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  Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 33  |  Issue : 5  |  Page : 43-45
 

Association of mycobacterium tuberculosis in the causation of Eales' disease: An institutional experience


1 Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
2 Department of Microbiology, ? All India Institute of Medical Sciences, New Delhi, India
3 Department of Microbiology, Vardhaman Mahavir Medical College and Safdarjung Hospital, New Delhi, India

Date of Submission01-Sep-2013
Date of Acceptance09-Apr-2014
Date of Web Publication6-Feb-2015

Correspondence Address:
U B Singh
Department of Microbiology, ? All India Institute of Medical Sciences, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.148829

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 ~ Abstract 

Background: Eales' disease is an idiopathic retinal vasculitis characterized by retinal inflammation, ischemia, and neo-vascularisation. It frequently causes massive vitreous haemorrhage and retinal detachment leading to blindness. Although the exact etiology is unknown, this condition is considered to be a consequence of hypersensitivity reaction to tubercular protein due to previous Mycobacterium tuberculosis (M. tuberculosis) infection. This study is aimed at the detection of association of M. tuberculosis in patients with Eales' disease. Materials and Methods: A prospective case-control study was undertaken in 65 clinically diagnosed cases of Eales' disease. Patients with proliferative diabetic retinopathy, neo-vascular proliferation, macular oedema, premacular fibrosis and tractional retinal detachment were taken as controls. M. tuberculosis DNA was detected (MPT64 gene by polymerase chain reaction, PCR) in patients with Eales' disease. Clinical symptoms along with tuberculin skin test (TST) and erythrocyte sedimentation rate (ESR) were used as gold standard for comparing results of PCR. Result: PCR positivity was found in 12 (38.7%) patients with Eales' disease. The PCR positivity was significantly associated with the patients with high TST reading and high ESR values. Conclusion: Patients with a high TST reading and ESR value and a positive PCR in vitreous samples have a high likelihood of having M. tuberculosis as an etiology.


Keywords: Eales′ disease, Mycobacterium tuberculosis, retinal detachment, vitreous haemorrhage


How to cite this article:
Rajpal, Singh U B, Mohapatra S, Wagh V K, Porwal C, Kaushik A. Association of mycobacterium tuberculosis in the causation of Eales' disease: An institutional experience . Indian J Med Microbiol 2015;33, Suppl S1:43-5

How to cite this URL:
Rajpal, Singh U B, Mohapatra S, Wagh V K, Porwal C, Kaushik A. Association of mycobacterium tuberculosis in the causation of Eales' disease: An institutional experience . Indian J Med Microbiol [serial online] 2015 [cited 2019 Aug 18];33, Suppl S1:43-5. Available from: http://www.ijmm.org/text.asp?2015/33/5/43/148829



 ~ Introduction Top


Eales' disease is a well-established clinical entity for more than a century. The disease is predominantly an obliterative vasculitis, which results in peripheral and retinal hypoxia leading to neo-vascularisation. Vitreous haemorrhage with or without tractional rhegmatogenous retinal detachment is the common sequelae of this disease. [1] However, the exact etiopathogenesis and treatment of Eales' disease is still elusive. Of the several causes suggested, Mycobacterium tuberculosis (M. tuberculosis) is the commonest one. Its role in the etiopathogenesis of Eales' disease as an active infection or hypersensitivity reaction to tubercular protein has been discussed in literature. [2] It is primarily a disease of healthy adult males. Approximately 35% of patients with Eales' disease have been associated with pulmonary tuberculosis. In India, the incidence of Eales' disease was found to be 1 in 135 ophthalmic patients, in a referral centre, and 1 in 200 in a general eye hospital. [3] Various studies have been carried out using immunological parameters to demonstrate a correlation with tuberculosis, but the results are inconclusive. Thus, the etiopathogenesis of Eales' disease is still poorly understood, and the role of M. tuberculosis is not yet clear. Because of nonavailability of tests with a high predictive value, it is difficult to prove conclusively the role of M. tuberculosis in Eales' disease. However, the increasing use of polymerase chain reaction (PCR) for various ocular diseases has helped reaching a final diagnosis. MPT64 gene in M. tuberculosis translates into a highly immunogenic protein. PCR for this gene has been successfully used in our laboratory for diagnostic purposes for over 18 years. We designed a case-control study for the detection of M. tuberculosis DNA in vitreous samples of patients with Eales' disease, by PCR targeting this gene.


 ~ Materials and Methods Top


Sample collection

A total of 65 patients (31 cases with clinical diagnosis of Eales' disease and 34 controls) were enrolled for this study. Patients with a complication of Eales' disease such as non-resolving vitreal haemorrhage for ≥4 months, vitreal haemorrhage <4 months with tractional retinal detachment, vitreal haemorrhage with rubeosis iridis and recurrent/bilateral vitreous haemorrhage were taken as cases. The control specimens were collected from patients who underwent vitreous surgery for proliferative diabetic retinopathy, neo-vascular proliferation, macular oedema, premacular fibrosis and tractional retinal detachment. Vitreous samples were collected with proper aseptic precaution. Epiretinal membranes (ERM) were also collected and sent to the laboratory along with vitreous in heparinized sterile vials for PCR analysis.

Sample processing

The samples were decontaminated using N-acetyl L-cysteine-NaOH solution. Decontaminated sample (200 μl) was incubated in equal amount of lysis buffer containing 20 mM Tris-HCL (pH 8.3), 10% Tween 20 and 5 mM NaOH for 2 hours at 60°C followed by heat lysis at 95°C for 15 minutes. The DNA was extracted using chloroform followed by precipitation in 0.3 M sodium acetate and absolute ethanol overnight at −20°C. A 240-bp long region of MPT64 gene of M. tuberculosis was amplified using primers MPT1 (5′-TCCGCTGCCAGTCGTCTTCC-3′) and MPT2 (5′- GCTCTCGCGAGTCTAGGCCA-3′). The PCR was carried out in 25 μl volumes comprising 10× PCR buffer containing 1.5 mM MgCl 2 , 200 μM each of the four deoxynucleotide triphosphates (Perkin-Elmer, Applied Biosystems Division, California, USA), 0.4 μM of each primers, 1.25 U Taq DNA Polymerase (Perkin-Elmer) and 5 μl extracted DNA. Using Amplitron Thermocycler (Barnstead/thermolyne, IA, USA), amplification was carried out for 30 cycles, each consisting of denaturation at 94°C for 1 minute, annealing at 60°C for 1.15 minutes and extension at 72°C for 1.15 minutes. Each series of PCR had a positive control (100 pg of H37 Ra DNA) and several negative controls (sterile distilled water) interspersed with the samples. The amplified products were fractionated along with the molecular weight marker, ΦX174 DNA digested with HaeIII (Promega, WI, USA) in 1.5% agarose gel and electrophoresed in Tris-HCl/boric acid/EDTA buffer for 1 hour. Using transilluminator (Chemlmager, Alpha Innotech, USA), amplified products having a 240-bp band were identified as positive. The DNA from PCR-negative samples was spiked with 100 pg and 10 ng of DNA from the H37Ra strain of M. tuberculosis and re-amplified to check for amplification inhibitors giving false-negative results. All the patients were subjected to tubercular skin test (TST) and erythrocyte sedimentation rate (ESR). An erythema of ≥10 mm after 72 hours of tuberculin injection was considered as positive TST. Statistical analysis was done using Chi-square statistical test (χ2 ) and Fisher's exact test for the comparison of the results of PCR in patients with Eales' disease and controls.


 ~ Results Top


All the samples were subjected to PCR for M. tuberculosis. The statistical analysis of data was done by χ2 and Fischer's exact test wherever applicable. Most of the patients with Eales' disease belonged to an age group of 21-30 years. Only two (6.5%) casesamong the diseased group had a past history of pulmonary tuberculosis. Three patients had a history of antitubercular drug intake. The major patho-anatomic finding among the diseased group was posterior vitreous detachment [Table 1]. It was incomplete in 29 (93.5%) and complete in 2 (6.5%) of the patients. Maximum numbers of patients were found to have tractional retinal detachment as their commonest etiology. Fourteen out of 31 (45%) cases were positive for TST in the test group, in comparison to 6 (20%) out of 35 in the control group. Eleven (35.5%) cases among the diseased group had ESR value ≥20 mm/h by Westergren method in comparison to two in controls.   ERM were found in the eyes of 10 (32.3%) patients and were sent along with vitreous for PCR analysis. PCR positivity was found to be more in samples containing ERM 50% (5 out of 10) in comparison to vitreous only 33.3% (7 out of 21; P = 0.373, statistically not significant). PCR was positive in 12 (38.7%) cases of Eales' disease while 6 (17.64%) in controls (P = 0.058). Four out of five cases of Eales' disease with history of pulmonary tuberculosis and history of antitubercular intake were found positive for PCR and ESR value ≥20 mm/h, whereas three out of five showed TST >10 mm. The results correlated well with the TST reading [Table 2]. Moreover, in patients with tractional retinal detachment, secondary rhegmatogenous retinal detachment and diffuse vasculitis, the PCR positivity is higher than in the control group. However, the number of patients in each group is not significant to prove its statistical significance. In the test group, patients with high TST reading (>10 mm) showed a higher PCR positivity, whereas those with a lower TST reading (<10 mm) were associated with high PCR negativity (P < 0.001). No such association was observed in the control group. Moreover, more number of PCR positives were found in patients with higher ESR as compared to patients with low ESR values in the test group (P = 0.03)  [Table 2].
Table 1: Relationship of different pathoanatomic finding with Eales' disease

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Table 2: Correlation of PCR results with TST and ESR results

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 ~ Discussion Top


The etiopathogenesis of Eales' disease is still poorly understood, and the role of M. tuberculosis is still not confirmed. It has been difficult to prove conclusively because of the risk involved in obtaining the tissue sample from patients for microbiologic and histopathology studies. In the present study, we collected ERM along with vitreous which are proven to be important samples for studies regarding etiology. In our study, PCR was positive in 12 (38.7%) of the Eales' disease patients who had undergone vitrectomy. PCR was positive in 50% of samples containing ERM with vitreous and 33% in samples containing vitreous only. The result was found to be same in patients with tractional retinal detachment, secondary rhegmatogenous retinal detachment and diffuse vasculitis. This supports that ERM along with vitreous will be a better choice for PCR. Six out of 34 controls were also positive by PCR. Madhavan et al., have evaluated the role of nested PCR for MPT64 gene in 23 patients of Eales' disease in the ERM. Positivity of nested PCR was 47.18% in patients of Eales' disease as compared to 11.1% in controls. [4] In another study, the positivity of nested PCR for MPT64 gene was found to be 20.8% in the vitreous samples of patients of Eales' disease as compared to 4.2% of controls. [5] PCR for MPT64 gene is a highly sensitive technique and capable of picking up 3-5 viable as well as nonviable bacilli. In developing countries, the prevalence of latent infection with M. tuberculosis is very high. Hence, the presence of nonviable bacilli in healthy individuals with latent infection may give rise to positive results as found in the present study. [6] Though 19 samples were negative by PCR, the strong correlation of the PCR positivity with TST and ESR showing statistical significance further supports the tubercular etiology of Eales' disease. PCR was found to be positive in patients with higher TST and ESR values. Sensitivity of PCR improved in samples with both vitreous as well as ERM samples. It would be useful to include both ERM and vitreous samples for the laboratory diagnosis. The hypothesis of an immunological pathogenesis for the Eales' disease seems to be truly seconded here. Patients with a clinical diagnosis of the disease without laboratory evidence of M. tuberculosis DNA seems to be of poor immunological response, as evidenced by a negative TST. The immunological involvement of the eye as part of a systemic disease may also explain the absence of DNA in local samples. The high endemicity of disease in our country is often reflected as varied presentations of clinical disease or latency. Hidden/overt disease could sometimes be detected by circulating DNA in the patients enrolled as controls.


 ~ Conclusion Top


present study substantiates the hypothesis that infection with M. tuberculosis has a role in the pathogenesis in at least a subgroup of patients with Eales' disease. To the best of our knowledge, this is the first study emphasizing correlation of PCR results with TST and ESR values. Eales' disease with a positive PCR, high reading on TST, high ESR value, tractional retinal detachment, secondary rhegmatogenous retinal detachment, epiretinal membrane and diffuse vasculitis have more likelihood of having M. tuberculosis as an etiology.

 
 ~ References Top

1.
Patnaik B, Pran N, Nagpal PN, Kalsi R. Eales disease: Clinical features, pathophysiology etiopathogenesis. Ophthal Clin North Am 1998;11:601-17.  Back to cited text no. 1
    
2.
Therese KL, Deepa P, Therese J, Bagyalakshmi R, Biswas J, Madhavan HN. Association of mycobacteria with Eales' disease. Indian J Med Res 2007;126:56-62.  Back to cited text no. 2
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3.
Das T, Biswas J, Kumar A, Nagpal PN, Namperumalsamy P, Patnaik B, et al. Eales' disease. Indian J Ophthalmol 1994;42:3-18.  Back to cited text no. 3
[PUBMED]  Medknow Journal  
4.
Madhavan HN, Therese KL, Gunisha P, Jayanthi U, Biswas J. Polymerase chain reaction for detection of Mycobacterium tuberculosis in epiretinal membrane in Eales' disease. Invest Ophthalmol Vis Sci 2000;41:822-5.  Back to cited text no. 4
    
5.
Madhavan HN, Therese KL, Doriaswamy K. Further investigations on the association of mycobacterium tuberculosis with Eales' disease. Indian J Ophthalmol 2002;50:35-9.  Back to cited text no. 5
[PUBMED]  Medknow Journal  
6.
Richeldi L, Barnini S, Saltini C. Molecular diagnosis of tuberculosis. Eur Respir J Suppl 1995;20:689-700s.  Back to cited text no. 6
    



 
 
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  [Table 1], [Table 2]



 

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