Eales' disease is an idiopathic condition of unknown aetiology presenting with primary retinal perivasculitis predominantly affecting the peripheral retina of young and otherwise healthy adults aged 15-40 years. The disease is currently more common in the Indian subcontinent and in our tertiary referral eye institute, the disease is reported one in 135 to 200 ophthalmic patients referred from various parts of the country.
The aetiopathogenesis of Eales' disease still remains unclear. Our first report in 1977 confirmed the higher incidence among males in the age group 10-45 years with complaints of spots and floaters and painless diminution of vision (Abraham C, Baig SM, Badrinath SS. Proc All Ind Ophthalmol Soc 1977;33:223-29). Our laboratory studies showed statistically significant higher phenotype frequencies of HLA B5 (B51), DR1 and DR4 among patients with Eales' disease compared to controls. Since the most favoured aetiologies were tuberculosis and hypersensitivity to tuberculoprotein, we performed Mantoux testing and lymphocyte proliferation assay to purified protein derivative (PPD) on patients with Eales' and age and gender-matched volunteers with normal fundus findings. We did not find any significant differences between them. Therefore, we applied polymerase chain reaction (PCR) using IS 6110 primers on vitreous fluid (VF) of patients with Eales' disease and controls and M. tuberculosis DNA was present in a statistically significant number of specimens of Eales' patients. We have also demonstrated statistically significant presence of M. tuberculosis DNA in the epiretinal membrane (ERM) of Eales' patients compared to the controls by nested PCR (nPCR) using primers for the gene coding for MPB 64 protein of M. tuberculosis.
In another study, we showed that PCR for the gene coding for MPB 64 of M. tuberculosis was several times more sensitive than that using IS 6110 primers to detect the bacterial genome in clinical specimens. Therefore, for further confirmation of the association of M. tuberculosis with Eales' disease, we applied PCR using primers for the gene coding for MPB 64 protein of M. tuberculosis on VF samples from a large number of Eales' patients and controls. We also applied PCR using IS 6110 combined with dot-blot hybridisation on VF specimens that were positive for M. tuberculosis DNA by primers coding for MPB 64 protein.
Materials and Methods
Patients and specimens
A total of 69 VF samples from 69 patients (24 Eales' disease patients and 45 non-Eales' disease patients as controls) were included in this study. The control specimens were collected from patients undergoing vitreous surgery for proliferative diabetic retinopathy and vitreous haemorrhage. The VF was aspirated by a syringe connected to the suction port of the vitreous cutter at the beginning of the vitrectomy surgery procedure; a sterile disposable 23G needle was fixed to the syringe and transported to the microbiology laboratory with aseptic precautions within 30 minutes of collection.
All vitreous fluid specimens were processed by standard bacteriological procedures for detection of acid-fast bacilli. Two to three drops of the aspirated fluid were inoculated into Lowenstein-Jensen slopes in duplicates and cytospined smears were prepared and stained by the Zeihl-Neelsen method, and observed for the presence of acid-fast bacilli. The remaining specimen was transferred into sterile PCR vials and preserved at -20°C for extraction of DNA for PCR amplification.
Polymerase chain reaction
DNA extraction from bacterial strains
To confirm the specificity of the nPCR, DNA extracted from standard strains of Mycobacteria and known isolates of M. tuberculosis were tested. DNA extraction from standard strains of Mycobacteria and the laboratory isolates was done by the method of Hermans et al as described elsewhere. [5,6]
DNA extraction from vitreous fluids
DNA was extracted from vitreous fluids by a modification of the method of Pitcher et al. In brief, the extraction of DNA from vitreous samples was done as follows: To 50µl of the VF in a 1.5 µl microfuge tube, 10µl of lysozyme (10mg/µl) was added and incubated at 37°C for one hour. 20µl of Proteinase K (20mg/µl) was added and incubated at 37°C for 30 minutes. DNA was extracted by addition of a solution containing 10M Guanidium thiocyanate and 150µl of 200mMEDTA (pH=7.0), followed by addition of 7.5M ammonium acetate (150µl) and Chloroform: isoamylalcohol 24:1 v/v (300µl). The two phases were separated by centrifugation at 10,000 rpm for 15 minutes. The aqueous phase was placed in another centrifuge tube; twice the volume of isopropanol was added and left at -20°C for one hour. The mixture was centrifuged in a microfuge at 10,000 rpm for 15 min. The precipitated DNA was then washed with 80% ethanol, dried and reconstituted with 30µl of sterile distilled water, 5 µl of the DNA template was used in a PCR reaction.
The primers and the probe were custom synthesised by Bangalore Genei, Bangalore, India; the PCR reagents used in the study also were obtained from them.
Amplification by nPCR of Mycobacterium tuberculosis-specific 200-bp DNA
The nPCR for the gene coding for MPB 64 protein of M. tuberculosis was done as described by Narita et al with minor modifications by us. The nPCR amplification was performed on all the 69 VFs obtained from Eales' and control patients.
Dot-blot hybridisation on the amplified products of IS6110 gene of M. tuberculosis complex
The PCR for the IS 6110 gene of M. tuberculosis complex was carried out by the method of Eisenach et al with minor modifications. The amplified product was further processed for dot-blot hybridisation for characterisation of the M. tuberculosis genome. For this purpose, the amplified product of only nPCR positive samples were chosen. 9 ml of the amplified product was blotted on to a positively charged nylon membrane (Hybond N+, Amersham, Buckinghamshire, England) in a dot-blot format. The membrane was placed between two Whatman No. 3 filter papers and baked in the hybridisation oven at 80°C for 90 minutes. Then it was placed on periplates of appropriate size, to facilitate free movement during strokes and it was pre-hybridised with the hybridisation buffer at 45° C for 30 minutes at 60 strokes/minutes. The membrane was probed with non-isotopically labeled probe (5'- CTGCCCAGGT CGACACAT-3') specific for IS 6110, using the 3' oligo labelling and detection system (Amersham, Burkinghamshire, England). This system utilised enhanced chemiluminiscence associated with horse radish peroxidase catalysed oxidation of luminol to detect the presence of the oligonucleotides tailed at the 3' end, with Fluorescein-11-dUTP hybridized to the target sequences on the membranes. The procedure was carried out according to the manufacturer's directions (ECL 3' oligolabelling and detection system) with the post-hybridisation stringency washing done at 55°C.
The signals on the membrane were then detected by exposing the membrane overnight to a blue light sensitive X-ray film. (Amersham, Buckinghamshire, England).
All the 69 vitreous fluids (24 Eales' and 45 controls) included in the study were negative for acid-fast bacilli by both direct smear and by culture.
Results of Polymerase chain reaction
The results of nPCR and PCR based DNA hybridisation for IS 6110 specific to M.tuberculosis DNA in 69 VF specimens (24 Eales' disease patients and the 45 controls) are shown in Table.
Vitreous fluid was positive for M.tuberculosis by nPCR in 5 (20.8%) of 24 Eales' disease patients (two of which are shown in [Figure - 1]) and 2 (4.2%) of 45 controls by using primers coding for MPB 64 gene. This difference was statistically significant (P= 0.02) by the Chi-square test. False positive result due to contamination of the reagents used for amplification, especially Taq DNA polymerase was ruled out since adequate negative controls and the extraction control were included in each nPCR reaction and nonspecific or nonspecific amplified products were encountered in this study.
All the 69 VFs were negative by PCR using IS 6110 primers [Figure - 2]. By applying DNA dot blot hybridisation on the IS 6110 PCR products of the 7 vitreous samples proven positive for M. tuberculosis by nPCR for MPB 64 gene, an attempt was made to increase the sensitivity of the IS 6110 PCR assay for M. tuberculosis DNA. Only 2 of the 5 Eales' patients were positive and both VF samples of the control group were negative [Figure - 3].
The present prospective study was undertaken to reconfirm the role of M. tuberculosis in Eales' disease. We applied nPCR for MPB 64 gene of the bacterium on the vitreous fluid of Eales' patients since it was several times more sensitive than the PCR for IS 6110. We demonstrated the presence of IS 6110 of Mycobacterium tuberculosis complex DNA in a significant number of the vitreous fluid of Eales' patients. nPCR demonstrated the presence of MPB 64 gene of M. tuberculosis in significant number of epiretinal membranes of Eales' disease patients. The results of the present study further confirm the presence of M. tuberculosis genome in significant numbers of the vitreous fluid of Eales' disease patients.
The IS 6110 PCR assay is universally accepted for detection of M. tuberculosis DNA, but studies in Chennai, South India, have shown that 40% of the local clinical isolates of M.tuberculosis have only a single copy of IS 6110 and 4% did not carry even a single copy of IS 6110 [12,13] Populaire et al has also found PCR using IS 6110 primers to be insensitive. Our attempts to increase its sensitivity by modifications in amplifying procedures or changes in volume of PCR product electrophoresed in the agar gel have not been sucessful. In the present study, we attempted to increase its sensitivity by applying DNA dot-blot DNA hybridisation on the IS 6110 PCR products of the 7 VFs positive by nPCR for MPB 64 gene. It could detect M. tuberculosis DNA in 2 samples and both these were from Eales' cases. In this study we found that dot-blot hybridisation also did not significantly improve the sensitivity of detection of M.tuberculosis complex genome by using the primers coding for IS 6110 sequence. Hence, It is not advisable to use PCR based on IS 6110 for detection of M. tuberculosis DNA in clinical specimens in India.
The conventional bacteriological methods to detect acid-fast bacilli (AFB) in VFs were not successful though use of more sensitive methods such as BACTEC™ (Becton and Dickenson, NJ, USA) may be done in future. Failure to demonstrate the tubercle bacilli from the VF of the Eales' disease patients in the present and earlier studies suggests that the mycobacterial antigen could be in a sequestered state in the eye leading to an immunological inflammatory manifestation with retinal vasculitis, particularly in HLA-prone individuals. [2,5] The other possibility is that the mycobacterium may be localised in some part of the body and the circulating antigen may stimulate an inflammation in HLA-prone individuals, resulting in perivasculitis. These findings further emphasise the role of M.tuberculosis in the aetiopathogenesis of Eales' disease. Since non-tuberculous Mycobacteria (NTM) are commonly prevalent in India, further molecular studies on them are needed to understand their role in Eales' disease
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Conflict of Interest:
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