Most studies that have monitored for trachoma after 3 mass azithromycin distributions have reported only the clinical signs of trachoma. However, there is poor agreement between the clinical signs of trachoma and ocular chlamydial infection after mass antibiotic treatments, likely because clinical signs persist for months after ocular chlamydia has been cleared [2]. The few studies that have monitored ocular chlamydia after 3 repeated mass antibiotic distributions have done so using commercially available nucleic acid amplification tests (NAATs) targeting chlamydial DNA [3, 4]. Recently, studies in both the trachoma and sexually transmitted disease literature have demonstrated that RNA-based NAATs are more sensitive than these DNA-based tests for detecting chlamydia in an individual [5–7]. RNA-based tests have an additional target capture step and a more abundant genetic target, either of which may contribute to the increased sensitivity. It is possible that communities without any DNA evidence of ocular chlamydia could nonetheless have infection detectable by an RNA-based test—although evidence for this was not found in a previous study [8]. In this report, we use both RNA-based and DNA-based tests to monitor 12 trachoma-hyperendemic communities that have been treated with 3 annual mass azithromycin distributions.

We performed monitoring in a randomly selected sentinel community from each subkebele. In each of the 12 sentinel communities, 50 randomly chosen children aged 0–9 years underwent conjunctival examination and swabbing at baseline and month 36 (ie, 1 year after the third mass treatment). Separate random samples of children were selected at each time point, based on the most recent census. At each monitoring visit, 1 of 13 trained examiners assessed the upper right tarsal conjunctiva for follicular trachomatous inflammation (TF) and intense trachomatous inflammation (TI) according to the WHO simplified grading system [9]. The examiner then passed a Dacron swab 3 times over the upper right tarsal conjunctiva. At month 36, an additional swab for chlamydial RNA was collected in a similar fashion, using swabs and transport media from the APTIMA-CT Unisex Swab Specimen Collection Kit (Gen-Probe). RNA swabs were always collected after Dacron swabs. Examiners used the same pair of gloves for the Dacron and RNA swabs for each study subject, changing into a new pair of gloves for each new subject. We collected negative control swabs on 5 randomly selected children per community for each test by passing a swab within 1 inch of, but not touching, the tarsal conjunctiva.

Dacron swab specimens were stored and transported frozen, and then tested for C. trachomatis DNA using the AMPLICOR PCR assay (Roche Diagnostics). Dacron swab specimens from baseline were analyzed as pools of 2 swabs randomly selected from the same community, with maximum likelihood estimation used to determine the number of positive individual tests most likely to have resulted in the pooled results. Dacron swab specimens from month 36 were analyzed as pools of 5 swabs, with individual testing of any positive pools. RNA swab specimens were stored and transported to San Francisco at room temperature; swabs were processed using the APTIMA-CT assay to detect C. trachomatis ribosomal RNA, also in pools of 5 with individual testing of positive pools.

Follow-up monitoring was performed on 583 children from 370 households at month 36 (1 year after the last azithromycin distribution; Table 1). In total, 25 children from 20 households and 7 communities had DNA evidence of ocular chlamydia (mean prevalence, 4.2% [95% CI, .3%–8.0%]; P = .002 compared to baseline), and 41 children from 35 households and 8 communities had RNA evidence (mean prevalence, 6.9% [95% CI, .4%–13.3%]). For both tests, the distribution of infection was highly skewed, with 80% of infected children living in just 3 of the 12 communities. Although the DNA- and RNA-based tests were closely correlated (Spearman ρ = 0.95, P < .001), the ocular chlamydia prevalence estimate was higher for the RNA-based test compared to the DNA-based test (P = .02). The correlation between the community prevalence of ocular chlamydia and the prevalence of clinically active trachoma (TF and/or TI) was similar when using the RNA-based test (ρ = 0.62, P = .03) or the DNA-based test (ρ = 0.64, P = .02). All swabs that were positive for DNA were also positive for RNA. No negative control swabs collected at month 36 tested positive for chlamydial DNA (n = 60) or RNA (n = 59).

Of the 8 communities with evidence of chlamydial infection, 6 showed a higher prevalence with the RNA test compared to the DNA test. One community was classified as having eliminated infection by the DNA-based test but not by the RNA-based test. In this discrepant community, the RNA-based test detected only a single case of ocular chlamydia. Thus, although the RNA-based test appears to be a more stringent classifier of elimination, it is unclear whether the increased sensitivity of this test would be meaningful to trachoma programs.

According to WHO guidelines, communities that have already received 3 rounds of mass azithromycin should continue receiving mass treatments until the prevalence of TF is <5% [1]. In this study, all communities would have been eligible for continued mass treatment, even though one-third of the communities had no evidence of ocular chlamydia. Continued mass treatments may nonetheless be wise, because ocular chlamydial infection can return rapidly after discontinuation of mass azithromycin distributions in areas with hyperendemic trachoma [11]. Alternatively, programs may wish to minimize the disadvantages of continued mass treatments, such as potential antibiotic resistance and cost, by targeting antibiotic treatments to those most likely to be infected. As this study and others have shown, ocular chlamydia clusters by household, suggesting that strategies to target infected households could be helpful for trachoma elimination.