Purchase this article with an account.
Soumyava Basu, Manas Ranjan Barik, Praveen Kumar Balne, Soveeta Souravee Rath, Mamatha Reddy, Savitri Sharma; Evaluation of real-time PCR for the diagnosis of intra-ocular tuberculosis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4062.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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.
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).
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.
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.
This PDF is available to Subscribers Only