Fool's gold: Why imperfect reference tests are undermining the evaluation of novel diagnostics: a reevaluation of 5 diagnostic tests for leptospirosis

Abstract

Background: We observed that some patients with clinical leptospirosis supported by positive results of rapid tests were negative for leptospirosis on the basis of our diagnostic gold standard, which involves isolation of Leptospira species from blood culture and/or a positive result of a microscopic agglutination test (MAT). We hypothesized that our reference standard was imperfect and used statistical modeling to investigate this hypothesis.

Methods: Data for 1652 patients with suspected leptospirosis recruited during three observational studies and one randomized control trial that described the application of culture, MAT, immunofluorescence assay (IFA), lateral flow (LF) and/or PCR targeting the 16S rRNA gene were reevaluated using Bayesian latent class models and random-effects meta-analysis.

Results: The estimated sensitivities of culture alone, MAT alone, and culture plus MAT (for which the result was considered positive if one or both tests had a positive result) were 10.5% (95% credible interval [CrI], 2.7%-27.5%), 49.8% (95% CrI, 37.6%-60.8%), and 55.5% (95% CrI, 42.9%-67.7%), respectively. These low sensitivities were present across all 4 studies. The estimated specificity of MAT alone (and of culture plus MAT) was 98.8% (95% CrI, 92.8%-100.0%). The estimated sensitivities and specificities of PCR (52.7% [95% CrI, 45.2%-60.6%] and 97.2% [95% CrI, 92.0%-99.8%], respectively), lateral flow test (85.6% [95% CrI, 77.5%-93.2%] and 96.2% [95% CrI, 87.7%-99.8%], respectively), and immunofluorescence assay (45.5% [95% CrI, 33.3%-60.9%] and 96.8% [95% CrI, 92.8%-99.8%], respectively) were considerably different from estimates in which culture plus MAT was considered a perfect gold standard test.

Conclusions: Our findings show that culture plus MAT is an imperfect gold standard against which to compare alterative tests for the diagnosis of leptospirosis. Rapid point-of-care tests for this infection would bring an important improvement in patient care, but their future evaluation will require careful consideration of the reference test(s) used and the inclusion of appropriate statistical models.

Figure 1.

Schematic illustration of the use of Bayesian latent class model to obtain unbiased estimates of accuracy of diagnostic tests. A, Overview of all possible outcomes of diagnostic tests, based on true disease status, if 3 diagnostic tests are applied to 1 study population. Broken lines represent unknown parameters. Patients under evaluation could be either diseased or nondiseased, and prevalence represents the probability that a patient is diseased. Solid lines represent the application of all 3 diagnostic tests (t₁, t₂, and t₃) to every patient in the study. Test results are conditional on the sensitivity and specificity of each test. True disease status (diseased or nondiseased) is a latent variable, as it is not directly observed but can be estimated as the prevalence in the Bayesian latent class model. B and C, Comparison of how to estimate the accuracy of diagnostic tests, using the gold standard model (B) and the imperfect gold standard model (C).

Figure 2.

Study flow diagram.

Figure 3.

Forest plot of sensitivity and specificity of culture (A), microagglutination test (MAT; B), and the combination of culture and MAT (culture + MAT; C and D) for leptospirosis estimated by Bayesian latent class models (LCMs) and random-effects meta-analysis models. Squares represent median estimates of the sensitivities and the specificities, and the size of the square represents the size of the study. Horizontal lines represent 95% credible intervals of the estimates. Bayesian LCM assuming conditional dependence between culture and PCR and between MAT and lateral flow (LF) was used for study A, and Bayesian LCM assuming conditional dependence between MAT and immunofluorescence assay was used for study B. Bayesian LCM assuming conditional independence between tests and consistency of test accuracies between study sites was used for studies C and D. Meta-analysis was performed by application of random-effects variables into the combined data set of all 4 studies, assuming conditional dependence between culture and PCR, between MAT and LF, and between MAT and IFA. Abbreviations: IFA, immunofluorescence assay; LCM, latent class model; LF, lateral flow; MAT, microagglutination test; PCR, polmerase chain reaction.