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J. G. Parikh, L. M. Mosqueda, N. A. Rao; Correlation of Mycobacterium Genome Levels With Histopathologic Changes in Ophthalmic Tissue. Invest. Ophthalmol. Vis. Sci. 2008;49(13):497. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Quantitiative PCR (qPCR) was recently introduced for the diagnosis of mycobacterium tuberculosis (mTB). The current study is undertaken to correlate the amplified mTB genome with histopathologic changes and results of acid fast Ziehl-Neelsen staining results.
Thirty-one cases with clinical and or histologic features suggestive of mTB or atypical mycobacterial (ATB) infection were identified from files of the Doheny Eye Institute Pathology Laboratory. Formalin-fixed and paraffin-embedded ophthalmic tissues were processed for acid-fast staining and submitted for standardized qPCR using specific forward primer IS6 (5' AGGCGAACCCTGCCCAG-3') and reverse primer IS7 (5' GATCGCTGATCCGGCCA-3') for the IS6110 gene. Cases were divided into 5 groups based on the levels of the amplification product (bacterial load) and size of ceseous necrotic area. The area of caseous necrosis was measured using Leica DM LB2 digital microcope and SPOT digital camera software.
Seven (22.5%) cases were positive for mycobacterium with acid-fast staining, and all seven were also positive on qPCR. Three of the seven were ATB infection. Eight (25.8%) cases revealed amplified genome of mTB but were negative for acid-fast staining; two of the eight were ATB infection. The bacterial genome load varied from as low as 7 per 1 µg of total extracted tissue DNA to 217,425 per 1 µg of total extracted tissue DNA. The area of necrosis ranged from 0 to 32.2 mm2. In those cases with high genome levels, the acid-fast organisms could be readily detected and the necrotic areas were more prominent. Cases that had a low bacterial genome level and were negative for acid-fast staining showed tiny foci of necrosis or no necrosis. All mTB infections showed histopathologic features of granulomatous inflammation with or without caseation necrosis, whereas two of ATB infections showed nongranulomatous inflammation.
The extent of necrosis in the tuberculosis granuloma depends on the bacterial genome load. Larger genome loads are associated with large area of necrosis. The specific diagnostic method of qPCR is more sensitive than acid-fast staining for detection of infectious agents with low bacterial load with or without caseous necrosis. The nucleic acid amplification procedure, however, cannot distinguish mTB from ATB infections.
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