Manas Ranjan Barik

Factors influencing polymerase chain reaction outcomes in patients with clinically suspected ocular tuberculosis.

BackgroundPolymerase chain reaction (PCR) assay can be a useful method for definitive diagnosis in paucibacillary infections such as ocular tuberculosis (TB). In this study, we have evaluated factors affecting PCR outcomes in patients with clinically suspected ocular TB. Patients with clinically suspected ocular TB were investigated by PCR of aqueous or vitreous samples. Three control groups were also tested: group 1 included culture-proven non-tuberculous endophthalmitis, group 2 culture-negative non-tuberculous endophthalmitis, and group 3 patients undergoing surgery for uncomplicated cataract. PCR targeted one or more of following targets: IS6110, MPB64, and protein b genes of Mycobacterium tuberculosis complex. Multiple regression analysis (5% level of significance) was done to evaluate the associations between positive PCR outcome and laterality of disease, tuberculin skin test (TST)/interferon-gamma release assay (IGRA), chest radiography, and type of sample (aqueous or vitreous). The main outcome measures were positive PCR by one or more gene targets, and factors influencing positive PCR outcomes.ResultsAll 114 samples were tested for MPB64, 110 for protein b, and 88 for IS6110. MPB64 was positive in 70.2% (n = 80) of tested samples, protein b in 40.0% (n = 44), and IS6110 in only 9.1% (n = 8). DNA sequencing of amplicons from four randomly chosen PCR reactions showed homology for M. tuberculosis complex. Of the 80 PCR-positive patients, 71 completed a full course of antitubercular therapy, of which 65 patients (91.5%) had complete resolution of inflammation at final follow-up. Among controls, 12.5% (3 out of 24) in group 1 and 18.7% (6 out of 32) in group 2 also tested positive by PCR. No PCR-positive outcome was observed in control group 3 (n = 25). Multiple regression analysis revealed significant association of positive PCR outcome with bilateral presentation, but not with a positive TST/IGRA, chest radiography, or type of sample (aqueous/vitreous) used.ConclusionsCareful selection of gene targets can yield high PCR positivity in clinically suspected ocular TB. Bilateral disease presentation but not any evidence of latent systemic TB influences PCR outcomes. False-positive results may be seen in ocular inflammation unrelated to ocular TB.

Development of a Loop-Mediated Isothermal Amplification Assay Targeting the mpb64 Gene for Diagnosis of Intraocular Tuberculosis

ABSTRACT A loop-mediated isothermal amplification (LAMP) assay targeting the mpb64 gene for the diagnosis of intraocular tuberculosis was highly specific (100%), sensitive (85.7%), rapid, and easy to perform. The LAMP assay can be an alternative to conventional PCR for the diagnosis of ocular tuberculosis in resource-limited settings.

Autoreactive T Cells in Immunopathogenesis of TB-Associated Uveitis

Purpose Intraocular inflammation in tuberculosis-associated uveitis (TBU) is usually widespread, and responds unpredictably to treatment. Herein, we analyze the intraocular T-cell response in TBU for its surface phenotype, antigenic specificity, and functional characteristics to explain the above observations. Methods We isolated T cells from vitreous humor samples of patients with TBU and non-TB uveitis (controls). These were directly stained for surface markers CD4, CD8, CD45RO, CD45RA, CCR7, as well as intracellular cytokines IFN-γ, TNF-α, and IL-17 and analyzed on flow cytometry. Antigenic specificity was determined by activating with Mycobacterium tuberculosis-specific antigen Early Secreted Antigenic Target-6 (ESAT-6) or retinal crude extract (RCE). Activation-induced cell death (AICD) characteristics of each T-cell population were analyzed by staining for PI-Annexin V, Fas-FasL, phospho-Akt, and phospho-Erk1/2. Results Immunophenotyping of vitreous humor samples demonstrated polyfunctional effector and central memory CD4+ T helper cells coexpressing IFN-γ, TNF-α, and IL-17. Both ESAT-6 and RCE (autoreactive) specificity was found in T cells extracted from TBU samples; however, the mycobacterial and autoreactive T-cell populations differed in their sensitivity to AICD. Autoreactive T cells appeared to resist AICD through decreased expression of apoptotic markers, FasL and caspase-3, sustained phosphorylation of Akt, and lowered Erk1/2 activity. Conclusions Autoreactive T cells are present in TBU eyes and are relatively resistant to AICD. An understanding of this epiphenomenon could be crucial in planning treatment of TBU patients, and interpreting response to anti-TB therapy.

Normalised quantitative polymerase chain reaction for diagnosis of tuberculosis-associated uveitis

Polymerase chain reaction (PCR)-based diagnosis of tuberculosis-associated uveitis (TBU) in TB-endemic countries is challenging due to likelihood of latent mycobacterial infection in both immune and non-immune cells. In this study, we investigated normalised quantitative PCR (nqPCR) in ocular fluids (aqueous/vitreous) for diagnosis of TBU in a TB-endemic population. Mycobacterial copy numbers (mpb64 gene) were normalised to host genome copy numbers (RNAse P RNA component H1 [RPPH1] gene) in TBU (n = 16) and control (n = 13) samples (discovery cohort). The mpb64:RPPH1 ratios (normalised value) from each TBU and control sample were tested against the current reference standard i.e. clinically-diagnosed TBU, to generate Receiver Operating Characteristic (ROC) curves. The optimum cut-off value of mpb64:RPPH1 ratio (0.011) for diagnosing TBU was identified from the highest Youden index. This cut-off value was then tested in a different cohort of TBU and controls (validation cohort, 20 cases and 18 controls), where it yielded specificity, sensitivity and diagnostic accuracy of 94.4%, 85.0%, and 89.4% respectively. The above values for conventional quantitative PCR (≥1 copy of mpb64 per reaction) were 61.1%, 90.0%, and 74.3% respectively. Normalisation markedly improved the specificity and diagnostic accuracy of quantitative PCR for diagnosis of TBU.

A Novel Mutation in Helical Domain 2 of NOD2 in Sporadic Blau Syndrome.

ABSTRACT We report a 12-year-old girl who presented with bilateral granulomatous anterior uveitis accompanied by boggy arthritis of knee and ankle joints, intermittent fever, and nodular skin rash. She was diagnosed with sporadic Blau syndrome (early-onset sarcoidosis) based on above clinical signs and presence of non-necrotising granuloma on iris biopsy. DNA sequencing revealed a previously unreported heterozygous mutation consisting of a G>A transition in exon 4 of the NOD2 gene. This resulted in a glutamic acid to lysine substitution in helical domain 2 of the nucleotide binding and oligomerization (NACHT) region, possibly reducing efficiency of auto-inhibition in NOD2 signaling. Interestingly, the ocular inflammation resolved completely following therapeutic vitrectomy in both eyes whereas the systemic symptoms of fever and arthritis continued to wax and wane while on treatment with oral methotrexate and corticosteroids.

Gelatinous drop-like corneal dystrophy: a review

Gelatinous drop-like corneal dystrophy (GDLD) is a rare autosomal recessive form of corneal dystrophy characterised by subepithelial and stromal amyloid deposits. It is relatively common in Japan. It usually presents in the first two decades of life with subepithelial nodular lesions that later coalesce to form mulberry-like opacities. Although various surgical modalities have been attempted, recurrence remains a major challenge.

Orbital tuberculosis: Clinicopathological correlation and diagnosis using PCR in formalin-fixed tissues

ABSTRACT This article describes the clinicopathological correlation and challenges encountered in the diagnosis of orbital tuberculosis (TB). Retrospective, interventional case series. A chart review of six patients who underwent biopsy for an orbital mass between January 2012 and December 2013 was performed. Institutional review board approval was obtained for the study. Clinical, radiological, and laboratory findings were documented, and response to antitubercular therapy (ATT) was noted. Age at presentation ranged from 18 to 64 years and duration of complaints varied from 2 weeks to 2 months. Pain, redness, and swelling with an orbital mass on computerized tomography (CT) were the common features. None of the patients was clinically suspected to have orbital TB at presentation. History of prior contact with a patient of TB and history of prior TB were present in one case each. Chest radiography (CXR) showed pulmonary consolidation in one case. Tuberculin skin test (TST) readings ranged from nonreactive to 23 mm. The presence of sputum acid fast bacilli (AFB) was tested in three cases and was negative. Polymerase chain reaction (PCR) for MPB64 gene was positive in all six cases. All cases responded to ATT. Clinical presentation of orbital TB can be variable and can simulate conditions like inflammatory disease and malignancy. Biopsy with histopathology plays a key role in diagnosis. Supportive laboratory investigations are necessary to clinch the diagnosis in cases where histopathology is suggestive of TB. In this regard, PCR for Mycobacterium tuberculosis (MTB) on tissue biopsy specimens may be a sensitive diagnostic tool.