assay. Sensitivity and specificity estimates from the best fitting latent class models were similar to estimates derived from those previously obtained using a nucleic acid amplification test as a gold standard for sensitivity and non-endemic pediatric specimens for specificity, although the estimates from latent class models had wider confidence intervals. The modeling process and evaluation highlighted the importance of including as many antibody tests as possible when fitting a latent class model to ensure that as many patterns as possible are available for evaluation. Future studies designed to evaluate antibody test performance in the absence of a gold standard should utilize as many tests as possible.

Antibody responses to Chlamydia trachomatis (CT) antigens may be useful tools for surveillance of trachoma by estimating cumulative prevalence of infection within a population. Data were compared from three different platforms-multiplex bead array (MBA), enzyme-linked immunosorbent assay (ELISA), and lateral flow assay (LFA)-measuring antibody responses against the CT antigen protein plasmid gene product 3 (Pgp3). Sensitivity was defined as the proportion of specimens testing antibody positive from a set of dried blood spots from Tanzanian 1-9-year olds who were positive for CT nucleic acid of all nucleic acid amplification test (NAAT)-positive individuals ( N = 103). The sensitivity of the LFA could not be determined because of the use of dried blood spots for this test; this specimen type has yet to be adapted to LFA. Specificity was defined as the proportion of sera from U.S. and Bolivian 1-9-year olds that had previously tested negative by the Chlamydia microimmunofluorescence (MIF) assay testing negative to Pgp3-specific antibodies ( N = 154). The sensitivity for MBA and ELISA was the same-93.2 (95% confidence interval [CI]: 88.3-98.1). Specificity ranged across platforms from 96.1 (95% CI: 91.8-98.2) to 99.4% (95% CI: 98.2-100). ELISA performance was similar regardless of whether the plates were precoated or freshly coated with antigen. Sensitivity and specificity of control panels were similar if the cutoff was determined using receiver operator curves or a finite mixture model, but the cutoffs themselves differed by approximately 0.5 OD using the different methodologies. These platforms show good sensitivity and specificity and show good agreement between tests at a population level, but indicate variability for ELISA outcomes depending on the cutoff determination methodology.

Trachoma is caused by Chlamydia trachomatis (Ct). It is targeted for global elimination as a public health problem. In 2014, a population-based cross-sectional study was performed in two previously trachoma-endemic areas of The Gambia. Participants of all ages from Lower River Region (LRR) (N = 1028) and Upper River Region (URR) (N = 840) underwent examination for trachoma and had blood collected for detection of antibodies against the Ct antigen Pgp3, by ELISA. Overall, 30 (1.6%) individuals had active trachoma; the prevalence in children aged 1-9 years was 3.4% (25/742) with no statistically significant difference in prevalence between the regions. There was a significant difference in overall seroprevalence by region: 26.2% in LRR and 17.1% in URR (p < 0.0001). In children 1-9 years old, seroprevalence was 4.4% in LRR and 3.9% in URR. Reversible catalytic models using information on age-specific seroprevalence demonstrated a decrease in the transmission of Ct infection in both regions, possibly reflecting the impact of improved access to water, health and sanitation as well as mass drug administration campaigns. Serological testing for antibodies to Ct antigens is potentially useful for trachoma programmes, but consideration should be given to the co-endemicity of sexually transmitted Ct infections.

Purpose: Testing for antibodies to Chlamydia trachomatis has potential as a surveillance tool. Our evaluation compares lateral flow assays (LFAs) during surveillance surveys in Nepal with Multiplex bead array (MBA). Fifty children were randomly sampled from each of 15 random clusters in two districts of Nepal. Finger prick blood samples were collected from 1509 children and tested onsite for anti-Pgp3 antibodies by LFA. The LFA was read at 30min as negative, positive, or invalid. Tests results were also rated as difficult to read ("equivocal"). Blood was processed at Johns Hopkins University using the MBA., Results: The LFA had agreement of 40.0% for MBA-positive samples and 99.3% for MBA-negative samples. Inter-reader reliability was kappa=0.65 (95% CI=0.56-0.74). If the equivocal results (7%) could be decreased, reliability could be improved., Conclusions: Further optimization and testing of the LFA test are needed to improve agreement with MBA and the interpretation of the results., (Copyright © 2017. Published by Elsevier Inc.)

Background: Efforts are underway to eliminate trachoma as a public health problem by 2020. Programmatic guidelines are based on clinical signs that correlate poorly with Chlamydia trachomatis (Ct) infection in post-treatment and low-endemicity settings. Age-specific seroprevalence of anti Ct Pgp3 antibodies has been proposed as an alternative indicator of the need for intervention. To standardise the use of these tools, it is necessary to develop an analytical approach that performs reproducibly both within and between studies., Methodology: Dried blood spots were collected in 2014 from children aged 1-9 years in Laos (n = 952) and Uganda (n = 2700) and from people aged 1-90 years in The Gambia (n = 1868). Anti-Pgp3 antibodies were detected by ELISA. A number of visual and statistical analytical approaches for defining serological status were compared., Principal Findings: Seroprevalence was estimated at 11.3% (Laos), 13.4% (Uganda) and 29.3% (The Gambia) by visual inspection of the inflection point. The expectation-maximisation algorithm estimated seroprevalence at 10.4% (Laos), 24.3% (Uganda) and 29.3% (The Gambia). Finite mixture model estimates were 15.6% (Laos), 17.1% (Uganda) and 26.2% (The Gambia). Receiver operating characteristic (ROC) curve analysis using a threshold calibrated against external reference specimens estimated the seroprevalence at 6.7% (Laos), 6.8% (Uganda) and 20.9% (The Gambia) when the threshold was set to optimise Youden's J index. The ROC curve analysis was found to estimate seroprevalence at lower levels than estimates based on thresholds established using internal reference data. Thresholds defined using internal reference threshold methods did not vary substantially between population samples., Conclusions: Internally calibrated approaches to threshold specification are reproducible and consistent and thus have advantages over methods that require external calibrators. We propose that future serological analyses in trachoma use a finite mixture model or expectation-maximisation algorithm as a means of setting the threshold for ELISA data. This will facilitate standardisation and harmonisation between studies and eliminate the need to establish and maintain a global calibration standard., Competing Interests: SG is employed by the commercial company IHRC, Inc. and is a contractor at the Centres for Disease Control and Prevention. The authors have declared that no competing interests exist.

We describe here a lateral flow-based assay (LFA) for the detection of antibodies against immunodominant antigen Pgp3 from Chlamydia trachomatis, the causative agent of urogenital chlamydia infection and ocular trachoma. Optimal signal detection was achieved when the gold-conjugate and test line contained Pgp3, creating a dual sandwich capture assay. The LFA yielded positive signals with serum and whole blood but not with eluted dried blood spots. For serum, the agreement of the LFA with the non-reference multiplex assay was 96%, the specificity using nonendemic pediatric sera was 100%, and the inter-rater agreement was κ=0.961. For whole blood, the agreement of LFA with multiplex was 81.5%, the specificity was 100%, and the inter-rater agreement was κ=0.940. The LFA was tested in a field environment and yielded similar results to those from laboratory-based testing. These data show the successful development of a lateral flow assay for detection of antibodies against Pgp3 with reliable use in field settings, which would make antibody-based testing for trachoma surveillance highly practical, especially after cessation of trachoma elimination programs., (Published by Elsevier B.V.)

Vaccines to prevent Trypanosoma cruzi infection in humans or animals are not available, and in many settings, dogs are an important source of domestic infection for the insect vector. Identification of infected canines is crucial for evaluating peridomestic transmission dynamics and parasite control strategies. As immune control of T. cruzi infection is dependent on humoral and cell-mediated immune responses, we aimed to define a serodiagnostic assay and T cell phenotypic markers for identifying infected dogs and studying the canine T. cruzi-specific immune response. Plasma samples and peripheral blood mononuclear cells (PBMCs) were obtained from forty-two dogs living in a T. cruzi-endemic region. Twenty dogs were known to be seropositive and nine seronegative by conventional serologic tests two years prior to our study. To determine canine seroreactivity, we tested sera or plasma samples in a multiplex bead array against eleven recombinant T. cruzi proteins. Ninety-four percent (17/18) of dogs positive by multiplex serology were initially positive by conventional serology. The frequency of IFNγ-producing cells in PBMCs responding to T. cruzi correlated to serological status, identifying 95% of multiplex seropositive dogs. Intracellular staining identified CD4+ and CD8+ T cell populations as the sources of T. cruzi lysate-induced IFNγ. Low expression of CCR7 and CD62L on CD4+ and CD8+ T cells suggested a predominance of effector/effector memory T cells in seropositive canines. These results are the first, to our knowledge, to correlate T. cruzi-specific antibody responses with T cell responses in naturally infected dogs and validate these methods for identifying dogs exposed to T. cruzi.

Trojan objects share a planet's orbit, never straying far from the triangular Lagrangian points, 60° ahead of (L4) or behind (L5) the planet. We report the detection of a Uranian Trojan; in our numerical integrations, 2011 QF99 oscillates around the Uranian L4 Lagrange point for >70,000 years and remains co-orbital for ~1 million years before becoming a Centaur. We constructed a Centaur model, supplied from the transneptunian region, to estimate temporary co-orbital capture frequency and duration (to a factor of 2 accuracy), finding that at any time 0.4 and 2.8% of the population will be Uranian and Neptunian co-orbitals, respectively. The co-orbital fraction (~2.4%) among Centaurs in the International Astronomical Union Minor Planet Centre database is thus as expected under transneptunian supply.

We have developed epifluorescence filter sets and computer software for the detection and discrimination of 27 different DNA probes hybridized simultaneously. For karyotype analysis, a pool of human chromosome painting probes, each labelled with a different fluor combination, was hybridized to metaphase chromosomes prepared from normal cells, clinical specimens, and neoplastic cell lines. Both simple and complex chromosomal rearrangements could be detected rapidly and unequivocally; many of the more complex chromosomal abnormalities could not be delineated by conventional cytogenetic banding techniques. Our data suggest that multiplex-fluorescence in situ hybridization (M-FISH) could have wide clinical utility and complement standard cytogenetics, particularly for the characterization of complex karyotypes.

In 1978 and 1979 two infant formulas, Neo-Mull-Soy and Cho-Free, were found to be deficient in chloride. The Centers for Disease Control received reports that hypochloremic metabolic alkalosis (HMA) had developed in 141 children as a result of exposure to these formulas. Thirty-five of these children were examined at 9 and 10 years of age and compared with a group of 32 children who were exposed to the chloride-deficient formulas but were not reported to experience HMA and a group of 61 children who received chloride-sufficient soy formulas in infancy. The control children were matched to the HMA children on sex, race, age, and maternal education. Growth characteristics, performance on the Wechsler Intelligence Scale for Children-Revised (WISC-R), the Boston Naming Test, the Rey-Osterrieth Test, the Clinical Evaluation of Language Fundamentals-Revised (CELF-R), and subtests from several other speech and language tests were compared across the groups. After adjustment for family income and the level of the father's education, significantly lower scores were observed in the HMA children on the WISC-R Arithmetic subtest (mean = 10.5) compared with the soy control children (mean = 12.0, P less than .05) and on the WISC-R Coding subtest (mean = 9.0) compared with the soy control children (mean = 10.8, P less than .01). All the WISC-R subtest scores were, however, within the normal range. Although no significant differences occurred on the CELF-R between groups, the risk of an HMA child falling below the range expected for a standard population was increased on the CELF-R Composite Total, Receptive, and Expressive Language scores: risk ratios = 2.14, 2.14, and 3.03 respectively. Significant differences were observed between the children exposed, both HMA and non-HMA children, and the soy control children for behavioral problems as determined by the Achenbach Childhood Behavioral Checklist. It is concluded that as a group, children with documented HMA appear to have recovered from their growth failure and have normal cognitive development. They may, however, be at risk for deficits in language skills that require expressive language abilities.