Community-level chlamydial serology for assessing trachoma elimination in trachoma-endemic Niger

Background: Program decision-making for trachoma elimination currently relies on conjunctival clinical signs. Antibody tests may provide additional information on the epidemiology of trachoma, particularly in regions where it is disappearing or elimination targets have been met. Methods: A cluster-randomized trial of mass azithromycin distribution strategies for trachoma elimination was conducted over three years in a mesoendemic region of Niger. Dried blood spots were collected from a random sample of children aged 1-5 years in each of 24 study communities at 36 months after initiation of the intervention. A multiplex bead assay was used to test for antibodies to two Chlamydia trachomatis antigens, Pgp3 and CT694. We compared seropositivity to either antigen to clinical signs of active trachoma (trachomatous inflammation—follicular [TF] and trachomatous inflammation—intense [TI]) at the individual and cluster level, and to ocular chlamydia prevalence at the community level. Results: Of 988 children with antibody data, TF prevalence was 7.8% (95% CI 6.1 to 9.5) and TI prevalence was 1.6% (95% CI 0.9 to 2.6). The overall prevalence of antibody positivity to Pgp3 was 27.2% (95% CI 24.5 to 30), and to CT694 was 23.7% (95% CI 21 to 26.2). Ocular chlamydia infection prevalence was 5.2% (95% CI 2.8 to 7.6). Seropositivity to Pgp3 and/or CT694 was significantly associated with TF at the individual and community level and with ocular chlamydia infection and TI at the community level. Older children were more likely to be seropositive than younger children. Conclusion: Seropositivity to Pgp3 and CT694 correlates with clinical signs and ocular chlamydia infection in a mesoendemic region of Niger. Trial registration: ClinicalTrials.gov NCT00792922. The study methods have been previously reported in detail elsewhere [11–13]. Briefly, a cluster randomized trial of annual versus biannual mass azithromycin distribution for trachoma control was conducted in the Matameye district of the Zinder region of Niger from May 2010 until August 2013 [4–6]. Data on active trachoma and ocular infection were collected biannually on children aged 0–5 years; dried blood spots for serological analysis were collected only at the 36-month time point and only from children aged 1–5 years. Dried blood spots were shipped to CDC at ambient temperature and tested for antibodies from July to August 2014. Communities were chosen from among six different catchment areas for primary health care facilities and were eligible for inclusion if they met the following criteria: (1) contained a population between 250 to 600 persons, (2) were located more than 4 kilometers from the center of any semi-urban area, and (3) had a prevalence of active trachoma more than 10% in children aged 0–5 years [11]. 235 communities in the 6 health centers were deemed eligible, of which 48 were randomly selected for inclusion in the trial. Children aged 1–5 years were included in this analysis, due to the inability of antibody tests to differentiate between maternal-child antibodies in <1–year-olds. 48 communities were randomly divided into 4 treatment arms in a 2x2 factorial design (12 communities per arm), comparing two azithromycin coverage targets (standard versus enhanced coverage) and annual versus biannual treatment. Randomization of communities to treatment arms was done using RANDOM and SORT functions in Microsoft Excel (Version 2003). Only communities from the enhanced coverage arms were included in testing for antibodies (N = 24 communities) for logistical reasons. Trained study health workers conducted a full household census in all communities prior to the initial survey visit. During the baseline visit, adults in the household consented to census data collection, and study personnel recorded the name, sex, and age or date of birth for all individuals in the household. After consent was obtained, study participants were examined for the presence of TF and TI. Clinical grading of each everted superior tarsal conjunctiva was performed using a 2.5x binocular loupe and a torch light, if necessary, per the WHO grading system. Clinical grading was performed according to the WHO simplified grading system of TF being the presence 5 or more follicles >0.5 mm in diameter and TI as inflammation severe enough to obscure 50% of deep tarsal vessels in one or both eyes [14]. Prior to swabbing, a trained photographer took at least 2 photographs of the right eyelid of all participants using a Nikon D-series camera and a Micro Nikon 105 mm; f/2.8 lens (Nikon, Tokyo, Japan). After conjunctival examination, a Dacron swab was passed 3 times over the right upper tarsal conjunctiva, rotating the swab approximately 120 degrees between each pass. All samples were placed immediately on cold packs in the field and transferred to -20°C within 10 hours, then shipped on cold packs to University of California, San Francisco, CA, USA where they were stored in -80°C freezers until processing. PCR testing was performed for children aged 0–5 years. Samples from the same village, age in years, and visit were randomly pooled into groups of five for group testing, with a possible remainder pool of one to four samples [15]. Pooled samples were tested for the presence of Ct DNA using the Roche Amplicor qualitative PCR assay (Roche Molecular Systems, Indianapolis, IN, USA). Community prevalence was estimated from the pools as previously described [11,15]. In communities randomized to annual treatment, study participants age 6 months of age and older received a directly observed dose of oral azithromycin (20 mg/kg up to a maximum dose of 1 g in adults). In biannually treated communities, only children up to 12 years of age were offered treatment. Children under 6 months of age in all communities were offered topical tetracycline ointment (1%) to be applied to both eyes twice a day for six weeks. Pregnant women in the annual arm and individuals allergic to macrolides were offered topical tetracycline. All communities were visited up to four days in order to achieve 90% treatment coverage [12,13]. Children under 5 years of age were selected randomly in each village for blood sample collection via finger stick or heel stick, with a goal of 50 children per village. Blood spots were analyzed for antibody to Ct antigens Pgp3 and CT694 using a multiplex bead array assay on a Luminex 200 platform, as previously described [7]. Results were reported as median fluorescence intensity minus background (MFI-BG) where background is the signal from beads run with buffer only. Positivity cut-off for Pgp3 was greater than or equal to 1083, and CT694 cutoff was greater than or equal to 496 as determined by receiver operator characteristic (ROC) curve analysis from a pediatric U.S.-based negative panel (N = 117) and Tanzania based positive panel from children with ocular Ct infection (N = 40) [7]. Data were entered into a customized database (Microsoft Access v2007) developed at the Dana Center, Johns Hopkins University. To estimate associations between seropositivity, clinical trachoma, and age at the individual level, we used generalized linear models with a binomial distribution and log link to estimate prevalence ratios (PR). All standard errors were clustered at the community level, which was the randomization unit of the study. As individual-level PCR data were not available, associations between seropositivity to the Ct antigen and ocular chlamydia infection were conducted only at the community level. We additionally analyzed the association between seropositivity and clinical trachoma at the cluster level. We used linear regression models to evaluate relationships between trachoma indicators at the community level. All analyses were conducted in Stata 14.1 (StataCorp, College Station, TX). All procedures and protocols for this study were approved by the Committee for Human Research of UCSF, and le Comité Consultatif National d’Ethique du Minstère de la Santé Publique, Niger (Ethical Committee, Niger Ministry of Health). The study’s Data Safety and Monitoring Committee observed the study implementation during annual reviews of quality assurance, as appointed by the PRET study Executive Committee. All village leaders of communities within the study agreed to participate in the trial with written (thumbprint) consent. For children under the age of 16, consent was given by a parent or a guardian. All persons participating in the trial were given the opportunity to be treated according to their community’s random treatment assignment. Communities not included in the study were offered treatment through the national treatment program. CDC personnel did not have access to personal identifying information and were determined to be non-engaged in the study.