Microsoft PowerPoint - 20110319.pptxprogress progress
Preface
After a period of global acceleration in 2001–05, the case
detection rate worldwide decelerated in 2006 and 2007, reaching 63%
in 2007. Thus, the target of a case detection rate of at least 70%
bya case detection rate of at least 70% by 2005 has not yet been
achieved, and is unlikely to be met until 2014.
WHY
Even in 2010, national tuberculosis programmes in disease endemic
countries continue to rely largely on antiquated and inaccurate
methods such as direct smear microscopy solid cultureas direct
smear microscopy, solid culture, chest radiography, and tuberculin
skin testing.
WHY
There is no rapid, point-of-care test that allows early detection
of active tuberculosis at health clinics. Diagnosis of
smear-negative tuberculosis i d lt i f t d ith HIV d i hildin
adults infected with HIV and in children continues to pose
substantial clinical challenges.
Barriers to development of new tuberculosis diagnostics
Market failure has been an important factor hindering the
development of new diagnostics for tuberculosis health systems in
developing countries are
ll k ki th bl tgenerally weak, making them unable to take advantage
of tuberculosis diagnostics to achieve best possible performance,
and to introduce new advances in diagnostic technologies
The diagnostics pipeline and new WHO policies
Bill & Melinda Gates Foundation Foundation for Innovative New
Diagnostics (FIND) – develop and deliver a pipeline of tests that
are
appropriate for disease endemic countries.appropriate for disease
endemic countries. Global Laboratory Initiative—one of the Working
Groups of the Stop TB Partnership—plans are underway for a large
scale-up of laboratory services for tuberculosis.
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WHO policies and WHO policies and statements onstatements on
tuberculosistuberculosistuberculosis tuberculosis
diagnosticsdiagnostics
Liquid media for culture and DST (introduced in 2007)
• The use of liquid medium for culture and DST in middle-income and
low-income countries. • Rapid species identification to address the
needs
for culture and DST, taking into consideration thatculture and DST,
taking into consideration that implementation of liquid systems
will be phased, will
be integrated into a country-specific comprehensive
plan for laboratory-capacity strengthening, and will address
several issues including biosafety and training.
Rapid Speciation (2007)
Definition of a new sputum-smear-positive tuberculosis case
(introduced in 2007)
The revised definition of a new sputum- smear-positive case of
pulmonary tuberculosis is based on the presence of at least one
acid fast bacilli in at least one sputum sample in countries with a
wellsputum sample in countries with a well functioning external
quality-assurance system
Reduction of number of smears for diagnosis of pulmonary
tuberculosis (introduced in 2007)
WHO recommends the number of specimens to be examined for screening
of tuberculosis cases can be reduced from three to two, in places
where a well functioning external quality assurancefunctioning
external quality assurance system exists, where the workload is
very high, and human resources are scarce.
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Molecular line-probe assays for rapid screening of patients at risk
of MDR
tuberculosis (introduced in 2008)
• Adoption of line-probe assays for rapid detection of MDRdetection
of MDR quality-assured secondline antituberculosis drugs. Direct
use of line-probe assays on smear- negative clinical specimens is
not recommended.
Molecular line-probe assays for rapid screening of patients at risk
of MDR
tuberculosis (introduced in 2008) The use of commercial line-probe
assays, rather than inhouse assays, is recommended to ensure
reliability and reproducibility of results. Adoption of line probe
assays does notAdoption of line-probe assays does not eliminate the
need for conventional culture and DST capability; culture remains
necessary for definitive diagnosis of tuberculosis in
smear-negative patients, whereas conventional DST is needed to
diagnose XDR tuberculosis
LED-based microscopy (introduced in 2009–10)
conventional fluorescence microscopy be replaced by LED microscopy
using auramine staining in all settings where fluorescence
microscopy is currently used, LED i b h d iLED microscopy be phased
in as an alternative for conventional Ziehl-Neelsen light
microscopy in both high-volume and low-volume laboratories.
Fluorescence VS LED microscopy
inexpensive, robust, consume little electricity, are highly
sensitive, and need less technician time than does Ziehl- Neelsen
microscopy.
Non-commercial culture DST methods (introduced in 2009–10)
selected non-commercial culture and DST methods be used as an
interim solution in resource constrained settings, in reference
laboratories, or in those with sufficient culture capacity while
capacity forculture capacity, while capacity for genotypic and/or
automated liquid culture and DST are being developed.
Non-commercial culture DST methods (introduced in 2009–10)
Microscopically observed drug susceptibility (MODS)as direct or
indirect tests, for rapid screening of patients suspected of having
MDR tuberculosis.
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Non-commercial culture DST methods (introduced in 2009–10)
Nitrate reductase assay (NRA), as direct or indirect tests, for
screening of patients suspected of having MDR tuberculosis time to
detection of MDR tuberculosis in i di t li ti ld t b f tindirect
application would not be faster than conventional DST methods using
solid culture.
Non-commercial culture DST methods (introduced in 2009–10)
Colorimetric redox indicator methods (CRI), as indirect tests on
Mycobacterium tuberculosis isolates from patients suspected of
having MDR tuberculosis ti t d t ti f MDR t b l itime to detection
of MDR tuberculosis would not be faster (but would be less
expensive) than conventional DST methods using commercial liquid
culture or molecular line-probe assays.
Ninety-six-well microtitre plate for the susceptibility of M.
Martin A et al. J. Antimicrob. Chemother. 2006;59:175-183
© The Author 2006. Published by Oxford University Press on behalf
of the British Society for Antimicrobial Chemotherapy. All rights
reserved. For Permissions, please e-mail:
[email protected]
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Same-day diagnosis by microscopy (introduced in 2009–10):
WHO recommends that countries that have successfully implemented
the current WHO policy for a two-specimen case- finding strategy
consider a switch to the same day diagnosis approach
especiallysame-day diagnosis approach, especially in settings where
patients are likely to default from the diagnostic process.
Same-day diagnosis by microscopy (introduced in 2009–10):
Countries that are still using the three- specimen case finding
strategy consider a gradual change to the same-day-diagnosis
approach, once WHO-recommended external microscopy quality
assuranceexternal microscopy quality-assurance systems are in place
and good quality microscopy results have been documented.
Same-day diagnosis by microscopy (introduced in 2009–10):
Same-day-diagnosis ('spot-spot') vs the conventional strategy
('spot-morning'), using two specimens and direct ZN microscopy –
Same-day-diagnosis was on average 2.8% less
sensitive than the conventional approach (95CI -pp ( 5.2% -
+0.3)
– Patients assigned to the same-day-diagnosis scheme were more
likely to submit both specimens (drop-out 2%) than patients
screened with the conventional scheme (drop-out 5.8%).
Same-day diagnosis by microscopy (introduced in 2009–10):
Same-day diagnosis ('spot-spot'morning' vs the conventional
strategy ('spotmorning- spot') using three specimens and direct ZN
microscopy
Th ' t t i ' t t h d 3%– The 'spot-spot-morning' strategy showed 3%
higher sensitivity (71%; 95CI 65% - 77%) than the
'spot-morning-spot' approach (68%; 95CI 83% - 73%), although this
difference was not statistically significant
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Summary of findings from systematic reviews on tuberculosis
diagnostic test
Diagnosis of active tuberculosis Diagnosis of latent tuberculosis
Diagnosis of drug-resistant tuberculosis
Diagnosis of active tuberculosis Sputum-smear microscopy for
pulmonary tuberculosis NAATs for pulmonary and extrapulmonary
tuberculosis Serological antibody detection tests for pulmonary and
extrapulmonary tuberculosis ADA for tuberculosis pleuritis,
pericarditis, peritonitis Interferon γ for tuberculosis pleuritis
Phage amplification assays for pulmonary tuberculosis Automated
liquid cultures for pulmonary tuberculosis
Sputum-smear microscopy for pulmonary tuberculosis
• FM is on average 10% more sensitive than is conventional
microscopy. Specificity of both FM and conventional microscopy is
similar. FM is associated with improved time efficiencytime
efficiency.
• LED FM performs equivalently to conventional FM, with added
benefits of low cost, durability, and ability to use without a
darkroom.
AFB
• BLEACH
– Sputum processing methods to improve the sensitivity f i f t b l
i t tiof smear microscopy for tuberculosis: a systematic
review --Lancet Infect Dis. 2006 Oct;6(10):664-74. • Fluorescence
versus conventional sputum
smear microscopy for tuberculosis: a systematic review--Lancet
Infect Dis. 2006 Sep;6(9):570-81.
• LED Use of light-emitting diode fluorescence microscopy to detect
acid-fast bacilli in sputum. --Clin Infect Dis. 2008 Jul
15;47(2):203-7.
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Auramine O has two absorbance peaks: one at 432 nm and one at 370
nm. High power ‘Royal Blue’ LEDs have recently become available,
with peak emission in the 440–460 nm range and a spectral half
width of 25 nm.
Characteristics:
Fluorescence microscopy available at a small fraction of the usual
cost
Fits any microscope with DIN objectives
Emits cool, super blue-violet lightEmits cool, super blue violet
light
Bulb never needs to be replaced
Easy to install on existing microscopes
Objectives - 40x dry, 60x oil, or both
Power supply input from 100 to 240 VAC
Optional 12 V battery (solar rechargeable)
Optional solar panel for recharging battery
NAATs for pulmonary and extrapulmonary tuberculosis
NAATs have high specificity and positive predictive value. NAATs,
however, have relatively lower (and highly variable) sensitivity
and negative predictive value for all forms of
tuberculosispredictive value for all forms of tuberculosis,
especially in smear-negative and extrapulmonary disease. In-house
(so-called home brew) NAATs produce highly inconsistent results
compared with commercial, standardised NAATs.
(1)NAA (>95%)(2) 50%–80% NAA NAA
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Serological antibody detection tests for pulmonary and
extrapulmonary tuberculosis
Commercial serological tests for both pulmonary and extrapulmonary
tuberculosis produce highly inconsistent estimates of sensitivity
and specificity; none of the assays do well enough tonone of the
assays do well enough to replace microscopy.
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ADA for tuberculosis pleuritis, pericarditis, peritonitis
Measurement of ADA concentrations in pleural, pericardial, and
ascitic fluid has high sensitivity and specificity for
extrapulmonary tuberculosis
Interferon γ for tuberculosis pleuritis
Pleural fluid interferon-γ determination is a sensitive and
specific test for the diagnosis of tuberculosis pleuritis
Phage amplification assays for pulmonary tuberculosis
Phage-based assays have high specificity but lower and variable
sensitivity. Current commercial phage-based assays are limited by
high rates of indeterminate resultsresults.
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Automated liquid cultures for pulmonary tuberculosis
Automated liquid cultures are more sensitive than are solid
cultures; time to detection is more rapid than for solid
cultures.
RAPID ID
Capilia TB assay Medipro BD MGIT™ TBc Identification Test
Result By detecting MPB64 with specific monoclonal antibody Capilia
TB assay (TAUNS, Numazu, Japan), an immunochromatographic assay
(ICA), has been demonstrated as an easy and rapid diagnostic tool
for culture confirmation of M. tuberculosis in liquid q medium. The
results are readable within 15min . The overall sensitivity and
specificity of the Capilia TB assay were 98.6% and 97.9%,
respectively, The positive and negative predictive values for the
Capilia TB assay were 98.6% and 97.9%, respectively,
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MeDiPro TB antigen rapid test
enzyme-linked immunosorbent as say (ELISA) using anti-ESAT-6 and
CFP-10 antibodies (MeDiPro Mycobacterium tuberculosis Antigen
ELISA, Formosa Biomedical Corporation, Taipei, Taiwan) was
developed for the detection of M. tuberculosis in positive signals
of Mycobacterium Growth Indicator Tubes (MGIT) in the BACTECGrowth
Indicator Tubes (MGIT) in the BACTEC MGIT 960 system. The RD1
segment of M. tuberculosis is not shared with bacille
Calmette-Guerin (BCG) substrains and most environmental
mycobacteria (with the exception of M. kansasii, M. szulgai, M. fl
avescens and M. marinum
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TST for latent tuberculosis infection T-cell-based IGRAs for latent
tuberculosis infection
TST for latent tuberculosis infection
Individuals who had received BCG vaccination are more likely to
have a positive TST; the effect of BCG on TST results is less
ft 15 iti TST ithafter 15 years; positive TST with indurations of
>15 mm are more likely to be the result of tuberculosis
infection than of BCG vaccination.
TST for latent tuberculosis infection
The effect on TST of BCG received in infancy is small, especially
10 years after vaccination. BCG received after infancy produces
more frequent, more persistent, and larger TST reactions. g NTM
infection is not a clinically important cause of false-positive
TST, apart from in populations with a high prevalence of NTM
sensitisation and a very low prevalence of tuberculosis
infection.
T-cell-based IGRAs for latent tuberculosis infection
IGRAs have excellent specificity (higher than the TST), and are
unaffected by previous BCG vaccination. IGRAs cannot distinguish
between latent tuberculosis infection and active tuberculosis, and
have no role for active tuberculosisand have no role for active
tuberculosis diagnosis in adults. IGRAs correlate well with markers
of tuberculosis exposure in low-incidence countries. IGRA
sensitivity varies across populations and tends to be lower in
high-endemic countries and in HIV-infected individuals
Diagnosis of drug-resistant tuberculosis
Phage amplification assays for rapid detection of rifampicin
resistance Line-probe assays: INNO-LiPA Rif and GenoType MTBDR
assays for rapid detection of rifampicin resistance CRI methods and
NRA for rapid detection ofCRI methods and NRA for rapid detection
of rifampicin and isoniazid resistance MODS for rapid detection of
rifampicin and isoniazid resistance TLA for rapid detection of
rifampicin and isoniazid resistance
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Phage amplification assays for rapid detection of rifampicin
resistance
Commercial phage amplification assays produce variable results when
used directly on sputum specimens. Studies have raised concerns
about
t i ti f l iti lt dcontamination, false positive results, and
technical assay failures.
Line-probe assays: INNO-LiPA Rif and GenoType MTBDR assays for
rapid detection
of rifampicin resistance The INNO-LiPA Rif assay is a highly
sensitive and specific test for the detection of rifampicin
resistance in culture isolates. The test has lower sensitivity when
used directly on clinical specimens. GenoType MTBDR assays have
excellent sensitivity and specificity for rifampicin resistance,
even when directly used on clinical specimens.
Sample
6 hours
Rapid ID/DST
6 hours
Rapid ID
• Amplify specific gene fragment
• High sensitivity to distinguish the difference on only one
nucleotide
Manual TwinCubator® - 12 samples
Genotype® MTBDRplus
Rifampincin -- rpoB
Rifampincin Resistance Determining Region (RRDR)
Isoniazid – katG, InhA katG : detect the mutations in coden 315
inhA : detect the alterations in the promoter region (-8, -15,
-16)
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• PCR (2.5-3hrs)
• Evaluation
•
2009,03,03GenoType® MTBDRsl
rrs gene
170 GenoType® MTBDRslFluoroquinolones90.24% GenoType®
MTBDRslamikacin/ capreomycin83.33%/86.79% GenoType®
MTBDRslEthambutol58.97%
CRI methods and NRA for rapid detection of rifampicin and isoniazid
resistance
Colorimetric methods are sensitive and specific for the detection
of rifampicin and isoniazid resistance in culture isolates. CRIs
use inexpensive non- commercial supplies and equipment and have a
rapid turnaround time (7 days). NRA h hi h h d t d t tNRA has high
accuracy when used to detect rifampicin and isoniazid resistance in
culture isolates. – NRA is simple, uses inexpensive
non-commercial
supplies and equipment, and has a rapid turnaround time (7–14 days)
compared with conventional methods
CRI=colorimetric redox indicator. NRA=nitrate reductase
assays
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MODS for rapid detection of rifampicin and isoniazid
resistance
MODS has high accuracy when testing for rifampicin resistance, but
shows slightly lower sensitivity when detecting isoniazid
resistance. MODS seems to do equally well with use of direct
patient specimens and culture isolates.direct patient specimens and
culture isolates. MODS uses non-commercial supplies and equipment,
and has a rapid turnaround time (10 days) compared with
conventional methods. MODS=microscopically observed drug
susceptibility
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TLA for rapid detection of rifampicin and isoniazid
resistance
Data assessing TLA for the detection of drug susceptibility are
scarce; however, all studies so far have reported 100% concordance
with their reference standards. TLA uses inexpensive non-commercial
supplies and equipment, and has a rapid turnaround time (11 days)
compared with conventional methods. TLA=thin-layer agar.
Optimism for the future Xpert MTB/RIF--marked in 2009 – which was
co-developed by the Foundation for Innovative
New Diagnostics, Cepheid (Sunnyvale, CA, USA), and the University
of Medicine and Dentistry of New Jersey, NJ, USA.
– safety, LOW contamination, ease of use, etc, can be done by staff
with little training and can be used for case detection or MDR
screening– and can be used for case detection or MDR
screening
– On-demand , near-patient technology excellent performance
– both smear-positive and smear-negative patients – high accuracy
for determination of rifampicin resistance – detect M tuberculosis
directly from sputum in less than 2 h.
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Containment of Bioaerosol Infection Risk by the Xpert MTB/RIF Assay
and Its
Applicability to Point-of-Care Settings compared the bioaerosols
generated by the Xpert assay to acid-fast bacillus (AFB) microscope
slide smear preparation During the preparation of AFB smears,
sputum samples spiked with Mycobacterium bovis BCG atsamples spiked
with Mycobacterium bovis BCG at 5 x 108 CFU/ml produced 16 and 325
CFU/m3 air measured with an Andersen impactor or BioSampler,
respectively. In contrast, neither the sample preparation steps for
the Xpert assay nor its automated processing produced any
culturable bioaerosols.
Journal of Clinical Microbiology, October 2010, p. 3551-3557, Vol.
48, No. 10
Sensitivity and specificity of the Xpert assay with the culture
method as reference standard
Specimen type Sensitivity Specificity
Gastric fluid 87.5% 100% Pleural fluid Not calculable 98.1%
t l 100% 91 7%stool 100% 91.7% Urine 100% 98.6% Total 77.3%
98.2%
Optimism for the future
Several options are being explored for simpler, less expensive
point-of-care and multiplexed assay formats in the future,
including – manual molecular testing that can be done in
i h l ttiperipheral settings, – lab-on-chip approaches that can be
used to
detect several infections simultaneously, – antigen detection on
highly sensitive platforms, – And antibody detection with panels of
recently
identified antigens of diagnostic value.
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Optimism for the future Owing to growing interest and funding for
new methods and biomarkers, several agencies, industries, and
groups are working on developing point-of-care platforms for
tuberculosis, including – novel serological assays, g y , –
detection of volatile organic compounds in breath, – handheld
molecular devices, – microchip technologies, and – tests that
exploit approaches such as
microfluidics, nanotechnology, proteomics, and metabolomics
Conclusions
The need for a more accurate, inexpensive point-of-care
tuberculosis diagnostic test that is applicable in tuberculosis and
HIV endemic areas is greater nowadays than ever before andgreater
nowadays than ever before, and will be crucial for achieving global
tuberculosis control
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