Difference Between Skeletal Smooth and Cardiac Muscles
Invest to End TB. Save Lives
1. Laboratory updates
TB day 2022
Pavia 25 marzo 2022
Daniela Maria Cirillo
WHOCC Ita 98
FCSR Milano
'Invest to End TB. Save Lives.'
2. Outline
• From infection to diseases: improvements and gaps
• New recommendations by WHO (policies and documents
• Facing the reality: alignment of diagnostics with treatment options
• The future:
• Biomarkers
• Moving out from sputum for Tb diagnosis
3. Tuberculosis: from infection to disease
uninfected
infected
Incipient
TB
Subclinical
Active TB Immunological
evidence of infection
Absence of clinical
pictures, CXR
abnormalities,
bacterial proliferation
Probable progressive
infection, No
abnormalities,
signature profile
Clinical picture,
symptoms, bacterial
proliferation
Precipitating event
Current diagnostics for disease
5. New tests for infection
Skin tests
• C-Tb
• Diaskintest and
• EC test
IGRA tests
• Quantiferon plus
• QuantiFERON Access
• Standard E and F TB-feron
• Advansure TB-IGRA
VIDAS TB-IGRA, BioMerieux
Heparin blood 4ml
fully automated
17 Hs to results
Expected 2021
6. Diagnostic algorithms for screening and
diagnosis of TB disease
How to select
among the
WRMDs?
Infrastructures
Sample
transports
Workload
Multiple tests
7. New trends for MDR regimens
• Oral/in combination with injectables
• Short/long
• Standard/personalised
• Including new drugs such as Bedaquiline, Linezolid, Delamanid,
Pretomanid and repurposed in addition to Floroquinolones
• Limiting toxicity and long term side effects with reduced dosage of the
most toxic drugs
8. Tests availability for drugs in current and
future regimens
Grouping of drugs
Drugs
LA-NAAT MA-NAAT LPA tNGS WGS pDST pBMD
First line
RIF
INH
EMB
PZA
Group A
LFX/
MFX
BDQ
LZD
Group B
CFZ
DCS
Group C
DLM
IMP-CLN/
MPM
AMK
STR
ETO/
PTO
PAS
9. Pretomanid (Nitroimidazole class)
• New drug approved as part of BPaL regimen (Dovprela)
• No CC officially established and endorsed
• No molecular screening for testing mutations in ddn fbiC fgd1
• Pa powder available from TB Alliance, lyophilised panel of control strains with known
MGIT Pa MICs is available from the BCCM/ITM collection (
https://bccm.belspo.be/about-us/bccm-itm )
10. Rapid approved molecular assays for MDR-TB cases
NO assays for Bedaquiline, Delamanid,
Pretomanid and Linezolid
11. Mic plates containing new drugs (home made or
commercial)
• Ecoff determination for new drugs accord to EUCAST
protocol
• Proposed breakpoints from CRyPTIC consortium data
• 20,637 samples were collected and had their
susceptibility tested to 13 anti-TB drugs
• ECOFF proposed by ECOFF finder or interval regression
Testing options: phenotypic methods: BMD
Fowler P. et al CRyPTIC cons. 2022, ERJ in press
12. The current strategy for testing is disaligned with the
needs
• Phenotypic tests are generally to long to address the therapy
• Phenotypic tests are performed sequentially further dilating times to
response
• Phenotypic tests for the key drugs are performed in few centers (which
strategy? Decentralize and increase incompetence or centralize and
decrease cost and competence)
• Mic when and how? Current commercial lay out of TB plate is obsolete
compered to drugs in use
• Rapid commercial molecular test are also not fulfilling the scope
14. Building components:
1. High quality phenotypic DST data
2. High quality, standardized WGS for generating unbiased raw sequence data
3. A standardized bioinformatics pipeline for variant detection and annotation
4. A standardized and validated methodological approach for associating genotype-phenotype
CRyPTIC Consortium
ReSeqTB
WHO Surveillance Program Contributors
Multinational TB researchers
Public Health Bodies
38k pDST/WGS matching data (QA/QC passed) from >40 countries
AMI, BDQ, CFZ, DLM, EMB, ETH, INH, LEV, LZD, MXF, PZA, RIF, STM (CAP, KAN)
Clockwork pipeline
https://github.com/iqbal-lab-org/clockwork
Identification of “solo” mutations (i.e. single mutations within a set of genes of
interest that best explain the observed DR phenotype, once neutral mutations have
been excluded)
Data-driven grading based on odd ratio and associated p-value, and PPV 95% CIs
Additional criteria based on literature evidence and expert rules
1200 variants associated with resistance
246 variants associated with no resistance
First WHO catalogue of DR mutations in MTBC
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Proportion of resistant isolates with a category 1 or 2 mutation, or subject to
an expert rule, and of susceptible isolates without a category 1 or 2 mutation
and not subject to an expert rule
Total number of isolates for each drug and the proportion of resistant phenotypes
for which a category 1 or 2 mutation is present or for which an expert rule applies.
Each drug is weighted by the prevalence of phenotypic resistance to that drug in
this dataset
Few R strains
Multiple targets
Few R strains
single target
Categorization of mutation associated to new drugs still
suboptimal
18. NGS-based strategies for detection of known genes associated with resistance
Target sequencing
Whole genome sequencing
• Test for selected target on direct clinical specimen
• High coverage per sample, increased sensitivity
for mutation detection
• High number of samples/run, lower cost per
sample compared to standard Sanger sequencing
• detection of heteroresistance at low frequencies
• detection of the total genomic changes
• High volume of data
• Assembly and analysis challenging
• Expensive for higher coverage data
• No standardized kit available
Cabibbe et al, 2018 51: 1800387; DOI: 10.1183/13993003.00387-2018
20. Adaption and uptake of the lateral flow
urine LAM test in Europe and Central Asia:
current landscape and barriers
Christian Kraef et al 2021
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LAM assays
TAG report 2020
21. • Advantages: early diagnosis, active case finding
• BMGF funded study
Comparison of different kinds of oral swabs and three sampling locations in the
mouth: cheek, tongue and gums. Results compared to traditional methods including
sputum testing as well as clinical diagnosis.
Oral scraping
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Cangelosi et al
JCM 2019
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Absorbent strips in face masks
• Simple and acceptable
• Picks up Covid and TB
• Sensitivity mask < sputum
• Bi-direction screening?
Williams et al, Lancet ID 2020
www.AVELOlife.com
23. • Maximizing the quality of individual patient care
• Surveillance and monitoring of effectiveness of public health action
• Limitations for current test for TB treatment monitoring
• Smear -sub-optimal accuracy
• Culture –long turn-around-time and requires laboratory and logistical
capacity
• Both are sputum based –not easy to collect when people respond to
treatment
24. Use case Goal of the test Potential applications Characteristics Optimum Minimum
Triage To identify individuals who
need a confirmatory test or
those who are unlikely to
have TB
Systematic TB
screening in
community
settings.
Ruling out active
TB before TB
preventive
treatment
Sensitivity > 95% > 90%
Specificity > 80% > 70%
Cost < US$ 1.00/ test < US$ 2.00/ test
Diagnosis To diagnose both
pulmonary and
extrapulmonary TB using
non-sputum samples for the
purpose of initiating
treatment
Diagnosis of TB and
treatment
initiation on the
same day by health
workers with
limited training in
peripheral facilities
Sensitivity for pulmonary
TB in adults
≥ 98% for smear-positive
culture-positive pulmonary
TB
≥ 68% for smear-negative
culture-positive pulmonary
TB
Overall sensitivity should be ≥
65% but should be > 98%
among patients with smear-
positive culture positive
pulmonary
Sensitivity for
extrapulmonary TB in
adults
>=80% No lower range of sensitivity
defined
Sensitivity in children >= 66% No lower range of sensitivity
defined
Specificity >= 98%
Cost < US$ 4.00/ test < US$ 6.00/ test
Prediction for progression
to active TB
To identify people who
develop active TB and thus
benefit from TB preventive
treatment
To identify
individuals with or
without risk factors
who should be
given TB
preventive
treatment
Sensitivity for progression
to active TB in 2 years
>=90% >=75%
Specificity for progression
to active TB in 2 years
>=90% >=75%
Cost < US$ 5.00/ test < US$ 10.0-100/ test
Monitoring treatment
response
To identify people who are
not cured and at risk for
relapse.
Individualize
treatment duration
Evaluate the
efficacy of new
drugs quickly.
No target product profile is available
Test characteristics for new TB diagnostics set by the World
Health Organization
TPP 2020
25. Biomarkers tests for TB and treatment monitoring
• Sweeny 3 genes signature (Lancet RM 2016)
• 50ul finger stick blood
• explored for:
• Screening test for subclinical and clinical TB
• Treatment monitoring
• Risk of progression
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• 6 genes signature finger stick blood
• explored for:
– Screening test for subclinical and clinical TB
– Treatment monitoring
– Risk of progression
– Ability to predict cure at baseline
– Correlate with PET-CT
– Similar performances across regions
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Risk 6
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Track to performance target
for triage and diagnosis
Sweeney3 (Prototype GeneXpert PCR)
Achieved the minimal target when
compared to Xpert MTB/RIF as a
reference standard (Specificity of 85.9% at
near 90%
sensitivity of 65.7% (at 95% specificity)
against a comprehensive microbiological
reference standard
RISK11, RISK6, Roe1, Roe3 and Suliman4
met the optimal target in symptomatic
HIV-uninfected participants, and
Francisco2, Maertzdorf4, Sweeney3, and
Thompson5 met the minimal target for
PLHIV
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• Filmarray pouch assay on Biofire platform
• 30 biomarkers panel (Berry et al Nature 2010)
• Developed as:
• Diagnostic for TB (sensitivity 93%,
specificity 94%)
• Evaluated as predictor of cure
• Results under evaluation
• Can predict relapse free outcome at 1
mo)
Non sputum-based test for TB and treatment monitoring
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28.
29. To all my internal and external collaborators
To all people affected by TB and their families
To all health care staff working for them
To all scientists working for innovation
To all companies investing in this field
To all scientific society engaged
To all I may have forgotten!
To all of you for your attention and…
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TB DAY 2022
INVEST TO END TB SAVES LIVES
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