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: journals.permissions@oxfordjournals.org
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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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