June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Evaluation of real-time PCR for the diagnosis of intra-ocular tuberculosis
Author Affiliations & Notes
  • Soumyava Basu
    LV Prasad Eye Institute, Bhubaneswar, India
  • Manas Ranjan Barik
    LV Prasad Eye Institute, Bhubaneswar, India
  • Praveen Kumar Balne
    LV Prasad Eye Institute, Bhubaneswar, India
  • Soveeta Souravee Rath
    LV Prasad Eye Institute, Bhubaneswar, India
  • Mamatha Reddy
    LV Prasad Eye Institute, Bhubaneswar, India
  • Savitri Sharma
    LV Prasad Eye Institute, Bhubaneswar, India
  • Footnotes
    Commercial Relationships Soumyava Basu, None; Manas Barik, None; Praveen Balne, None; Soveeta Rath, None; Mamatha Reddy, None; Savitri Sharma, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4062. doi:
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      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.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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