After three rounds of mass azithromycin, the WHO simplified grading system appeared to have comparable or better sensitivity but lower specificity than a DNA-based laboratory test. In other words, clinical examination identified more infected persons than the DNA-based test but at the cost of more false positives. Both TF and TI significantly increase the posttest probability of infection in a person but to a lesser degree than the DNA-based test.
Consistent with two previous reports, ocular swabs from this trachoma-endemic region were more frequently positive using the test for chlamydial RNA compared with the test for chlamydial DNA.17,18
Although it is possible that results of the 16 excess RNA-positive tests are false positives, there are several reasons to think they represent a true measure of chlamydial infection. First, the Aptima RNA-based tests have consistently been shown to be more sensitive, and equally or more specific, than DNA-based tests in the sexually transmitted disease literature.7,10–15
Second, a latent class analysis, which makes no assumptions about the gold standard, suggested that the data were most consistent with a highly specific RNA-based test and a less sensitive DNA-based test. Third, none of the negative control swabs tested positive for RNA, arguing against the possibility of false positives through contamination in the field or in the laboratory. Finally, confirmatory testing with RNA-based tests demonstrated infection in 81% of discrepant cases using ACT and in 31% of cases using AC2. Although one might have expected a higher proportion of discrepant cases to have tested positive with the ACT test, this degree of discrepancy on repeat testing is consistent with the 90% reproducibility of chlamydial NAATs demonstrated in other studies.16,36
The lower number of positive confirmatory tests with the AC2 assay was expected given that this test has been shown to be less sensitive than the ACT assay.8,11,16
Compared with a gold standard of chlamydial RNA, the DNA-based chlamydial NAAT had a sensitivity of only 61%, albeit with wide confidence intervals. This may seem to be lower than expected. However, these results are consistent with those of other studies of these particular RNA- and DNA-based tests in which the DNA-based test detected 64% to 86% of the RNA-positive cases.11–13
RNA-based methods are thought to more easily detect chlamydia than DNA-based methods, likely because chlamydial organisms contain approximately 1000 copies of 16s rRNA but only 7 to 10 copies of the cryptic DNA plasmid.9,37–39
In addition, the target capture step in the Aptima assays probably contributes to enhanced sensitivity. This increased sensitivity of the RNA target may be most relevant in the setting of a low load of infectious organisms, which was likely the case in this study of communities monitored after repeated mass azithromycin treatments. A separate study in Ethiopia found evidence for this hypothesis: the sensitivity of the DNA-based test was lower in villages treated with mass azithromycin than in untreated villages.18
In the present study, the sensitivity of the DNA test was lowest in those without clinically active trachoma, perhaps because these persons have a lower infectious load of chlamydia.40,41
The increased sensitivity of the RNA-based test—in the face of nearly equivalent specificity—suggests that the RNA-based test is the NAAT of choice for monitoring ocular chlamydia, especially for researchers and trachoma programs interested in trachoma elimination.
The WHO recommends conjunctival examination of children aged 1 to 9 years in communities treated with three annual mass azithromycin treatments, with further mass treatments until the prevalence of TF in children falls below 5%.20,21
Although the WHO recommendations are widely implemented, few studies have assessed the diagnostic test performance of the WHO simplified grading system after mass azithromycin treatment (). Here, we showed that both TF and TI provide diagnostic information regarding chlamydial infection after three mass azithromycin distributions, yet TF and TI are not interchangeable tests. In this and other studies, TF appears to be a more sensitive test than TI for ocular chlamydia infection after mass azithromycin treatments.42
In this study, TF even had a higher sensitivity than a DNA-based laboratory test. TF is, therefore, a sensible metric if the goal is trachoma elimination and identification of all possible cases of ocular chlamydia is desired.
Sensitivity, Specificity, and Predictive Values of Various Tests for Ocular Chlamydia after Mass Azithromycin Treatments, from Published Studies
It is important to note that the positive predictive values for both TF and TI were far lower than those of the DNA-based NAAT. This indicates that basing treatment decisions solely on the simplified grading system will necessarily result in treatment of a considerable number of uninfected persons. For example, in these communities, three mass treatments had a dramatic effect on ocular chlamydia, reducing the prevalence among 0 to 9-year-old children from 42% to 4% (reported elsewhere).27
The prevalence of TF declined much more slowly, to approximately 35%, much higher than the <5% prevalence required for discontinuing mass antibiotic treatments. Thus, many uninfected persons would receive antibiotics if mass treatments were continued. This is probably appropriate in areas with hyperendemic trachoma, where reinfection can rapidly occur after the cessation of mass treatments, even when these treatments have reduced the prevalence of chlamydial infection to very low levels.44
In areas with less prevalent trachoma, however, reliance on the WHO simplified grading system will likely result in the overtreatment of communities with low levels of ocular chlamydia. Use of NAATs for trachoma monitoring could reduce unnecessary antibiotic treatments. The perceived costs of NAAT are likely to prevent its use by many trachoma programs though, especially because the expenses of using the simplified grading system are so low. More expensive nucleic acid amplification tests may be worth the added cost in certain situations, such as in communities with a low pretreatment prevalence of trachoma, where reinfection is less likely to occur after mass antibiotics have reduced ocular chlamydia to low levels.42
Formal cost-effectiveness studies might be helpful in more clearly defining the role of laboratory tests after mass antibiotic treatments.
A limitation of this study is its cross-sectional nature. We had data for the WHO simplified grading system, the DNA-based NAAT, and the RNA-based NAAT only at a single time point, which did not allow us to comment on the clinical significance of infections detected only by the RNA-based test but not the DNA-based test (e.g., whether these infections are new, almost resolved, or low-level persistent).
In conclusion, we report the diagnostic test characteristics of the WHO simplified grading system and a DNA-based nucleic acid amplification test after three repeated mass antibiotic distributions in a hyperendemic region of Ethiopia compared with an RNA-based gold standard test. TF was the most sensitive test and appears to be an appropriate test for detecting the maximum amount of ocular chlamydia. However, both TF and TI had much lower positive predictive values than the DNA-based laboratory test, indicating that use of the WHO simplified grading system could result in overtreatment. The positive predictive value of the NAATs remained high even after repeated mass treatments, suggesting these tests would minimize unnecessary antibiotic distributions. If resources allow for the use of NAAT, the RNA-based test, given its superior sensitivity, is the test of choice.