“TB -LAMP: New validation study and its importance to findthe “missing millions”

Dr. Sunil Sethi
Professor, Department of Medical Microbiology
Post Graduate Institute of Medical Education & Research, Chandigarh

Introduction• Tuberculosis- very high morbidity and mortality among infectious disease• 10 million cases globally in 2018.

• India has the highest burden of TB with ~25% of global TB (~2.8 million new cases/year).
WHO TB Report 2017

• In India, problem is more complex due to- Higher incidence- Inadequate diagnostic service in difficult to reach areas

Need an accurate diagnostic test which is simple, cost effective, rapid and can be performedat peripheral settings.

The Problem of “Missing the Millions”• Though, patients enrolled under DOTS program increased, yet it is notreaching to 100% target• Many people are escaping diagnosis due to insufficient capacity even after
instituting ACF campaign• In 2018, the diagnostic coverage was able to screen ~30 Million suspects• 15-20 million more suspects which need to be screened for TB than what
the Public/Private Program is not able to reach

Total Suspects: 40-45 Mn
SSM~20
millionXpert(3-5Mn)

TruNat(2-3Mn)(Missing 10-15 Mn)

India’s End TB 2025
Which test is actually required for TB diagnosis ?

1. Can be performed at peripheral settings
2. Minimal requirement of training
3. Better or equal sensitivity and specificity than the existing tests at peripheral healthsettings
4. Rapid and cost effective , simple - help in treatment initiation under TB control
programmeCurrent TB diagnostics tests• Smear Microscopy - 50-60% sensitive and misses almost every alternate case

• Culture - 2 to 4 weeks to become positive• The NAAT’s(PCR based ) like Xpert MTB/RIF/ True Nat(Mol bio)-- Needs a costly infrastructure- Trained staff- Limited to tertiary and secondary care centers

Test

Sensitivity

Essential Requirements

Penetration

Limitations

Cost /
instrument

Cost ofDiagnosis

Smear
Microscopy

50-60% (LEDMicroscopy is
not widelyused)

Sputum Processing, Staining,
Manual intervention,(LOD 10000 cfu/ml)

Until the CHC(approx. 20000DMC) covering ~15-20 Million
suspects•Subjective ,• tedious sample processing requires two samples

Rs 1-1.5
lakhs

Rs 75-100/test

GeneXpert~ 95%

Dedicated infrastructure,cold chain, highly trainedmanpower(LOD 130 cfu/ml)

At the District level(~ 1500 sites)
3-5 Million suspects

Specialized infrastructure,
Transportation of samples ,High cost of instrumentation ,Recalibrated issue
Cost of the test

Rs 15 lakhs

Rs 1100/test(with RIFresistance)

CB NAAT(TruNat)~ 95%

Cold chain, complex sputum
processing, trainedmanpower(LOD 130-150 cfu/ml)

District level(2-3 Millionsuspects)

Complex sputum processing,
single test per run, samples
have to be brought toinstallation site, cold chain
reqt, low thruput (6-8samples/day)Rs 6-8 lakhs

Rs 800/test(with RIFresistance)

TB LAMP~95%

Similar infrastructure as
microscopy, minimal trained
manpower, no sputum
processing (raw sputum canbe used directly), highthroughput (1 - 60-70
samples/day)(LOD 100 cfu/ml)

As alternative to of
Smear microscopy(WHOrecommendation),
until DMC level(can cover 20-25
Million suspects)

Don’t detect RIF resistanceRs 3 lakhs

Rs 800/test

Current available tests(which willcome down tohalf as volumes
build up)

The Proposed Solution: TB-LAMP Assay• Isothermal amplification Method• High sensitivity and specificity• Visible detection of positive samples (throughfluorescence)• No cold chain requirement and can be
performed in room temperature conditions

Our Experience
Evaluation of In-House Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Diagnosisof M. tuberculosis in Pulmonary Specimens
Sethi S et al. JCLA, 2013

First Indian Study on in-house TB-LAMP assay

Combination of adenosine-deaminase and nucleic acid amplification assays
for diagnosing tuberculous pleural effusion
Sethi S et. al. J Infect. 2014

Loop-mediated isothermal amplification assay for detection of Mycobacterium tuberculosis
complex in infertile women
Sethi S et al. Indian J Med Microbiol. 2016

• Sensitivities of ZN smear, culture, HPE, PCR and LAMP were
2.94%, 10.29%,8.82%, 95.59% and 66.18%, respectively.
• Concordance between PCR and LAMP was 63%, whichshows a good agreement.

A loop-mediated isothermal amplification assay for the diagnosis of pulmonary tuberculosis
Lett Appl Microbiol. 2019

• A LAMP assay based on the mpt64 gene sequence wasdeveloped• Analytical Sensitivity of 1 pg per ml (or 200 copies of M.tbgenome) in a 40-min reaction

The WHO Endorsed TB-LAMP Assay (Eiken, Japan)• TB-LAMP (Eiken, Japan) – WHO endorsed in 2016
• Based on isothermal amplification and strand displacement principle,
no requirement of costly PCR based instrument• High sensitivity (>95%) and specificity (~98%)- (LOD of 100 CFU/ml)• 15% more patients than Smear Microscopy, 40% additionalsmear negative cases- Can replace or add on test to sm microscopy.

• High throughput- 14 samples in 1.5 hours, easy to perform

TB-LAMP Workflow – Quick and Reliable!
1

2
Sampletransfer& lysis

3
Pure DNA
extraction

4
Reaction mix
preparation

LAMP reaction

ResultreadingTime to result
approx.1-1,5h
16 tests/run

How TB-LAMP will help in diagnosis of Missing millions• Can reach up to CHC level, making it the only option which gives fast and accurate diagnosis• Through battery/solar power, can reach to even remote areas (thus making access/samplecollection easy)• Increase of scale will reduce the cost/test to < Rs 600/test, much lower than any other similar test

TB-LAMP Test Performance -WHO Policy Guideline 2016

Gray et al. study featured 2 Indian sites – PGIMER, Chandigarh and MGIMS, Wardha

TB-LAMP Test Performance -WHO Policy Guideline 2016

Pooled sensitivity-77-80%
Specificity-97-98%

Determination of diagnostic accuracy of TB - LAMP assay in pulmonary tuberculosis
patients from North India• Total samples(n)= 234• 120 TB suspects+ 117 pulmonary disease(other than TB)

GroupName of the Test

Sensitivity

Specificity

Overall

Xpert MTB/RIF

98.1% (89.799.9%)

TB LAMPNo TB cases

97.2 %( 93.199.2%)

Positive
predictive value
92.7%(82.997.1%)

Negative
Predicative Value
99.3 % (97.899.8%)

94.2% (84.198.8%)

96.6% (92.198.9%)

90.7% (80.595.9%)

97.9% (93.999.3%)

Xpert MTB/RIF---

TB LAMP-

99.2% (95.499.98%)
99.2%(95.499.98%)--

Unpublished data

TB-LAMP assay for diagnosis of pediatric tuberculosis: A prospective cohort study• Respiratory samples(n)= 187• (GA/GL/BAL/Sputum)
Sensitivity

Specificity

PPV

NPV

TB-LAMP

80% (95% CI,59.3-93.2%)

96.1% (95% CI,91.6-98.5%)

76.9%(95% CI,59.8-88.2%)

96.7%(95% CI, 9398.5%)

Xpert MTB/RIF
ultra

84%(95% CI, 63.9- 97.4% (95% CI,95.5%)
93.4-99.3%)

84%(95% CI, 66.3- 97.3%(95% CI,93.3%)
93.7-98.9%),

Unpublished data

TB-LAMP is part of WHO EDL

Still need high quality studies
under programmatic conditions inIndia at peripheral level“Multi-centric validation of WHO approved TB-LAMP assay for
detection of Mycobacterium tuberculosis in suspects of Pulmonary
tuberculosis in peripheral settings”
Objectives of the study1. To elucidate the diagnostic accuracy of TB-LAMP assay in sputum samples ofsuspected tuberculosis patients at peripheral centres in different pats of India

2. To assess the feasibility of TB-LAMP assay at the peripheral level for diagnosis oftuberculosis.

Methodology• A total of 2400 TB suspects will be enrolled at 5 peripheral sites in different states.
( The sample size was calculated taking the sensitivity of 78% and specificity of 98% with a desired precision of 7% at 95% confidence
interval. Using these parameters, the sample size is 2251. If we took 5% of sample loss during the study the total sample size would be2364. so we are taking a sample size of 2400, and will be divided into equal for each site)• The samples will be tested as per standard protocols (as per WHO guidelines) for- Smear Microscopy- Liquid Culture- Gene Xpert- TB-LAMP

Participating Sites•

PGIMER, Chandigarh (Parent Institute)•MGIMS, Wardha•

Lucknow•

Guwahati, Assam•

Kochi, Kerala•

Punjab

Level of Training required• TB-LAMP can be operated by a minimally trained technician – could be paramedic,lab technician or a physician.
• Such people need to be trained on sputum sample collection and handling in a biosafety environment• 3-day hands-on training which will be imparted by the PGIMER on the use of TB
LAMP and running the test

Thanks