Ask A Doctor Appointment
Sankara Nethralaya
Sankara Nethralaya
Home About Us Patient Care Education Research LASIK Donate Now Career Contact Us
Back to Microbiology
Department of Microbiology
RESEARCH ACTIVITIES

SIGNIFICANT CONTRIBUTIONS OF THE DEPARTMENT

This centre is the first in the country to standardize and apply molecular biological methods “In-house Polymerase Chain Reaction (PCR) techniques” for the detection of Eubacterial, Panfungal genomes from body fluids of patients with infectious etiology and specific genomes of Eubacterial genome, Panfungal genome, Propionibacterium acnes, Mycobacterium tuberculosis, Chlamydia trachomatis, Chlamydia pneumoniae , Herpes simplex virus , Varicella Zoster virus, Cytomegalovirus virus, Adenovirus, Rubella virus, Enterovirus and Coxsackie virus , Toxoplasma gondii and Acanthamoeba from ocular specimens.

These techniques have become the standard laboratory procedures to institute early specific therapy. Currently, the in-house PCR techniques standardized in this department are being utilized for rapid detection of infectious agents in clinical specimens by clinicians from 105 hospitals / clinics in and around Chennai through Vision Research Foundation Referral Laboratory ( Unit of Medical Research Foundation). The laboratory also caters to the ophthalmologists from five other states of India.

SAIL Molecular Microbiology Research Centre which is a part of L & T Microbiology Research Center was inaugurated by Shri P. Kulshrestha, Shri G.P. Shrivastava Regional Managers Southern region SAIL/Central Marketing Organisation Chennai on May 28th 2007.

Inauguration of SAIL Molecular Microbiology
LARSEN & TOUBRO MICROBIOLOGY RESEARCH CENTRE IS SHIFTED TO THE VI FLOOR OF NEW RESEARCH BUILDING (KNBIRVO BLOCK) FROM NOVEMBER 2009

MAJOR CONTRIBUTIONS

1. THE FIRST PATENT CERTIFICATE FOR VISION RESEARCH FOUNDATION

The first Patent certificate for Vision Research Foundation has been granted to the patentee Vision Research Foundation by the Patent office Chennai, Government of India for an invention entitled “A Semi nested Polymerase Chain Reaction (PCR) For Rapid Detection And Specific Identification Of Mycobacterium fortuitum” on 29th May 2009 for the term of 20 years from the 12 day of July 2005 in accordance with the provisions of the Patents Act 1970 (The Patent No is 232658 934/ CHE/ 2005). The inventors are Dr. K. Lily Therese, Ms. Jasmine Therese and Dr. H.N. Madhavan. This is the first patent received by scientists in Vision Research Foundation.
2. THE FIRST PATENT CERTIFICATE FOR VISION RESEARCH FOUNDATION
Vision Research Foundation has been granted to the patentee Vision Research Foundation by the Patent office Chennai, Government of India for an invention entitled “A Semi nested Polymerase Chain Reaction (PCR) Method For Rapid Detection And Specific Identification Of Mycobacterium Chelonae” on 12th November 2009 for the term of 20 years from the 13 day of July 2005 in accordance with the provisions of the Patents Act 1970 (The Patent No is 236495, 937/ CHE/ 2005). The inventors are Dr. K. Lily Therese, Ms. Jasmine Therese and Dr. H.N. Madhavan.
3. THE THIRD PATENT CERTIFICATE FOR VISION RESEARCH FOUNDATION
THE THIRD Patent Certificate for Vision Research Foundation has been granted to the patentee Vision Research Foundation by the Patent office Chennai, Government of India for an invention entitled “A method for Cultivating Cells derived from Corneal Limbal Tissue and Cultivation method using Mebiol gel (Thermoreversible polymer)” provided by Professor Y. Mori of Waseda University Tokyo, Japan” on 14th June 2010 for the term of 20 years from the 28 day of March 2005 in accordance with the provisions of the Patents Act 1970 (The Patent No is 239350, 4226/CHENP/2007) Dr. H. N. Madhavan is the inventor of this patent
4. THE FOURTH PATENT CERTIFICATE FOR VISION RESEARCH FOUNDATION
THE FOURTH Patent Certificate for Vision Research Foundation has been granted to the patentee Vision Research Foundation by the Patent office Chennai, Government of India for an invention entitled “A multiplex Polymerase chain reaction for rapid detection and specific identification of Cytomegalovirus” in accordance with the provisions of the Patents Act 1970 (The Patent No is) Dr. H. N. Madhavan is the inventor of this patent
2. “MACRO DNA VISION CHIP” FOR AETIOLOGICAL DIAGNOSIS OF OCULAR INFECTIONS

The team led by Prof. H N Madhavan from L & T Microbiology Research Centre, Vision Research Foundation worked for 4 ½ years on the project with a grant of Rs 3.5 crores under the New Millennium Indian Technology Leadership Initiative (CSIR- NMITLI) programme on Ocular Infectious Diseases group for a Novel molecular diagnostics

“Xcyto-screen DNA chip” is a diagnostic product of collaborative research of four Ophthalmic centres viz. Vision Research Foundation, Chennai; L.V. Prasad Eye institute, Hyderabad; Dr. Rajendra Prasad Centre for Ophthalmic Sciences, AIIMS, New Delhi; Centre for Cell and Molecular biology, Hyderabad and Industrial partner, Xcyton Diagnostics Pvt., Ltd., Bangalore. The DNA chip aids in the diagnosis of the following four ocular clinical conditions:

Clinical validation of the X cyto-Screen kits was carried out by the team at L & T Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, Chennai. The product was launched on 22nd September 2007 in Bangalore.

The total time taken for the test to be performed is eight hours. The cost per test is Rs. 2, 500/-. The validation of the DNA chip was carried out under personal supervision of Dr. H. N. Madhavan at L & T Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, 18, College Road, Chennai – 600-006.

The advantages of the vision chip are

A Single chip can detect probable infectious etiology of a given clinical condition in a Multiplex format.
A significant reduction in cost, time and labour.
Reduced risk of cross-contaminations as nested round is avoided.
Sensitivity is equivalent to individual nested PCRs by an improved detection system
No carcinogenic chemicals are used in the test.
Detection of the amplified product does not require ultraviolet radiation or Radiographic or Spectrophotometric methods.
The DETECTION IS DONE BY NAKED EYE.
Though this is Polymerase Chain Reaction-based technique, a trained medical laboratory technician can perform the test with ease. Even laboratories at the district level hospital can introduce this test.
DR. S.S. BADRINATH, CHAIRMAN EMERITUS, SANKARA NETHRALAYA RECEIVING THE FIRST VISION CHIP KIT FROM T. RAMASAMI, SECRETARY, DST DURING THE LAUNCH OF THE KIT
Dr. H.N. MADHAVAN, DIRECTOR OF RESEARCH, SANKARA NETHRALAYA FELICITATED BY
DR. T. RAMASAMI, SECRETARY, DST
The kit was launched on Saturday 22nd September 2007 by our beloved Chairman Emeritus, Sankara Nethralaya, Dr. S. S. Badrinath and Dr. T. Ramasami, Secretary, Department of Science and Technology. The kit was officially launched on behalf of CSIR, the Research Scientists at Vision Research Foundation, Sankara Nethralaya, Chennai; LVPEI, Hyderabad; RP centre for Ophthalmology, AIIMS, New Delhi and Centre for Cellular and Molecular Biology, Hyderabad and X Cyton Diagnostics Ltd, Bangalore (the Industrial partner) at Taj Gateway Residency in Bangalore. Dr. G. Padmanabhan of Indian Institute of Science presided over the function.
3. COMMERCIAL KIT FOR DETECTION OF ADENOVIRUS FROM OCULAR SPECIMENS BASED THE PUBLICATION FROM THIS CENTRE
Development and use of nested polymerase chain reaction (PCR) for the detection of adenovirus from conjunctivitis specimens. J. Clin. Virol 11: 77-84, 1998.
4. The first report of Isolation of Vesicular virus belonging to the family Rhabdoviridae from the aqueous humor of a patient with bilateral corneal endotheliitis
Ultrastructure features of Electron Microscopic picture of the isolated vesiculovirus from aqueous humor
Madhavan HN, Goldsmith CS, Rao SK, Fogla R, Malathi J and Priya K. Isolation of a vesicular virus belonging to the family rhabdoviridae from the aqueous humor of a patient with bilateral corneal endothelilitis, Cornea; 21 : 333-335, 2002.
Translational research contributions for laboratory diagnosis of ocular infections – In house PCR Techniques for rapid detection of aetiological agents

I. Indigenous PCRs for Rapid detection of infective agents in endophthalmitis

Detection of eubacterial / Propionibacterium acnes (P. acnes) / panfungal genome in intra-ocular specimens from clinically suspected endophthalmitis patients by application of in house nested Polymerase Chain Reaction (nPCR)

Polymerase chain reaction in the diagnosis of bacterial endophthalmitis. Br J Opthalmol. 82: 1078-1082, 1998.

Agarose gel electrophoretogram of amplified products of P.acnes from intraocular fluids
 
Agarose gel electrophoretogram of amplified products of Panfungal genome
 
Agarose gel electrophoretogram of Aspergillus PCR showing the amplified products of the four species of Aspergillus
 
Development of multiplex PCR for etiological diagnosis of infectious endophthalmitis
 
Detection of Non sporulating moulds (NSM) identified by Polymerase chain reaction (PCR) based DNA sequencing technique targeting internal transcribed spacer (ITS) region
 
II. Infectious Etiology of Uveitis
A. Detection of Mycobacterium tuberculosis genome in intraocular fluids of granulomatous uveitis and other ocular specimens (1999 - 2004):
Mycobacterium tuberculosis DNA was detected in 18% (51 of 284) intraocular specimens including choroidal biopsies, subretinal mass aspirations, by application of in house nPCR using primers targeting MPB64 gene.
B. Association of mycobateria with Eales’ disease
Agarose gel electrophoretogram showing the amplified products of Mycobacterium tuberculosis
III. RAPID DETECTION OF VIRAL OCULAR INFECTIONS

Association of herpes viruses in the aqueous humor of patients with serpiginous choroiditis & Progressive Outer Retinal Necrosis (PORN)

PCR for CMV, HSV and VZV was carried out on nine patients with serpiginous choroiditis. Five were positive for VZV, one was positive for HSV suggesting the role of these viruses in pathogenesis. CMV DNA was detected by PCR in an individual with clinical diagnosis of PORN (HIV positive ) indicating the possible association of CMV in PORN.

 
Prevalence of HSV, VZV and CMV in HIV positive and HIV negative patients with of viral retinitis in India.
PCR for the detection of HSV, VZV and CMV was applied on intraocular fluids collected from 34 HIV positive and 99 HIV negative individuals. HSV infection was more prevalent among HIV negative patients, VZV was equally prevalent CMV infection was statistically more prevalent among the HIV positive patients.
Eur J Clin microbial Infect Dis 2004
Standardization and application of semi nested PCR for detection of the serotypes HSVI and HSV2 directly from ocular specimens.
Primers targeting the Glycoprotein D gene of HSV was standardised and applied onto a total of 18 intraocular fluids of Acute Retinal Necrosis (ARN) of which 4 were identified as HSV-2 and 3 as HSV-1.and in 7 cases of keratouveitis one was positive for HSV-2.
IJMM, 2005
Agarose gel electrophoretogram showing the amplified products of Herpes Simplex Virus serotypes 1 & 2
 
Genotyping of HSV – isolates:
 
Multiplex PCR for etiological diagnosis of ocular herpetic infections

IV. DETECTION OF HUMAN CYTOMEGALOVIRUS (HCMV) FROM CLINICAL SPECIMENS

1. Development of multiplex PCR for etiological diagnosis and quantitation of Human Cytomegalovirus infections

2. Standardization of pp65 antigenemia assay for semi-quantitation of late antigen of Cytomegalovirus in blood and Standardization of 5’ nuclease assay for Quantitation of viral load in clinical specimens positive for CMV.

3. Standardization of Multiplex PCR for genotyping of gB gene of Human Cytomegalovirus
4. Standardization of RFLP for identification of strain variations in gB, gH, gO, gL and gN regions of Cytomegaloviru
5. Analysis of mixed infections with multiple genotypes of Human Cytomegalovirus by RFLP and DNA sequencing
6. Analysis of mixed infections by multiple genotypes of Human Cytomegalovirus in Immunocompromised patients
ACHIEVEMENTS OF TRANSLATIONAL RESEARCH CLINICAL SPECIMENS PROCESSED BY APPLICATION OF INHOUSE POLYMERASE CHAIN REACTION (PCR) DURING JAN 2010 – DEC 2010 AT VRF REFERRAL LABORATORY
COMPLETED RESEARCH PROJECTS IN COLLOBORATON WITH INTERNATIONAL LABORATORIES
I. Alcon Laboratories Inc., USA: Nucleic acid based methods (including ribotyping) for direct species level detection of bacterial genome(s) and ribotyping of bacterial isolates from eyelid /lash swabs of Blepharitis patients.
Clinical trials and microbiological studies
A Clinical evaluation of efficacy and safety of 3% Ciprofloxacin Ophthalmic Solution in treating bacterial corneal ulcer.
A clinical and microbiological evaluation of efficacy and safety of 3% Ciprofloxacin Ophthalmic ointment relating bacterial corneal ulcers.
A study on bacterial corneal ulcers treated with “standard antibacterial therapy”
A double masked pilot study comparing Ciprofloxacin and Tetracycline ophthalmic ointments for treating conjunctivitis caused by Chlamydia trachomatis.
Double masked comparison of Ciloxan to Fortified Tobramycin/Cefazolin for treating bacterial Corneal Ulcers.
Efficacy and Safety of 1.0% Rimexolone Ophthalmic suspension versus Pred Forte for treatment of uveitis
An evaluation of the Safety and Efficacy of Moxifloxacin Ophthalmic Solution 0.5% Versus OCUFLOX Ophthalmic Solution 0.3% in the Treatment of Bacterial Conjunctivitis in India.
Microbiology of Healthy Eyes in India
Comparison of Ciprofloxacin 0.3% Dexamethasone 0.1% suspension , versus Ciprofloxacin ).3% Solution for treatment of Chronic Blepharitis
II. Waseda University, Tokyo, Japan:

In-vitro studies on the applications of Mebiol gel in cultivation of cells from eye tissues and growth of viruses in these cells and their susceptibility to antiviral drugs

IJMR, 2006
Current science, 2004

Ex vivo cultivation of corneal Limbal epithelial cells in a Thermo-reversible polymer (Mebiol Gel) and their transplantation in Rabbits – an animal model.

COMPLETED RESEARCH PROJECTS IN COLLOBORATON WITH NATIONAL LABORATORIES

Cadila Pharmaceuticals Ltd, India:

Synergic activity of SN-198 and Amphotericin B SN-198 and Ketaconazole

“Synergistic activity of SN-198 and / or its modified product with the Amphotericin B and Fluconazole using the fungal strains which are resistant to Amphotericin B and SN-198 in our earlier study”
INTRAMURAL COMPLETED FUNDED PROJECTS
Effect of Mitomycin C on Fibroblast cell cultures grown from sub-conjunctival tissue from glaucoma patients. (Ophthalmic Surgery, 1995)
To study the aetological role of tubercle bacillus in Eales' disease from the serum, vitreous paraffin section of epiretinal, vitreous and subretinal membrane. (BJO 1999, IOVS 2000, IJO 2002)
Ampicillin and Sulbactum Assay in Aqueous Humor and Serum. (IJO, 1998)
Comparison of In-vitro activities of Tetracycline and Ciprofloxacin against C. trachomatis isolates for conjunctivitis. (IJMR, 1996)
Long term in-vitro effects of Mitomycin C treated human Tenon's capsule fibroblasts and their correlation with surgical outcome in trabeculectomy in Glaucoma patients. (IJMR, 1997)
Evaluation of direct diagnosis with culture confirmation using modified shell vial method for diagnosis of Chlamydia, Adenovirus and Herpes Simplex Virus (HSV) infection of cornea and conjunctiva in patients attending Sankara Nethralaya. (Afro-Asian J Ophthalmol, 1992)
Use of Polymerase chain reaction (PCR) and DNA probe Hybridization to Determine the Gram reaction of the infecting bacterium in the intraocular fluids of patients with Endophthalmitis (J Infection; 41: 221 -226)
Polymerase Chain Reaction to find the association of Chlamydophila (Chlamydia pneumoniae) in optic neuritis and papilledema - A pilot study (IJPM, 2007)
Standardisation Of Western Blotting Technique For Detection Of Anti-IgG and IgM against Reactive Antigens of Toxoplasma Gondii Tachyzoites.
Pilot Study - Etiology of Fuchs’ heterochromic uveitis A pilot study has been undertaken to evaluate the role of rubella, herpes zoster, herpes simplex and Toxoplasma gondii in the pathogenesis of Fuchs’ heterochromic uveitis and PCR is positive for Toxoplasma gondii genome in 3 and HSV genome in one of the 40 AH from patients with clinically suspected Fuchs’ heterochromic uveitis.
Study on the antiviral property of isatin derivatives and acyclovir prodrugs against isolates of Human Herpes Simplex Virus”.
COMPLETED PROJECTS THROUGH RESEARCH GRANTS EXTERNAL FUNDING AGENCIES
I. INDIAN COUNCIL OF MEDICAL RESEARCH (ICMR)
1. Title of the project : “Polymerase chain reaction for the detection and genotyping of Chlamydia trachomatis in conjunctivitis” (5/3/3/17/98 – ECD – I) (1999 – 2001)

Primers targeting plasmid and major outer membrane protein (MOMP) gene of Chlamydia trachomatis (C. trachomatis) were evaluated for their sensitivity and specificity in detecting the organism among conjunctival specimens. Plasmid primers were more sensitive in detecting C. trachomatis in 11(13.8%) whereas MOMP primers were able to amplify C. trachomatis DNA in only 3 among the 11 positive specimens.

A total of 486 conjunctival specimens collected from individuals suspected to have inclusion conjunctivitis were processed for detecting of C. trachomatis. The overall rate of C. trachomatis positivity was 5.6 % . Isolation techniques were not successful. The predominate genotype was determined as Ba.

2. Molecular biological and virological study of intraocular fluids from patients with viral retinitis and choroiditis - correlation with clinical features and visual outcome. ( 5/4/6/1/2000 – NCD – II) (2001 – 2003)

PCR is a rapid diagnostic tool for aetiological diagnosis of viral retinitis. VZV was the most commonest cause of viral retinitis based on PCR and was detected in 28, CMV in 20 and HSV in 14 of 100 viral retinitis patients.

VZV DNA was detected in 5, and HSV in one among 9 patients with serpiginous choroiditis, pathogenesis of this disease.

PCR based RFLP is a reliable, less laborious and cost effective molecular biological tool for the determination of HSV serotypes both for the clinical isolates and culture negative PCR positive specimens.

3. Title of the project : Detection and molecular characterization of mycobacteria by Polymerase Chain Reaction (PCR) Restriction Fragment Length Polymorphism (RFLP) and DNA sequencing technique from ocular and other clinical specimens. ( 5/8/5/11/2000 – ECD - I ) (2000 – 2002)

4. Title of the project: “Prevalence of Rubella virus associated congenital cataract: detection of the virus in the lens aspirates ( 5/4/6/2/03 - NCD- II) (2005 – 2008)

Significant contributions: Congenital Rubella syndrome (CRS) occurs only when the mother acquires a primary Rubella infection during the first trimester. 20% of the children born after such an infection suffer the severe congenital abnormalities associated with CRS.

Reverse transcriptase Polymerase chain reaction (RT-PCR) was applied on 100 lens aspirates and peripheral blood and 23 (23%) were positive by nRT-PCR).

Among the 23 lens aspirates positive by nRT-PCR 13 were isolated on SIRC cell line.

Sequencing of the 13 culture isolates reveals two genotypes, Genotype I and Genotype II. 11 of these isolates belong to Genotype I and two belong to Genotype II. The genotyping was also confirmed by PCR –RFLP technique using Taq I enzyme and found to be in concordance with the results of the DNA sequencing.

5. Polymerase Chain Reaction for detection of Mycobacterium genus specific genome, Mycobacterium fortuitum and Mycobacterium chelonae genomes in vitreous and epiretinal membranes and blood of Eales’ disease patients.( 5/3/3/3/2004 – ECD - I) (2005 – 2008)

The aetiopathogenesis of Eales’ disease is not clearly understood because of lack of appropriate specimens for microbiologic study.

In this prospective study, 4 out of 45 (8.9%) vitreous aspirates from Eales’ disease patients were positive by PCR for Mycobacterium fortuitum (M. fortuitum), one for Mycobacterium chelonae (M. chelonae) by PCR and 3 for M. tuberculosis by PCR.

8 intraocular clinical specimens from Eales patient were positive for mycobacterium either / or by PCR and culture showed a statistically significant (P <0.001) association of Mycobacetrium species with Eales’ disease.

Agarose gel electrophoretogram showing amplification of M. chelonae DNA targeting ITS gene from Eales’ patients Vitreous DNA

This is the first report mentioning the isolation of rapid growing nontuberculous mycobacteria (RGNTM) from the Vitreous aspirate of Eales’ patients with probable confirmation of their aetiopathogenic role in the disease process.

6. Application of Polymerase chain reaction (PCR) for the detection of Toxoplasma gondii in intraocular fluids from suspected cases of chorioretinits ( 5/3/3/3/2003- ECD - I ) (2005 – 2008)

Of the five different nPCRs standardized in this study- 2 targeting the B1 gene, one SAG1 and 2 for SAG2 (3’ end and 5’ end), T. gondii DNA was detected by at least one of the nPCR in intraocular fluids/peripheral blood leucocytes from 39.7% of 131 TRC patients including 74.1% of 27 HIV positive patients.

The clinical diagnosis of ocular toxoplasmosis was supported by laboratory tests-WDC and nPCR in 68.8% of 77 TRC patients including 85.1% of 27 HIV positive TRC patients.

In 52 congenital cataract patients included in this study, T. gondii DNA was detected by at least one of the 4 nPCRs (2 targeting B1 gene and 2 targeting SAG2 gene) in 59.6% of lens aspirate and/or peripheral blood leucocytes. This is the first report in literature on the detection of T. gondii DNA by nPCR in lens aspirate of congenital cataract patients.

The association of T. gondii with Fuchs Heterochromic Iridocyclitis (FHI) was detected for the first time in India in this study by the presence of T. gondii DNA by nPCR targeting B1 gene in 18.2% of 33 AH. Genotyping of T. gondii by nPCR-RFLP and DNA sequencing: Genotype I in 41 patients consisting of 13 TRC patients ( including 7 HIV positive), 23 congenital cataract patients, and 5 FHI patients. This is the first study of genotyping of T. gondii from India.

7. Comparison of taqman probe based Real-Time polymerase chain reaction (RT-PCR) and pp65 antigenemia assay for the quantitation of human cytomegalovirus (HCMV) in clinical specimens and a study on ganciclovir resistance of HCMV (Order No 5/8/7/17/2006-ECD-I)

The objectives of the study were optimization of
Taqman probe based real time PCR for detection and quantification of HCMV genome along with the application of pp65 antigenemia assay on to the clinical specimens obtained from immunocompromised patients ie. HIV patients, post renal transplant recipients with HCMV manifestations including HCMV retinitis. The results of pp65 antigenemia assay and Real-time PCR by applying the techniques prospectively on peripheral blood specimens obtained from these immunocompromised patients were correlated. Finally the novel ganciclovir resistance mutation by sequencing the UL-97 gene and UL-54 gene in the patients who do not respond to the treatment with ganciclovir or valgancyclovir as decided by the nephrologists or ophthalmologists were determined.

Qualitative nested PCR:

A total of 82 Peripheral blood specimens were collected from post renal transplant recipients and 82 healthy kidney donors, 20 HIV infected individuals with HCMV retinitis. DNA from the clinical specimens was extracted using Bioneer genomic extraction kits and stored at -20oc till further use. Qualitative nested PCR targeting the mtr II region of HCMV has been applied onto the DNA extracted from these specimens and the amplified product was detected by electrophoresis on 2% agarose gel incorporated with ethidium bromide. CMV AD-169 (ATCC-VR 538) serially passaged in WI-38 diploid cell line was used as positive control.

Observation & Results:

Fifty nine (71.95 %) of 82 post renal transplant patients, 11 (13.41 %) of 82 kidney donors and all 20 (100%) of 20 HIV patients with HCMV retinitis (either by blood DNA or by Aqueous humour DNA or by both) were positive for HCMV DNA, the copy number ranging from 1 copy/ml. to 8462847 copies/ml. The clinicians were prompted with the HCMV results of the donor for the further management and decision. In case of pp65 antigenemia assay thirty eight (46.34 %) of 82 post renal transplant patients, 1 (1.22 %) of 82 kidney donors and 13 (65 %) of 20 HIV patients with HCMV retinitis were positive by pp65 antigenemia assay. The clinicians were prompted with the HCMV results of the donor for further management and decision.

Comparative analysis of results of pp65 antigenemia assay with real time PCR

  Total No. of specimens Specimens positive
Real Time PCR pp65
Post transplant recipients 82 59 (71.95 %) 38 (46.34 %)
Kidney Donors 82 11 (13.41 %) 1 (1.22 %)
HIV patients with HCMV retinitis 20 20 (100%)* 13 (65 %)
Blood donors 20 0 (0 %) 0 (0 %)
Total 204 90 (44.11 %) 52 (25.49 %)+
 
Among the 204 specimens processed 52 were positive for both pp65 antigenemia and real time PCR and 38 more were positive only by real-time PCR.
The specimens were positive for HCMV DNA either for blood DNA or Aqueous humour DNA.s
Agarose Gel Electrophoretogram of PCR of Region I- Round II on Clinical Specimens

II. COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH (CSIR)

CSIR- NMITLI PROJECT

The project was aimed to develop a Xcyto-screen DNA chip as a diagnostic product of collaborative research of Ophthalmic centres viz. Vision Research Foundation, Chennai; L.V. Prasad Eye institute, Hyderabad; Dr. Rajendra Prasad Centre for Ophthalmic Sciences, AIIMS, New Delhi; Centre for Cell and Molecular biology, Hyderabad and industrial partner, Xcyton Diagnostics Pvt., Ltd., Bangalore. Multiplex Polymerase chain reaction based hybridization technique for simultaneous detection of infectious etiology of Keratoconjunctivitis (external ocular infections), viral Retinitis, Uveitis, infectious Endophthalmitis (intraocular infections) was standardized. The infective agents includes human herpes simplex virus, Chlamydia trachomatis and Adenovirus for external ocular infections and HSV, CMV, VZV for viral retinitis, gram positive, gram negative bacteria, Propionibacterium acnes and fungal genome for endophthalmitis, Mycobacterium tuberculosis, Mycobacterium fortuitum and Mycobacterium chelonae and Toxoplasma gondii genome in for uveitis. Internal control for Human beta globin is also incorporated to check for inhibition of the amplification. The hybridization reaction was later made as a DNA macro- chip in collaboration with X-Cyton, Bangalore. Clinical validation of the was carried out by L & T Microbiology Research Centre, Chennai. The use of the kit does not require Ultraviolet radiation or Radiographic or Spectrophotometric methods. The detection is done by naked eye. The kit was launched on Saturday 22nd September 2007 by our beloved Chairman Emeritus, Sankara Nethralaya, Dr. S. S. Badrinath and Dr. T. Ramasami, Secretary, Department of Science and Technology.

III. DEPARTMENT OF SCIENCE AND TECHNOLOGY (DST)

TITLE: Standardization of nucleic acid based amplification techniques in detection and identification of fungi in clinical specimens from clinically suspected cases of infectious keratitis and endophthalmitis ( SR/SO/HS-63/2003)

OBJECTIVES:

To optimize and apply a multiplex Polymerase Chain reaction (mPCR) targeting 18SrRNA, ITS and 28SrRNA for detection of Panfungal genome from clinically suspected cases of infectious keratitis and endophthalmitis.
To identify the non-sporulating moulds (NSM) by PCR based DNA sequencing and PCR-RFLP.
To apply PCR, PCR based Restriction Fragment length Polymorphism (PCR-RFLP) and DNA sequencing to identify Fusarium species.
I. Optimization of mPCR: mPCR was optimized targeting the 18SrRNA, ITS and 28SrRNA region. The sensitivity of mPCR was 1 picogram and it was specific only the for fungal DNA.. mPCR detected the presence of panfungal genome in 10 corneal scrapings and 10 intraocular fluids in 40 ocular specimens processed.
II. PCR-RFLPand DNA sequencing to identify Fusarium species.
Ten isolates of Fusarium species were identified based on colony morphology, microscopic morphology and PCR-RFLP targeting ITS region.

III. PCR based RFLP and DNA sequencing targeting ITS region for the identification of NSM to species level.

A total of 10 NSM isolates were subjected to DNA sequencing. The results of DNA sequencing identified 5 isolates as Botryosphaeria rhodina, 2 as Pythium insidiosum, 1 Macrophomina phaseolina, 1 Rhizoctonia bataticola. The 3 genera consisting of Botryosphaeria, Macrophomina, Rhizoctonia were found to be the potential aetiological agents causing fungal keratitis in Indian population. The 5 isolates of Botryosphaeria rhodina recovered from the same patient exhibited the same type of nucleotide polymorphisms in the ITS region.

Current Eye Research, 2008

IV. DEPARTMENT OF BIOTECHNOLOGY (DBT)

TITLE: Phylogenetic analysis of Human Herpes Simplex Virus (HSV) isolates based on DNA sequencing of genes coding for glycoproteins G, I and E, C regions and to determine novel targets to identify the genotypes of HSV – Funded by DBT (Project No. : DBT/PR9975/MED/29/54/2007) (July 2008 - August 2010)

SUMMARY AND CONCLUSIONS:
Phylogenetic analyses of envelope glycoprotein genes HSV provided insight into the existence of genotypes circulating in our population.

Glycoprotein G of HSV1 which did not split into geno-groups by PCR based RFLP as described previously for European strains (mentioned in first year annual report) split into 3 clades, with each clade bearing strains with common variations throwing possibilities of genotypes which are different from European strains.

Glycoprotein I-1 on the other hand, conformed to the European classification, but the VRF strains described as Genotype A, were not seen closely associated, hence showing the possible existence of sub genotypes within genotype A region. Genotype C was not encountered in this study.

Glycoprotein C-2 showed the existence of geno groups, with four distinct clades separating out.

Phylogenetic analyses of Glycoprotein I -2 proved existence of genotypes in an earlier thought conserved genome.

Phylograms of glycoprotein genes E of HSV1 and HSV2 and gC of HSV1 were highly divergent showing them to be rapidly evolving.

Analyses of the thymidine kinase regions showed the existence of 1 resistant strain (HSV2 isolate - vesicle swab) among the fifty tested.

HSV1 ocular isolates 922 and 1105 were seen to be associated closely in the Unique Short region and thymidine kinase region.