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Research Letter |

Loop-Mediated Isothermal Amplification for Rapid Diagnosis of Tubercular Uveitis FREE

Kusum Sharma, MD1; Reema Bansal, MD2; Aman Sharma, MD3; Nikhil Beke, MD2; Amod Gupta, MD2
[+] Author Affiliations
1Department of Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
2Department of Ophthalmology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
3Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India
JAMA Ophthalmol. 2014;132(6):777-778. doi:10.1001/jamaophthalmol.2014.330.
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Published online

Polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis in tubercular uveitis is limited by low sensitivity, expensive equipment, sophisticated laboratory facilities, and expertise. We evaluated loop-mediated isothermal amplification (LAMP) assay1 targeting the IS6110 region of M tuberculosis in intraocular samples from patients with tubercular uveitis.

The Institute Ethics Committee of the Post Graduate Institute of Medical Education and Research approved this study, and written informed consent was obtained from the patients. A total of 29 vitreous fluid samples from eyes undergoing pars plana vitrectomy and 1 iris biopsy sample were subjected to LAMP assay under the following groups: (1) group 1 (10 patients) with tubercular uveitis confirmed by positive multitargeted PCR for M tuberculosis from intraocular samples2; (2) group 2 (10 patients) with intraocular inflammation due to nontubercular etiology (negative controls); and (3) group 3 (10 patients) with vitreoretinal disorders without any intraocular inflammation (normal controls).

After DNA extraction, LAMP was performed using 6 primers specific for the IS6110 region of M tuberculosis3: (1) F3, 5′-AGACCTCACCTATGTGTCGA-3′; (2) B3, 5′-TCGCTGAACCGGATCGA-3′; (3) FIP, 5′-ATGGAGGTGGCCATCGTGGAAGCCTACGTGGCCTTTGTCAC-3′; (4) BIP, 5′-AAGCCATCTGGACCCGCCAACCCCTATCGTATGGTGGAT-3′; (5) FLP, 5′-AGGATCCTGCGAGCGTAG-3′; and (6) BLP, 5′-AAGAAGGCGTACTCGACCTG-3′.

The entire reaction could be conducted under isothermal conditions, requiring only a water bath or heating block at 60°C to 65°C. The needs for electrophoresis, specialized equipment (thermocycler), and technical expertise were eliminated. Amplification products were detected by the following: (1) agarose gel–based analysis showing a typical ladder pattern in positive reaction (Figure 1); or (2) naked eye using 0.1% SYBR Green 1 (Life Technologies) and observing the color of the solution under UV light, turning green in the presence of a LAMP amplicon but remaining orange with no amplification (Figure 2).

Place holder to copy figure label and caption
Figure 1.
Results of Loop-Mediated Isothermal Amplification by Agarose Gel–Based Analysis Showing a Typical Ladder Pattern in a Positive Reaction

Lane 1 (L1) is a 100–base pair molecular marker; L2, positive control with bending pattern on gel; L3 and L4, positive clinical samples; L5, negative control; and L6 and L7, negative clinical samples.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.
Results of Loop-Mediated Isothermal Amplification Using 0.1% SYBR Green 1 (Life Technologies) as Seen by Naked Eye

Tube 1 (T1) is a positive control (green color); T2 through T6, clinical samples with positive amplification; T7, negative control (SYBR Green I dye with no change in color); and T8 through T10, negative clinical samples.

Graphic Jump Location

The mean (SD) ages were 42.5 (9.8) years (range, 30-50 years) in group 1 (7 male, 3 female), 34.4 (20.2) years (range, 3-60 years) in group 2 (5 male, 5 female), and 40.6 (17.1) years (range, 8-63 years) in group 3 (7 male, 3 female). Seven samples in group 1 (70%) tested positive by LAMP. All samples in groups 2 and 3 were negative by both LAMP and multitargeted PCR. The sensitivity, specificity, positive predictive value, and negative predictive value of LAMP were 70%, 100%, 100%, and 86.9%, respectively.

The LAMP assay has a reported sensitivity of 96.1% (smear-positive and culture-positive cases) in extrapulmonary tuberculosis, 85% (smear-negative and culture-positive cases) in pulmonary tuberculosis,4 and 88.23% in tubercular meningitis.5 In our study, the possible reason for lower sensitivity of LAMP could be a low bacillary load.

We used IS6110 primers for the LAMP assay. The negative LAMP results in 3 patients in group 1 could be due to low copy numbers of IS6110 in some M tuberculosis strains,6 low number of bacteria, or poor lysis of bacteria in the ocular fluids. To our knowledge, this is the first study to report the utility of LAMP in tubercular uveitis. In the absence of mycobacterial culture, we used multitargeted PCR positivity as the gold standard, which has a sensitivity of 77.8% in tubercular uveitis.2 The LAMP assay had lower sensitivity than PCR because only 1 primer (IS6110) was used in LAMP, whereas 3 primers were used in multitargeted PCR (IS6110, MPB64, and protein B). The 3 cases missed by LAMP were positive for MPB64 and negative for IS6110 by multitargeted PCR. A small sample size is another limitation of our study. However, the major advantages of LAMP are low cost, ease in conducting the test, and rapid results.5 Although the cost of LAMP is much lower than that of conventional PCR, it is higher than the cost of a Mantoux test. While positive results of a Mantoux test merely suggest latent tuberculosis anywhere in the body, a positive result on LAMP may nearly confirm tubercular uveitis. The LAMP assay needs to be evaluated in studies with bigger sample sizes. It can be useful in diagnosing tubercular uveitis in rural or resource-poor settings.

Corresponding Author: Amod Gupta, MD, Department of Ophthalmology, Post Graduate Institute of Medical Education and Research, Chandigarh, India 160012 (eyepgi@sify.com).

Author Contributions: Dr K. Sharma had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: K. Sharma, A. Sharma, Gupta.

Acquisition, analysis, or interpretation of data: K. Sharma, Bansal, A. Sharma, Beke.

Drafting of the manuscript: K. Sharma, Bansal, A. Sharma, Beke.

Critical revision of the manuscript for important intellectual content: K. Sharma, Gupta.

Statistical analysis: K. Sharma.

Administrative, technical, or material support: A. Sharma, Gupta.

Study supervision: Bansal, Gupta.

Conflict of Interest Disclosures: None reported.

Notomi  T, Okayama  H, Masubuchi  H,  et al.  Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):E63.
PubMed   |  Link to Article
Sharma  K, Gupta  V, Bansal  R, Sharma  A, Sharma  M, Gupta  A.  Novel multi-targeted polymerase chain reaction for diagnosis of presumed tubercular uveitis. J Ophthalmic Inflamm Infect. 2013;3(1):25.
PubMed   |  Link to Article
Aryan  E, Makvandi  M, Farajzadeh  A,  et al.  A novel and more sensitive loop-mediated isothermal amplification assay targeting IS6110 for detection of Mycobacterium tuberculosis complex. Microbiol Res. 2010;165(3):211-220.
PubMed   |  Link to Article
Pandey  BD, Poudel  A, Yoda  T,  et al.  Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients. J Med Microbiol. 2008;57(pt 4):439-443.
PubMed   |  Link to Article
Nagdev  KJ, Kashyap  RS, Parida  MM,  et al.  Loop-mediated isothermal amplification for rapid and reliable diagnosis of tuberculous meningitis. J Clin Microbiol. 2011;49(5):1861-1865.
PubMed   |  Link to Article
Chauhan  DS, Sharma  VD, Parashar  D,  et al.  Molecular typing of Mycobacterium tuberculosis isolates from different parts of India based on IS6110 element polymorphism using RFLP analysis. Indian J Med Res. 2007;125(4):577-581.
PubMed

Figures

Place holder to copy figure label and caption
Figure 1.
Results of Loop-Mediated Isothermal Amplification by Agarose Gel–Based Analysis Showing a Typical Ladder Pattern in a Positive Reaction

Lane 1 (L1) is a 100–base pair molecular marker; L2, positive control with bending pattern on gel; L3 and L4, positive clinical samples; L5, negative control; and L6 and L7, negative clinical samples.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.
Results of Loop-Mediated Isothermal Amplification Using 0.1% SYBR Green 1 (Life Technologies) as Seen by Naked Eye

Tube 1 (T1) is a positive control (green color); T2 through T6, clinical samples with positive amplification; T7, negative control (SYBR Green I dye with no change in color); and T8 through T10, negative clinical samples.

Graphic Jump Location

Tables

References

Notomi  T, Okayama  H, Masubuchi  H,  et al.  Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):E63.
PubMed   |  Link to Article
Sharma  K, Gupta  V, Bansal  R, Sharma  A, Sharma  M, Gupta  A.  Novel multi-targeted polymerase chain reaction for diagnosis of presumed tubercular uveitis. J Ophthalmic Inflamm Infect. 2013;3(1):25.
PubMed   |  Link to Article
Aryan  E, Makvandi  M, Farajzadeh  A,  et al.  A novel and more sensitive loop-mediated isothermal amplification assay targeting IS6110 for detection of Mycobacterium tuberculosis complex. Microbiol Res. 2010;165(3):211-220.
PubMed   |  Link to Article
Pandey  BD, Poudel  A, Yoda  T,  et al.  Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients. J Med Microbiol. 2008;57(pt 4):439-443.
PubMed   |  Link to Article
Nagdev  KJ, Kashyap  RS, Parida  MM,  et al.  Loop-mediated isothermal amplification for rapid and reliable diagnosis of tuberculous meningitis. J Clin Microbiol. 2011;49(5):1861-1865.
PubMed   |  Link to Article
Chauhan  DS, Sharma  VD, Parashar  D,  et al.  Molecular typing of Mycobacterium tuberculosis isolates from different parts of India based on IS6110 element polymorphism using RFLP analysis. Indian J Med Res. 2007;125(4):577-581.
PubMed

Correspondence

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