We performed a clustered form of LCA to estimate the sensitivity and specificity of three tests of ocular chlamydia infection (TF and TI for clinical trachoma and PCR for chlamydial DNA) and provide village-level estimates of the trachoma prevalence. In making these estimates, we did not assume a gold standard. We estimated TF to be 87.3% sensitive and 36.6% specific, TI to be 53.6% sensitive and 88.3% specific, and PCR to be 87.5% sensitive and 100% specific.
TF, the diagnostic test used in the WHO trachoma guidelines, had its advantages. It is inexpensive, yields instant results, and is sensitive; 87.3% of latent class positives would be expected to be TF positive. However, this analysis suggests that it is poorly specific (36.6%) and tends to overestimate infection rates (), which could lead to unnecessary treatment. Low specificity may be the result of the kinetics of infection in which follicles may persist long after the infection has been cleared23
or in which follicles may reoccur without the presence of chlamydia.14
Despite low specificity, the advantages of TF—namely, low cost and immediate results—ensure that it will remain critical in disease surveys.
TI is not currently used in the WHO treatment protocols. TF is the preferred test, because it is more closely related to the historical MacCallan classification and because TI is often overdiagnosed in the presence of redness or scarring (Taylor H, personal communication, 2011). Nevertheless, this study suggests that TI is far more specific (88.3%) than TF (36.6%). TI has been shown to correlate more strongly with PCR results10,15
and to have higher chlamydial loads by quantitative PCR.24
Our results suggest that it lacks sensitivity (53.6%) and tends to underestimate prevalence (). Again, this may be due to infection kinetics; TI tends to resolve sooner in the course of infection than does TF.25
Another trachoma study used a hidden Markov model, which shares traits with latent class models, to analyze longitudinal data. Briefly, their model had a hidden true disease state, analogous to our latent class, and diagnostic tests approximated this disease state with parameterized sensitivity and specificity. They estimated clinical activity (presence of TF and/or TI) to be 97% sensitive and 93% specific.26
Their results are difficult to directly compare with ours for two main reasons. First, their model used longitudinal data and was designed with the goal of estimating duration of infection, not test characteristics. Second, we used PCR as a diagnostic test, whereas they used an immunoassay.
Our estimates of PCR sensitivity (87.5%) and specificity (100%) closely agree with the best current estimate in a recent review in which sensitivity was estimated at 90% to 100% and specificity at 95% to 100%.3
Although the Amplicor test (Roche Diagnostics) is officially indicated only for urogenital use, it is commonly used in trachoma studies.11,18
In the urogenital literature, similar performance has been reported: sensitivities of 90% to 92% and specificities of 99 to 100%.27–29
Our estimates come from macrolide naive areas with high prevalence. The temporal relationship between infection and clinical examination could lead to different results in other settings, particularly in recently treated or low-prevalence areas. Our estimates should be re-evaluated, as posttreatment data accumulate from ongoing mass-treatment studies.
PCR has limitations, particularly that it may have a false-negative rate as high as 20% (). The Amplicor test evaluated here detects the cryptic plasmid present in most C. trachomatis
. Others have isolated C. trachomatis
that lacks the plasmid,30
suggested that other species of Chlamydia
can cause trachomatous inflammation,31
and shown that a broadened spectrum of PCR targets increases sensitivity.32
The Amplicor test evaluated here would fail to detect other species or variants with altered or missing, which could be critical in the face of selection pressure. Differences between conjunctival and epithelial specimens, human conjunctival cell yield, DNA extraction efficiency, and removal of molecular inhibitors may also affect test performance33
; those factors were not examined in this study and may partially account for PCR's imperfect sensitivity.
Determining test characteristics in the absence of a gold standard is difficult, and latent class analysis has several limitations.34,35
The most relevant limitation in our case is that we assume diagnostic tests are independent, given their underlying latent class disease state. Positive correlation between tests may cause LCA to overestimate sensitivity and specificity,36
and so it is possible that estimates are optimistic. There are methods that directly calculate correlation between diagnostic tests.37–39
These methods all require more than three diagnostic tests for the model to be identifiable. For our data, we were able to perform a two-test LCA comparing TF versus PCR and TI versus PCR (comparative analyses 1 and 2). By ignoring one of the clinical examinations in each of these comparative analyses, we eliminated any possible effect of correlation between TF and TI. The two-test LCA comparing TF and PCR produced estimates for the sensitivity of PCR and the specificity of TF that lay just outside the 95% CIs for the primary analysis. Neither of these differences was significant when accounting for error in each estimate. The results of the two-test LCA comparing TI and PCR lay within the primary analysis CIs. Taken together, these findings suggest that correlation between TF and TI may be playing a small role in our model. Quantifying the relationship further requires more diagnostic tests.
LCA may be particularly well suited to trachoma because of the unclear definition of a case of trachoma. The WHO defines cases based on clinical examination, whereas research studies, such as the source of these data, frequently use laboratory tests such as PCR. These two approaches are fundamentally different; examination detects inflammation whereas PCR detects the causative organism. Examination and laboratory tests are frequently discordant,14,23
possibly due to infection kinetics23
and age-dependent manifestations of infection.40
Latent class is appropriate for trachoma because the trachoma latent class in the model is never specifically defined. Instead, it acts as an unbiased composite of all available data, which is more appropriate than defining cases based on unverified assumptions. This begs clarification of what the latent trachoma class actually represents. In our case, the high sensitivity and almost 100% specificity of PCR suggest that the trachoma class represents chlamydial infection as determined by PCR more than examination findings.
Our LCA approach both reaffirms and challenges some traditionally held views about trachoma. PCR appears to have the specificity of a true gold standard (100%) but lacks sensitivity (87.5%). TF, the diagnosis used in the WHO protocol, is quite sensitive (87.3%) but poorly specific (36.6%). Although TI is no longer used in the WHO protocol, our findings suggest that it could play a role due to its specificity (88.3%) and its strong correlation with LCA prevalence (0.74). The next step is to compare these estimates with those from ongoing clinical trials, particularly those in areas of different prevalence and in areas after treatment.