WHO RECOMMENDED TESTS FOR TB

-DR. PRAPULLA CHANDRA

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.

• 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.

TESTS ENDORSED BY WHO

• MICROSCOPY -light and LED microscopy -same day diagnosisCULTURE 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

WHO RECOMMENDATIONS ON NEW TECHNOLOGIES 2006-2010

Year Policy Recommended

2007 Commercial Liquid Cultures (Automated and Manual) and DSTRapid Speciation –using immuno-chromatographic assays

2008 Line Probe Assays (LPA)

2009 LED based FM and Dual Mode Systems including low load settingsNon-Commercial Methods for Culture & DST (MODS, CRI, NRA etc)

2010 Automated Cartridge Based Nucleic acid technologies – Xpert

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

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.

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

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: homogenizationNaOH: decontaminantNeutralization by phosphate bufferTransportation: 1% cetyl pyridium chloride

Homogenization

Sample mixing

Phosphate buffer

Centrifugation

Pellet

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

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

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)

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, reportexact number

10-99 per 100 oil immersion fields 1+

1-10 per oil immersion field 2+

> 10 per oil immersion field 3+

FLUORESCENT MICROSCOPY

Fluorescent dye (Auramine O and Rhodamine B)

• Good for labs with high workload. • Auramine O- Bright yellow • Rhodamine B- Yellow orange.

Reporting in flouroscent techniqueNo of bacilli per HPF GRADE

<6 per field 1+

6-100 per field 2+

>100 per field 3+

LED FLUORESCENCE MICROSCOPY (light emitting diode fluorescent microscopy)

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

COMPARISON -overall sensitivity is 93% and specificity is 99% -6% more sensitive than Z-N microscopy & 5% more sensitive than conventional flouroscent microscopyRECOMMENDATIONS• 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.

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

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

CULTURE METHODS

Less common media

• Powlowski potato media• Tarshish blood media• Dubos media• Sulas media• Soutons media

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

Colony charecteristics

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

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)

• 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

MB/BacT SYSTEM

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

MGIT +ve tubes MGIT 960 instrument

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

Liquid Culture Systems MGIT 960

STRENGTH WEAKNESSAutomated 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

Results available within 7-14 days

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

MODS – MICROSCOPIC OBSERVATION DRUG SUSCEPTIBILITY• Microscopic colonies (micro-colonies) of M. tuberculosis are

observed in the culture media using an inverted microscopeUses 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

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.

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.,

STRIP SPECIATION

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

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

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

• 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

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

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.

• 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

Two commercial assays availableGenotype 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 /runGT Blot – 48/run

rpoB for rifampicin resistance (InnoLiPA)rpoB for rifampicin, katG and inhA for isoniazid resistance (MTBDRplus)

Commercial Assays

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%

Same technology as Hains MDRplusTarget genes identified are:• FQ – gyrA (oflox, Moxi)• Aminoglycosides and Polypeptides –rrs (Kana, Amik, Vio,

Capreo)• EMB- embB (Ethambutol)

LPA for diagnosis of XDR TB

Use of LPA under the programIntegrated national plan for LPAswith MDR-TB management and lab capacity strengthening

Use of LPAs on smear-positivesputum and from cultures if smear negative (insufficient evidence for direct testing in smear negatives)

LPAs do not replace conventional culture + DST

Commercial assays recommendedLab infrastructure, procedures andbiosafety

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

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

Xpert MTB/RIF assay & GeneXpert instrument

• diagnostic molecular test that uses modern technology:

• Extraction andpurificationDNA/RNA

&

• Amplification

&

• Multiplex detection

(automated)

Xpert MTB/RIF assay & GeneXpert instrument

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

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

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

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

• 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

Drug Susceptibility Testing

7 - 10 days

3 - 4 weeksSolid MediaLöwenstein-Jensen(Middlebrook)

Liquid MediaBACTEC 460 TBMGIT

Molecular based MethodsInnoLipaGenoTypeMTBDRXpert MTB‚home made‘- methods

Hours – 1day

Indirect tests

• ANTIBODY DETECTION• TB stat PAK• ELISA• INSTA TB TEST• ANTIGEN DETECTION• TB MPB 64 PATCH TEST

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.

INTERFERON GAMMA RELEASE ASSAY

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

WHO RECOMMENDATIONS

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

Sensitivity (cfu/ml) of pulmonary TB diagnostics

Solidculture

Phage based tests

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

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

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

• CULTURE BASED TECHNOLOGIES -BNP middlebrook -TREK sensititre MYCOTB mic plateOTHER TECHOLOGIES -TB rapid screen -TBDx

OVERVIEW OF WHO RECOMMENDATIONS

• 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.

• 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.

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.

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

THANK YOU