Publications by authors named "Pasupathi Aarthi"

3 Publications

  • Page 1 of 1

Optimization and application of a reverse transcriptase polymerase chain reaction to determine the bacterial viability in infectious endophthalmitis.

Curr Eye Res 2012 Dec 3;37(12):1114-20. Epub 2012 Jul 3.

Larsen and Toubro Microbiology Research Centre, Kamal Nayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.

Purpose: To develop RNA based assay - reverse transcriptase polymerase chain reaction (RT-PCR) to detect viable bacteria in intraocular specimens obtained from patients with infectious endophthalmitis.

Materials And Methods: Thirty-five intraocular specimens (19 vitreous fluid and 16 aqueous humor) collected from patients with typical infectious endophthalmitis were subjected to conventional and molecular microbiological investigations. Culture negative, eubacterial genome PCR positive intraocular specimens were subjected to denaturing high performance liquid chromatography (dHPLC) for separation of mixed genomes and subsequently identified by PCR based DNA sequencing. In parallel, RT-PCR was performed to detect the presence of viable bacteria in intraocular specimens.

Results: Among 35 intraocular specimens, single bacterial genome was detected in 9 (25.7%) and two or more genomes in 26 (74.28%) intraocular specimens. Eubacterial genome was detected by RT-PCR in 29 (82.85%) specimens. PCR based dHPLC followed by PCR based DNA sequencing revealed the presence of 65 bacterial genomes in 35 intraocular specimens. Five novel genera namely Terrabacter species, Facklamia species, Xylella fastidiosa, Duganella species and Synechococcus species were detected.

Conclusion: RT-PCR serves as a rapid and reliable tool to detect viable bacteria causing endophthalmitis.
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December 2012

Identification of bacteria in culture negative and polymerase chain reaction (PCR) positive intraocular specimen from patients with infectious endopthalmitis.

J Microbiol Methods 2011 Apr 22;85(1):47-52. Epub 2011 Jan 22.

L & T Microbiology Research Center, Sankara Nethralaya, 18, College Road, Chennai 600006, India.

A novel Denaturing High-Performance Liquid Chromatography (dHPLC)-based technique allows rapid high-resolution analysis of PCR products. We show the application of this PCR/dHPLC approach for direct detection and identification of bacterium from the Eubacterial PCR amplified products of aqueous and vitreous aspirates from patients with endopthalmitis and to differentially identify the culture negative cases and initiate appropriate therapy. The aim of this study is to identify culture negative PCR positive cases by the application of PCR based DNA sequencing. A total of 116 intraocular specimens were subjected for the study. Sixty-nine different bacteria were identified using dHPLC based DNA sequencing of which predominant ones were Gram-positive bacteria and cannot be cultured by conventional methods. Forty eight different bacteria detected in this study is being reported for the first time in infectious endopthalmitis.
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April 2011

Homocysteinethiolactone and paraoxonase: novel markers of diabetic retinopathy.

Diabetes Care 2010 Sep 14;33(9):2031-7. Epub 2010 Jun 14.

Biochemistry and Cell Biology Department, Sankara Nethralaya Hospital, Chennai, Tamil Nadu, India.

Objective: Paraoxonase (PON) exhibits esterase activity (PON-AREase) and lactonase activity (PON-HCTLase), which prevent LDL oxidation and detoxify homocysteine thiolactone (HCTL). The role of HCTL and PON-HCTLase as a risk factor for the microvascular complication in diabetic retinopathy at the level of vitreous has not been investigated.

Research Design And Methods: Undiluted vitreous from patients with proliferative diabetic retinopathy (PDR) (n = 13) and macular hole (MH) (n = 8) was used to determine PON-HCTLase and PON-AREase activity spectrophotometrically. HCTL levels were detected by liquid chromatography-tandem mass spectrometry. In vitro studies were done in primary cultures of bovine retinal capillary endothelial cells (BRECs) to determine the dose- and time-dependent effect of HCTL and homocysteine (Hcys) on PON-HCTLase activity, as well as to determine mRNA expression of PON by RT-PCR.

Results: A significant increase in HCTL and PON-HCTLase activity was observed in PDR compared with MH (P = 0.036, P = 0.001), with a significant positive correlation between them (r = 0.77, P = 0.03). The in vitro studies on BRECs showed a dose- and time-dependent increase in the PON-HCTLase activity and mRNA expression of PON2 when exposed to HCTL and Hcys.

Conclusions: This is the first study showing elevated levels of vitreous HCTL and PON-HCTLase activity in PDR. These elevations are probably a protective effect to eliminate HCTL, which mediates endothelial cell dysfunction. Thus, vitreous levels of HCTL and PON activity can be markers of diabetic retinopathy. The bioinformatics analysis reveals that the structure and function of PON that can be modulated by hyperhomocysteinemia in PDR can affect the dual-enzyme activity of PON.
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September 2010