loop mediated isothermal amplification (lamp) for rapid...
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Loop Mediated Isothermal Amplification (LAMP) for rapid diagnosis of Tuberculous meningitis(TBM) in HIV positive patients
Kusum Sharma
Associate Professor
Department of Medical Microbiology ,
PGIMER, Chandigarh, India
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Introduction
• Most common presentation of neurotuberculosis in Indian subcontinent is tuberculous meningitis (TBM)
• Early and rapid diagnosis is crucial for successful disease management -neurological sequelae in 20-25% due to delay in diagnosis
J Neurol Sci 2007;252: 163-168
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Introduction
• Microscopy and culture(Gold standard)- quite inadequate
• IGRAs can not differentiate latent from active infection
• Nucleic acid-based amplification (NAA) tests: important tools-high specificity but low sensitivity in TBM (Metaanalysis)
Lancet infect Dis 2003; 3:633-643
• Real time PCR is beneficial but requires expensive equipment and expertise, limiting its use to highly sophisticated facilities
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LAMP (loop mediated isothermal amplification )
• Highly sensitive and specific
• Six primer pairs recognize eight distinct regions in the target DNA
• Reaction takes only one hour, and can be conducted under isothermal conditions (ranging from 60-650C), eliminating the need for specialized equipment or expertise
• Can be performed even in rural setting
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• Positive reaction detected by a color change of the reaction mixture in ambient light when DNA binding dye(Sybergreen. HNB) is used or by ladder like banding pattern on gel
• The present study was undertaken to-
explore the utility LAMP for rapid diagnosis of TBM
compare LAMP with IS6110 PCR for diagnosis of TBM
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Material & Methods:
• CSF samples from total of 105 patients received in Mycobacteriology lab of
Medical microbiology, PGIMER, Chandigarh were tested
• The relevant history and other details of patients were noted and they were
divided into 3 groups on the basis of following criteria.
Patients groups
n=105
TBM patients
N=65
Disease Control
N=20
Non TBM
Non-infectious
N=20
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Group I: Tuberculous meningitis group (n= 65)
(a) Confirmed TBM (n=5): culture/smear positive.
(b) Suspected TBM (n= 60): Smear/culture negative , Fever , S/S meningeal irritation,
CSF: Increased Protein level (>40 mg/ dl), Deceased glucose,>10WBC/ml & 80% lymphocytes,
Good response to ATT.
GroupII: Non TBM infectious meningitis (n=20) Pyogenic meningitis (n=10 Viral meningitis (n=8 ) Fungal meningitis (n=2)
Group III: Non-infectious neurological disorders(n=20) HI-10 LGBS-5 MS-3 Tumors-2
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Processing of CSF: 200µl of samples were used for Culture, smear LAMP, PCR with IS611O
DNA Extraction: QIAGEN KIT
LAMP :Primers specific for IS6110 (123bp), MPB 64 were used
PCR:IS6110
Results of LAMP were compared with simple IS6110PCR,
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Results of LAMP
L1 L2 L3 L4 L5 L6 L7
L1- MM, L2- Positive control with bending pattern on gel, l3-l4: Positive samples, l5- Negative control, L6-L7; negative samples
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T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11
T1- Positive Control(Green color) , T2-T6: Clinical samples with positive amplification, T7- Negative control ( Sybers green dye with no change in color), T8-T10: negative clinical samples with no amplification
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
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Improved LAMP test for diagnosis of TBM
T1 T2 T3 T4 T5
T1 –Purple color no amplification by using HNB dye , T2-T6 – Blue
color amplification and positive for MTB DNA. T2- Positive control, T2-6,
-clinical samples
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TYPE SUB TYPE
NO. OF PATIENT
S
SMEAR (+) %
CULTURE (+)%
IS6110 PCR
N (%)
LAMP N
(%)
GROUP-1
TBM
Confirmed TBM Patients
5
- 5
(100)
4
(80)
5
(100)
Suspected TBM
Patients
60
- - 44
(73.33)
52
(86.6)
Total
65
- 18 48
(73.84)
57
(87.69)
Control group
Non TB 40 - - - - -
Results of ZN/Culture and Multiplex PCR
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Sensitivity & Specificity of LAMP, IS6110 PCR compared to culture /AFB smears
Test Test Results TBM cases
(N=65)
Control
Group
(N=40)
Sensitivity Specificity PPV NPV
LAMP Positive
Negative
57
8
-
40
87.69%
100%
100%
66.44%
IS6110 PCR Positive
Negative
48
17
-
40
74.16% 100% 100% 49.18%
Culture Positive
Negative
5
60
- 40
7.69% 100% 100% 21.92%
Microscopy Positive
Negative
-
65
-
40
18.33% 100% 100% -
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Comparison of LAMP & IS6110 PCR Results with response to ATT in cases of TBM
Response to ATT IS6110 PCR n (%) LAMP (%)
N=65
48 (74.16%)
57(87.69%)
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Study PCR target No. of cases Sensitivity Specificity
Halder et al,2009 IS6110
Dev R
167 84.6% -
Dil-Afrose et al,2008 MPB64 27 77.7% 100%
Sumi et al,2002 IS6110 37 75.67% 40.5%
Sharma K et al 2011 MPB64,
Protein b,
IS611O
MPCR
210 86.36% 100%
Nagdev et al ,2011 IS6110 LAMP
27 86% 90%
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Conclusions:
• LAMP is more sensitive than IS6110PCR in diagnosing TBM
• HNB is better than Syber green in diagnosing TBM
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Acknowledgments
• Dr Paul Fiorella, Jacksonville
• Prof. S. Varma
• Prof. S. Prabhakar
• Prof. P. Ray
• Dr M. Modi
• Dr Aman Sharma
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Thank you