Abstract
Purpose:
Definitive diagnosis of intraocular tuberculosis has remained challenging despite recent advances in molecular diagnostic techniques. Here we report the development of a real-time polymerase chain reaction (PCR) assay for detection of Mycobacterium tuberculosis complex in aqueous and vitreous samples from eyes with intraocular tuberculosis.
Methods:
Aqueous or vitreous humor samples were collected from patients with clinically suspected ocular tuberculosis (based on previously published diagnostic criteria; Gupta et al, Surv Ophthalmol' 2007) and non-uveitis eyes undergoing vitrectomy or cataract surgeries (controls). mpb64 gene of M. tuberculosis genome and human RPPH1 (RNase P RNA component H1) were amplified from the extracted DNA and detected real-time by customized FAM-labeled probes. The ratio of copy numbers of mpb64 and RPPH1, obtained from each test and control sample was used to generate Receiver Operating Characteristic (ROC) curves. The optimum cut-off value of real-time PCR was identified from the experimental data that had the highest Youden index (Youden index = sensitivity+specificity-1).
Results:
M. tuberculosis complex genome was detected in 33 of 47 test samples (70.2%) and 2 of 18 healthy controls (11.2%) based on optimum cutoff value of copy number ratios (0.025) obtained from ROC curve having highest Youden index number, 0.727. At this cutoff value the sensitivity was 81.0% and specificity 91.7%. The copy number ratios varied widely in different clinical samples, with the highest median value seen in intermediate uveitis sub-group (0.387±0.664). The numbers were not sufficient to compare aqueous and vitreous samples.
Conclusions:
We could develop a highly specific and sensitive PCR assay for detection of M. tuberculosis complex in aqueous and vitreous samples. There was wide variation in copy numbers in different disease sub-types that need to be analyzed in larger studies.