The target end date for the global elimination of trachoma as a public health problem is 2020. As countries begin the process for submitting their dossier for the validation of elimination of trachoma as a public health problem, strategies for post-validation surveillance must be considered. Seroprevalence of antibodies against antigens from the causative bacteria Chlamydia trachomatis (Ct) in young children has been shown to reflect trachomatous inflammation-follicular (TF) rates in both endemic and previously endemic settings. However, none of these studies has directly compared age seroprevalence in the same communities before and after mass drug administration (MDA) for trachoma. Here we report a marked shift in age seroprevalence curves in four villages in Kapilvastu District, Nepal, before and after MDA. Clinical examinations were performed and blood was taken before (N = 659) and 5 years after (N = 646) MDA. Rates of TF decreased from 17.6% in ≤ 9-year-olds before MDA (N = 52) to 0% in ≤ 9-year-olds (N = 73) after MDA. Positive antibody responses to Ct in the entire population decreased from 82.1% pre-MDA to 35.8% post-MDA, whereas those among ≤ 9-year-olds decreased from 59.6% to 4.1%. These data show that the postintervention decrease in TF was reflected in a drop in anti-Ct antibody responses, suggesting that antibody responses could be useful indicators for post-validation surveillance.

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.

Background

Serological surveys with multiplex bead assays can be used to assess seroprevalence to multiple pathogens simultaneously. However, multiple methods have been used to generate cut-off values for seropositivity and these may lead to inconsistent interpretation of results. A literature review was conducted to describe the methods used to determine cut-off values for data generated by multiplex bead assays.

Methodology/principal findings

A search was conducted in PubMed that included articles published from January 2010 to January 2020, and 308 relevant articles were identified that included the terms "serology", "cut-offs", and "multiplex bead assays". After application of exclusion of articles not relevant to neglected tropical diseases (NTD), vaccine preventable diseases (VPD), or malaria, 55 articles were examined based on their relevance to NTD or VPD. The most frequently applied approaches to determine seropositivity included the use of presumed unexposed populations, mixture models, receiver operating curves (ROC), and international standards. Other methods included the use of quantiles, pre-exposed endemic cohorts, and visual inflection points.

Conclusions/significance

For disease control programmes, seropositivity is a practical and easily interpretable health metric but determining appropriate cut-offs for positivity can be challenging. Considerations for optimal cut-off approaches should include factors such as methods recommended by previous research, transmission dynamics, and the immunological backgrounds of the population. In the absence of international standards for estimating seropositivity in a population, the use of consistent methods that align with individual disease epidemiological data will improve comparability between settings and enable the assessment of changes over time.

The MORDOR trial in Niger, Malawi, and Tanzania found that biannual mass distribution of azithromycin to children younger than 5 years led to a 13.5% reduction in all-cause mortality (NCT02048007). To help elucidate the mechanism for mortality reduction, we report IgG responses to 11 malaria, bacterial, and protozoan pathogens using a multiplex bead assay in pre-specified substudy of 30 communities in the rural Niger placebo-controlled trial over a three-year period (n = 5642 blood specimens, n = 3814 children ages 1-59 months). Mass azithromycin reduces Campylobacter spp. force of infection by 29% (hazard ratio = 0.71, 95% CI: 0.56, 0.89; P = 0.004) but serological measures show no significant differences between groups for other pathogens against a backdrop of high transmission. Results align with a recent microbiome study in the communities. Given significant sequelae of Campylobacter infection among preschool aged children, our results support an important mechanism through which biannual mass distribution of azithromycin likely reduces mortality in Niger.

The Trachoma Control Program in Amhara region, Ethiopia, scaled up the surgery, antibiotics, facial cleanliness, and environmental improvement (SAFE) strategy in all districts starting in 2007. Despite these efforts, many districts still require additional years of SAFE. In 2017, four districts were selected for the assessment of antibody responses against Chlamydia trachomatis antigens and C. trachomatis infection to better understand transmission. Districts with differing endemicity were chosen, whereby one had a previous trachomatous inflammation-follicular (TF) prevalence of ≥ 30% (Andabet), one had a prevalence between 10% and 29.9% (Dera), one had a prevalence between 5% and 10% (Woreta town), and one had a previous TF prevalence of < 5% (Alefa) and had not received antibiotic intervention for 2 years. Survey teams assessed trachoma clinical signs and took conjunctival swabs and dried blood spots (DBS) to measure infection and antibody responses. Trachomatous inflammation-follicular prevalence among children aged 1-9 years was 37.0% (95% CI: 31.1-43.3) for Andabet, 14.7% (95% CI: 10.0-20.5) for Dera, and < 5% for Woreta town and Alefa. Chlamydia trachomatis infection was only detected in Andabet (11.3%). Within these districts, 2,195 children provided DBS. The prevalence of antibody responses to the antigen Pgp3 was 36.9% (95% CI: 29.0-45.6%) for Andabet, 11.3% (95% CI: 5.9-20.6%) for Dera, and < 5% for Woreta town and Alefa. Seroconversion rate for Pgp3 in Andabet was 0.094 (95% CI: 0.069-0.128) events per year. In Andabet district, where SAFE implementation has occurred for 11 years, the antibody data support the finding of persistently high levels of trachoma transmission.

Background

The WHO seeks to control trachoma as a public health problem in endemic areas. Achham District in western Nepal was found to have TF (trachoma follicular) above 20% in a 2006 government survey, triggering 3 annual mass drug administrations finishing in 2010. Here we assess the level of control that has been achieved using surveillance for clinical disease, ocular chlamydia trachomatis infection, and serology for antibodies against chlamydia trachomatis protein antigens.

Methods

We conducted a cross-sectional survey of children aged 1-9 years in communities in Achham District in early 2014 including clinical examination validated with photographs, conjunctival samples for Chlamydia trachomatis (Amplicor PCR), and serological testing for antibodies against chlamydia trachomatis protein antigens pgp3 and CT694 using the Luminex platform.

Findings

In 24 randomly selected communities, the prevalence of trachoma (TF and/or TI) in 1-9 year olds was 3/1124 (0.3%, 95% CI 0.1 to 0.8%), and the prevalence of ocular chlamydia trachomatis infection was 0/1124 (0%, 95% CI 0 to 0.3%). In 18 communities selected because they had the highest prevalence of trachoma in a previous survey, the prevalence of TF and/or TI was 7/716 (1.0%, 95% CI 0.4 to 2.0%) and the prevalence of ocular chlamydia trachomatis infection was 0/716 (0%, 95% CI 0 to 0.5%). In 3 communities selected for serological testing, the prevalence of trachoma was 0/68 (0%, 95% CI 0 to 5.3%), the prevalence of ocular chlamydia trachomatis infection was 0/68 (0%, 95% CI 0 to 0.5%), the prevalence of antibodies against chlamydia trachomatis protein antigen pgp3 was 1/68 (1.5%, 95% CI 0.04% to 7.9%), and the prevalence of antibodies against chlamydia trachomatis protein antigen CT694 was 0/68 (0%, 95% CI 0 to 5.3%).

Conclusion/significance

This previously highly endemic district in Nepal has little evidence of recent clinical disease, chlamydia trachomatis infection, or serological evidence of trachoma, suggesting that epidemiological control has been achieved.

Background

Biannual mass azithromycin administration to preschool children reduces all-cause mortality, but the mechanism for the effect is not understood. Azithromycin has activity against malaria parasites, and malaria is a leading cause of child mortality in the Sahel. The effect of biannual versus annual azithromycin distribution for trachoma control on serological response to merozoite surface protein 1 (MSP-1₁₉), a surrogate for malaria incidence, was evaluated among children in Niger.

Methods

Markers of malaria exposure were measured in two arms of a factorial randomized controlled trial designed to evaluate targeted biannual azithromycin distribution to children under 12 years of age compared to annual azithromycin to the entire community for trachoma control (N = 12 communities per arm). Communities were treated for 36 months (6 versus 3 distributions). Dried blood spots were collected at 36 months among children ages 1-5 years, and MSP-1₁₉ antibody levels were assessed using a bead-based multiplex assay to measure malaria seroprevalence.

Results

Antibody results were available for 991 children. MSP-1₁₉ seropositivity was 62.7% in the biannual distribution arm compared to 68.7% in the annual arm (prevalence ratio 0.91, 95% CI 0.83 to 1.00). Mean semi-quantitative antibody levels were lower in the biannual distribution arm compared to the annual arm (mean difference - 0.39, 95% CI - 0.05 to - 0.72).

Conclusions

Targeted biannual azithromycin distribution was associated with lower malaria seroprevalence compared to that in a population that received annual distribution. Trial Registration Clinicaltrials.gov NCT00792922.

Little is known about enteropathogen seroepidemiology among children in low-resource settings. We measured serological IgG responses to eight enteropathogens (Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, enterotoxigenic Escherichia coli, Vibrio cholerae, Campylobacter jejuni, norovirus) in cohorts from Haiti, Kenya, and Tanzania. We studied antibody dynamics and force of infection across pathogens and cohorts. Enteropathogens shared common seroepidemiologic features that enabled between-pathogen comparisons of transmission. Overall, exposure was intense: for most pathogens the window of primary infection was <3 years old; for highest transmission pathogens primary infection occurred within the first year. Longitudinal profiles demonstrated significant IgG boosting and waning above seropositivity cutoffs, underscoring the value of longitudinal designs to estimate force of infection. Seroprevalence and force of infection were rank-preserving across pathogens, illustrating the measures provide similar information about transmission heterogeneity. Our findings suggest antibody response can be used to measure population-level transmission of diverse enteropathogens in serologic 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 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.