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PURPOSE: Although trachoma control programs frequently use the World Health Organization (WHO) simplified grading system for trachoma to monitor the clinical response after repeated mass azithromycin treatments, the programmatic relevance of this evaluation after multiple rounds of antibiotic treatments is unclear. METHODS: Three rounds of annual mass azithromycin were distributed to 12 villages in Ethiopia. Twelve months after the third treatment, children were assessed for follicular trachomatous inflammation (TF) and intense trachomatous inflammation (TI) using the WHO simplified grading system and for ocular chlamydial infection using DNA-based and RNA-based tests. Test characteristics for predicting chlamydial infection were computed assuming a chlamydial RNA-based gold standard. As a secondary analysis, test characteristics were also assessed using a latent class analysis. RESULTS: The prevalence of RNA evidence of ocular chlamydia was 7.1% (95% confidence interval [CI], 2.7-17.4). A DNA-based test and TF had sensitivities of 61.0% (95% CI, 47.1-73.3) and 65.9% (95% CI, 41.6-83.9), specificities of 100% (95% CI, 99.3-100) and 67.5% (95% CI, 61.0-73.5), and positive predictive values of 100% (95% CI, 86.3-100) and 13.4% (95% CI, 5.5-29.3) compared with an RNA-based gold standard. The latent class analysis confirmed that the RNA-based test was a reasonable choice for a gold standard, with a sensitivity of 100% (95% CI, 67.1-100) and specificity of 99.6% (95% CI, 98.1-100). CONCLUSIONS: Basing treatment decisions after mass azithromycin distributions on the WHO simplified grading system will maximize the treatment of infected persons compared with a DNA-based test but will also result in more uninfected persons being treated. The RNA-based test was considerably more sensitive, and almost equivalently specific, compared with a DNA-based test. (ClinicalTrials.gov number, NCT00322972.).