WHO recommended tests of tuberculosis

1. WHO RECOMMENDED
TESTS FOR TB
-DR. PRAPULLA CHANDRA

2. INTRODUCTION
• Tuberculosis (TB) remains one of the world’s deadliest communicable
diseases.
• In 2013, an estimated 9 million people developed TB and 1.5 million
died from the disease.
• Of the estimated 9 million people, india accounts for 24% 0f the
cases.

3. • In 2013, about 64% of the estimated 9 million people who developed
TB were notified as newly diagnosed cases. That is, the remaining 3
million cases were either not diagnosed, or diagnosed but not
reported to national TB programmes (NTPs).
• Early diagnosis of TB and initiating optimal treatment would not only
enable cure of an individual patient but also will curb the
transmission of infection and disease to others in the community.

4. TESTS ENDORSED BY WHO
• MICROSCOPY
-light and LED microscopy
-same day diagnosis
CULTURE BASED TESTS
-commercial liquid culture systems and rapid speciation
-non commercial culture and DST (MODS, NRA, CRI)
MOLECULAR TESTS
-line probe assays
-Xpert MTB/RIF

5. WHO RECOMMENDATIONS ON NEW TECHNOLOGIES
2006-2010
Year Policy Recommended
2007 Commercial Liquid Cultures (Automated and Manual) and DST
Rapid Speciation –using immuno-chromatographic assays
2008 Line Probe Assays (LPA)
2009 LED based FM and Dual Mode Systems including low load settings
Non-Commercial Methods for Culture & DST (MODS, CRI, NRA etc)
2010 Automated Cartridge Based Nucleic acid technologies – Xpert

7. DIRECT METHODS
• Direct microscopy ( ZN, KINYOUN, FLUOROCHROME)
• Culture ( traditional, rapid methods)
• Detection of DNA/RNA of mycobacterial origin
(PCR, LCR, LAMP, NAA, FAST PLAQUE)

8. DIRECT MICROSCOPIC EXAMINATION
• Hallmark of staining is Ziehl-neelsen staining slides
• Easiest & quickest diagnostic test
• Limited sensitivity (46-78%) but specificity is almost 100%
• Centrifugation & fluorochrome staining (auramine O) with UV
microscopy markedly increase the sensitivity & a large no. of slides
can be examined in a much shorter time.

9. CONCENTRATION METHODS FOR SMEARS
• Autoclaving
• 1% sodium hypochlorite
• Sputum treated with 2-4% NAOH
• Urine, c.s.f, pleural fluids centrifused and deposit

10. Decontamination
• Eliminate normal flora from the non-sterile samples
(micobacteria is acid and alkaline resistant)
• Homogenization to release the bacteria from the sample and allow access to the
nutrient present in the media
N-acetyl-cysteine: homogenization
NaOH: decontaminant
Neutralization by phosphate buffer
Transportation: 1% cetyl pyridium chloride
Homogenization
Sample mixing
Phosphate buffer
Centrifugation
Pellet

11. • Sputum Concentration and decontamination methods
are not recommended by WHO as there is no
sufficient evidence.

12. Z-N staining method
Primary stain: Carbol-fuschin solution 0.3%
• Decolourising agent: 3% alcohol, or 20% Sulphuric acid
• Counterstain: Methylene blue 0.3%
• Observe under microscope

14. Microscopy- precautions
• To use sterile containers/collection pots
• Sterile reagents, slides and equipment
• Do not use tap water for staining(saprophytic mycobacteria)
• New slides only(old slides may harbour fungal spores in scratches)

15. Reporting on AFB Microscopy
Number of bacilli seen Result reported
None per 100 oil immersion fields Negative
1-9 per 100 oil immersion fields Scanty, report
exact number
10-99 per 100 oil immersion fields 1+
1-10 per oil immersion field 2+
> 10 per oil immersion field 3+

16. FLUORESCENT MICROSCOPY
Fluorescent dye (Auramine O and Rhodamine B)
• Good for labs with high workload.
• Auramine O- Bright yellow
• Rhodamine B- Yellow orange.

17. Reporting in flouroscent technique
No of bacilli per HPF GRADE
<6 per field 1+
6-100 per field 2+
>100 per field 3+

18. LED FLUORESCENCE MICROSCOPY
(light emitting diode fluorescent microscopy)

20. LED FLUORESCENCE MICROSCOPY
• Increase the sensitivity of smear microscopy and improve the efficiency of
facilities.
• Much larger area of the smear to be seen,
• More rapid examination of the specimen (up to four times faster) and making it
easier to count bacilli.
• ADVANTAGES:
• More sustainable ,
• User-friendly than the quartz-halogen lamps or high-pressure mercury vapour
lamps typically used in FM

21. COMPARISON
-overall sensitivity is 93% and specificity is 99%
-6% more sensitive than Z-N microscopy & 5% more sensitive than
conventional flouroscent microscopy
RECOMMENDATIONS
• conventional fluorescence microscopy be replaced by LED microscopy with
auramine staining in all settings where fluorescence microscopy is currently
used.
• LED microscopy be phased in as an alternative to conventional Ziehl-Nelsen
light microscopy.
• the switch to LED microscopy be made according to a carefully phased
implementation plan.

22. SAME DAY DIAGNOSIS
• Same-day diagnosis ('spot-spot') versus the conventional strategy ('spot-
morning'), with two specimens and direct Ziehl-Neelsen microscopy
-only 2.8% less sensitive and similar specificity
•Same-day diagnosis ('spot-spot morning') versus the conventional strategy
('spot-morning-spot') with three specimens and direct Ziehl-Neelsen
microscopy
-3% more sensitive and similar specificity

23. WHO RECOMMENDATIONS
• countries that have implemented the current WHO policy for two-
specimen case-finding consider switching to same-day diagnosis,
especially in settings where patients are likely to default from the
diagnostic pathway
• countries that are still using the three-specimen case-finding
strategy consider a gradual change to same-day diagnosis, once WHO-
recommended external microscopy quality assurance systems are in
place and good-quality microscopy results have been documented

24. CULTURE METHODS

26. Less common media
• Powlowski potato media
• Tarshish blood media
• Dubos media
• Sulas media
• Soutons media

27. Conditions for better growth
• Temp b’n 25-40c. Ideally 37c
• 5-10% co2 enhances the growth
• Ph 6.4-7
• Addition of low percentage of glycerol to the medium enhances the
growth of human strains
• Minimum time for growth to appear 2 weeks and can take upto 6-8
weeks

28. Colony charecteristics
• M.TB: these are raised rough ,discrete, dry, wrinkled , creamy white
• M.bovis: smooth , moist, white, break up more readily when touched

30. Rapid culture techniques
• BACTEC system
• MB/BAC T system
• Mycobact growth indicator tube (MGIT)
• Septi chek AFB method
• ESP culture system
• Microscopic observation drug sensitivity(MODS)

31. • BACTEC
-Radiometric method
-growth is ascertained by liberation of 14CO2 as metabolised by
mycobacteria
-average time: 8days
-can also be used for drug susceptibility testing
DRAWBACKS:
-high cost
-disposal of radiactive waste

33. MB/BacT SYSTEM
• Non radiometric continuous monitoring system
• Based on calorimetric detection of co2
• Slightly longer time than BACTEC (14 days)
• Prone to contamination

36. MGIT +ve tubes MGIT 960 instrument

37. MGIT 320 System and Manual Reader
• Holds 320 tubes for an annual capacity of
approx 2700 specimens/year
• Optimal use of valuable lab space
• Flexible configuration as bench top or stand-
mounted
• Manual UV Reader as back up for MGIT
960/320
• few specimens (in low load labs)
• Power problems
• MGIT tubes to be incubated in regular
incubators

38. Liquid Culture Systems
MGIT 960
STRENGTH WEAKNESS
Automated system – growth &
detection (except Blood)
Capacity to incubate & monitor 960 tubes
every 60 minutes for
Increase in fluorescence
Can handle 8000 cultures/year
System alerts when tubes become +ve
No radioactive material used
BSL III/negative
Pressure Environment
Continuous Electricity
Reagent costs and AMC
High Contamination
rates
Training in handling
Liquid culture
Speciation if+ve*
Cold chain transport for
sputUM

40. MODS – MICROSCOPIC OBSERVATION
DRUG SUSCEPTIBILITY
• Microscopic colonies (micro-colonies) of M. tuberculosis are
observed in the culture media using an inverted microscope
Uses tissue culture plate
- the wells coated with different drugs in different concentration are used
- presence of growth with INH /RIF /SM/EMB can be detected.
• Time taken 7 to 14 days

41. MODS – MICROSCOPIC OBSERVATION DRUG SUSCEPTIBILITY
• difficulty distinguishing between the micro-colonies of TB and some
nontuberculous mycobacteria (NTM).
• requires experienced personnel.
• , biosafety of laboratory workers must be taken into consideration.

42. STRIP SPECIATION
• Detect a TB-specific antigen (MPB64) from positive liquid or solid cultures to confirm the presence of
organisms belonging to M.tuberculosis complex.
• Speciation is necessary to differentiate M.tuberculosis complex and other mycobacteria grown in
cultures.
• ADVANTAGES:
• Results in 15 min
• Sensitivity & specificity of 98.6% and 97.9%
• no additional equipment or consumables are needed to perform the test and can
detect TB even when mixed with nontuberculous mycobacteria.,

43. STRIP SPECIATION

44. Rapid Speciation of Positive Cultures
• Immunochromatographic tests - Rapid strip test that
detects a TB-specific antigen (MPB 64) from culture
• Bacterial growth derived from solid or liquid culture can be used
• Outputs:
• Control band
• Test band:
• Present = M.tb complex
• Absent = not M.tb complex
• Increased yield of NTMs with liquid cultures necessitates prompt identification
of mycobacterial growth as TB vs NTM

45. COLORIMETRIC REDOX INDICATORS
• Based on the reduction of an indicator solution added to a liquid culture medium after TB organisms
have been exposed to different antibiotics.
• Isoniazid and rifampicin resistance is detected by a change in colour of the indicator.
• ADVANTAGES:
• Highly sensitive (about 95%) and specific for the detection of MDR-TB
• Faster than conventional solid or liquid culture DST methods
• Results between 7 and 14 days after culturing.
• do not require sophisticated equipment

46. LIQUID CULTURE SYSTEM
• liquid culture medium, enriched with oxygen
• As bacteria grows in the culture, the oxygen is utilized, causing it to be fluorescent when placed under
UV light.
• ADVANTAGES:
• Faster
• High sensitivity &specificity nearly 100%
• Diagnosis in 7-14 days
• Both automated and manual systems perform well in detection of isoniazid and rifampicin
susceptibility.
• Not as effective for ethambutol and streptomycin

47. • Measures nitrate reduction to indicate resistance to isoniazid and rifampicin.
• Based on the property of TB to reduce nitrate to nitrite& color change of the culture media.
ADVANTAGES
NRA (NITRATE REDUCTION ASSAY)less expensive to than liquid culture techniques for DST
• Results are earlier than by eye examination of colonies in solid culture
• the specificity and sensitivity of NRA were comparable to traditional solid culture
methods for DST of isoniazid and rifampicin
• . NRA does not need sophisticated equipment, is not complex to perform
• Disadvantages
• The culture is killed by the mix reagent used to develop the assay, requiring that multiple
cultures be prepared if comparative testing will be performed. Only fresh cultures must
be used (<14 days).
NITRATE REDUCTION ASSAY

48. Detection of mycobacteria directly from
clinical samples
• Genotypic methods {NAA}
• PCR (polymerase chain reaction)
• LAMP (loop mediated isothermal amplification)
• TMA (transcription mediated amplification)
• Ligase chain reaction
• AMPLICOR assay
• MTD test (mycobacterium tuberculosis direct test)
• BD probe Tec MTB test
• Phenotypic methods
• FAST plaque TB method

52. LIMITATIONS OF NAA
• No drug susceptibility information
• Detects nucleic acid from both dead and living organisms
• May be falsely positive in persons having recent infection and
undergone treatment.

53. • Line-probe assays are designed to identify M. tuberculosis
complex and simultaneously detect mutations associated
with drug resistance
• They are a family of novel DNA strip-based tests that use
PCR and reverse hybridization methods for the rapid
detection of mutations associated with drug resistance
• Line-probe assay has high sensitivity and specificity when
culture isolates are used.
• The majority of studies had sensitivity of 95% or greater, and
nearly all were 100% specific
Line Probe Assays

54. Two commercial assays available
Genotype MTBDRplus (Hain LPA), INNO-LipA Rif.TB
Validated for use directly from smear-positive sputum
(MTBDRplus) or from TB cultures
Manual and automated systems
Twincubator – 10 /run
GT Blot – 48/run
rpoB for rifampicin resistance (InnoLiPA)
rpoB for rifampicin, katG and inhA for isoniazid resistance
(MTBDRplus)
Commercial Assays

55. Results of Hain MTBDRplus
•97% of smear-positive specimens gave results within
1–2 days (24-48 hrs)
•Good sensitivity and specificity
-Rifampin: sensitivity: 98%; specificity: 99%
-Isoniazid: sensitivity: 89%, specificity: 99%

56. Same technology as Hains MDRplus
Target genes identified are:
• FQ – gyrA (oflox, Moxi)
• Aminoglycosides and Polypeptides –rrs (Kana, Amik, Vio,
Capreo)
• EMB- embB (Ethambutol)
LPA for diagnosis of XDR TB

57. Use of LPA under the program
Integrated national plan for LPAs
with MDR-TB management and lab
capacity strengthening
Use of LPAs on smear-positive
sputum and from cultures if smear
negative (insufficient evidence for
direct testing in smear negatives)
LPAs do not replace conventional
culture + DST
Commercial assays recommended
Lab infrastructure, procedures and
biosafety

58. GENEXPERT® MTB/RIF
1. The new, rapid and fully automated Xpert®
2. MTB/RIF test is cartridge-based automated DNA amplification test
3. Highly sensitive for confirmation of both smear positive and smear negative
samples.
4. The Xpert® MTB/RIF assay uses 3 specific primers and 5 unique molecular probes
to ensure a high degree of specificity
5. Assay targets the rpoB gene, which is critical for identifying mutations associated
with rifampicin resistance

59. GENEXPERT® MTB/RIF
1. Advantages:
• highly accurate results in less than 2 hours.
• Simultaneous detection of both MTB and rifampicin resistance,
• up to 95% of rifampicin resistance strains are INH resistance

60. Xpert MTB/RIF assay & GeneXpert instrument
• diagnostic molecular test that uses modern technology:
• Extraction and
purification
DNA/RNA
&
• Amplification
&
• Multiplex
detection
(automated)

61. Xpert MTB/RIF assay & GeneXpert instrument

62. Xpert MTB/RIF assay & GeneXpert instrument
• Closed system – no contamination risk
• Controls – Positive control included in test kit
• Reagents – All reagents in self-contains kit, kit contains a pipette to transfer liquid sputum from
container to cartridge
• Storage/stability (including reagents) – Maximum shelf-life of 14 months

63. Xpert MTB/RIF assay & GeneXpert instrument
• Reagents stable at temperature 2-280
• Instrumentations – Requires annual maintenance
• Power requirement – Low power requirement compared with other PCR
system
• Training – Approximately 1 day of training required

64. Sensitivity for AFB+/culture+ 98.2%
• Sensitivity for AFB-/culture+ 72.5%
• Specificity 99.2%
Rifampicin resistance detection
Sensitivity – 98%
• Specificity – 99%
Xpert MTB/RIF assay & GeneXpert instrument
Sensitivity and specificity

65. WHO RECOMMENDATIONS ON
XPERT MTB/RIF
• SHOULD BE USED:
-as the initial diagnostic test in adults and children presumed to have
MDR-TB or HIV-associated TB
-as the initial diagnostic test in testing cerebrospinal fluid specimens
from patients presumed to have TB meningitis

66. • MAY BE USED:
-as the initial diagnostic test in adults and children presumed to have
TB
-as a follow-on test to microscopy in adults presumed to have TB but
not at risk of MDR-TB or HIV-associated TB, especially in further testing
of smear-negative specimens
-as a replacement test for usual practice (including conventional
microscopy, culture, and/or histopathology)
-for testing of specific non-respiratory specimens (lymph nodes and
other tissues) from patients presumed to have extrapulmonary TB

67. Drug Susceptibility Testing
7 - 10 days
3 - 4 weeksSolid Media
Löwenstein-Jensen
(Middlebrook)
Liquid Media
BACTEC 460 TB
MGIT
Molecular based
Methods
InnoLipa
GenoTypeMTBDR
Xpert MTB
‚home made‘- methods
Hours – 1day

69. Indirect tests
• ANTIBODY DETECTION
• TB stat PAK
• ELISA
• INSTA TB TEST
• ANTIGEN DETECTION
• TB MPB 64 PATCH TEST

70. INTERFERON GAMMA RELEASE ASSAY
• much less likely than the tuberculin skin tests (TST) to be confounded by exposure to
environmental mycobacteria or by prior BCG vaccination. Does not boost responses
• do not require a second clinical contact to evaluate the test result,
• better sensitivity for HIV-infected people and patients with extrapulmonary TB
• Disadvantages
• lower sensitivity than the TST for past infection. Assays do not discriminate between active and
latent TB infection.
• moderately complex and requires standard ELISA equipment
• . Blood samples have to be incubated within 16 hours of being collected, which may require the
use of portable incubators
• Requires that blood be drawn from the patient with a needle, which can lead to other infections.
• application of such tests in disease-endemic, developing countries is the subject of extensive
investigation.

71. INTERFERON GAMMA RELEASE ASSAY

77. There is insufficient data and low quality evidence on the
performance of IGRAs in low- and middle-income countries,
typically those with a high TB and/or HIV burden
• IGRAs and the tuberculin skin test (TST) cannot accurately
predict the risk of infected individuals developing active TB
disease
• Neither IGRAs nor the TST should be used for the diagnosis
of active TB disease

79. WHO RECOMMENDATIONS
RECOMMENDED NOT TO USE :
• Commercial TB serodiagnostic tests.
• Interferon-gamma release assays for detection of active TB (all
settings).

80. Sensitivity (cfu/ml) of pulmonary TB diagnostics
Solid
culture

81. Phage based tests

86. • Can also be used on urine samples
• Results available in 1 day
• As it is cheap can be used in resource poor countries

88. TESTS BEING REVIEWED BY WHO IN 2015
• TB LAMP (loop mediated isothermal amplication)
• URINARY LAM (lipoarabinomannan)
• MOLECULAR DST METHODS- hains MTBDR sl tests

89. TECHNOLOGIES IN DEVELOPMENT
• VOLATILE ORGANIC COMPOUNDS
-breathlink
-prototype breath analyser device
MOLECULAR TECHNOLOGIES
-alere Q
-B SMART
-genedrive MTB/RIF ID
-LATE PCR
-genepert XDR catridge
-trueArray MDRTB
-INFITINIMTB assay
-fluorotype MTB/ fluorotype MTB RNA

90. • CULTURE BASED TECHNOLOGIES
-BNP middlebrook
-TREK sensititre MYCOTB mic plate
OTHER TECHOLOGIES
-TB rapid screen
-TBDx

91. OVERVIEW OF WHO
RECOMMENDATIONS

92. • LED microscopy: For use at all laboratory levels as replacement of
conventional fluorochrome and light microscopy.
• Commercial liquid culture and DST systems: For use at
central/regional reference laboratory level, as current reference
standard.
• Rapid speciation strip technology: For use with conventional
culture and DST at central/regional reference laboratory level, to
identify Mycobacterium tuberculosis.
• Commercial molecular line probe assays for 1st-line anti-TB
drugs: For use at central/regional reference laboratory level for
rapid detection of rifampicin (alone or with isoniazid) resistance.
Suitable for use on smearpositive specimens or culture isolates.

93. • Selected non-commercial DST methods: MODS(microscopic
observation of drug susceptibility), NRA(nitrate reductase assay),
CRI(calorimetric redox indicator): For conditional use at
central/reference laboratory level for detection of rifampicin resistance
only. MODS and NRA suitable for use on smear-positive specimens or
culture isolates, CRI suitable for use on culture isolates only.
• Automated real-time nucleic acid amplification - Xpert MTB/RIF
system: For rapid detection of pulmonary and extrapulmonary TB and
rifampicin resistance in both adults and children at decentralised
laboratory and health care centres.

94. NOT RECOMMENDED DUE TO CURRENT INSUFFICIENT
EVIDENCE
• Sputum concentration and decontamination methods.
• Phage-plaque technology for rapid rifampicin resistance.
• Thin-layer agar methods for rapid culture and DST.
• Interferon-gamma release assays as replacement for the tuberculin skin
test for detection of latent TB in low- and middle-income (typically high TB
and/or HIV) settings.
• Molecular line probe assays for 2nd-line anti-TB drugs.
• Loop-mediated isothermal amplification test kit for TB.

95. RECOMMENDED NOT TO USE :
• Commercial TB serodiagnostic tests.
• Interferon-gamma release assays for detection of active TB (all
settings).

96. THANK YOU