Laboratory Diagnosis of Tuberculosis(TB)MODERATOR DR. ASHOK PANCHONIASpeaker Dr. Gaurav Shelgaonkar

Earlier known as Consumption (severe wt loss)/ phthisis pulmonaris and the white plaque( extreme pallor in infected) .Potts disease (Egyptian mummies spinal tb) by archaeologist.*

Mycobacterium tubercule bacilli demonstrated by Robert Koch(coke) in 1882. (Kochs bacilli)Nobel prize 1905.Every third person is infected.*

Mycobacteria TuberculosisNon sporing, Non-capsulated. Weakly Gram Positive.Strongly Acid Fast (due to mycolic acid in cell wall).Lipid Rich Cell Wall (confers resistance to disinfectants, detergents, common antibiotics and traditional stains).Long Generation time.

Capable to grow intracellularly in unactivated alveolar macrophages.Primarily disease caused from host response to infection.

Humans are the only natural reservoir.Person to person transmission, by infectious aerosols/droplets.

Generation timeis simply the time it takes for one cell to become two.M. Tb is slow growing, a nonchromogen, doesnt grow at 25c or in PNB medium.*

Lin P et al. J Immunol 2010ClearancePulmonary TBLow grade TBpercolatingDormant infectionSeptic TBMiliary TBExtrapulmonary TBLTBIactive TBSpectrum of M. tuberculosis infection

*Follows iceberg phemomenon.. As only tip of iceberg is visible.. Part below the iceberg indicates a lot of dormant infection in individuals, requiring urgent need of screening clinical findings and investigations for finding the dormant infections

Magnitude of the Problem Source: WHO Geneva; WHO Report 2008: Global Tuberculosis Control; Surveillance, Planning and FinancingGlobal annual incidence = 9.1 millionIndia annual incidence = 1.9 millionIndia is 17th among 22 High Burden Countries (in terms of TB incidence rate)

Chart1

1933IndiaIndiaIndiaIndia

1311ChinaChinaChinaChina

534IndonesiaIndonesiaIndonesiaIndonesia

450NigeriaNigeriaNigeriaNigeria

454South AfricaSouth AfricaSouth AfricaSouth Africa

351BangladeshBangladeshBangladeshBangladesh

292PakistanPakistanPakistanPakistan

306EthiopiaEthiopiaEthiopiaEthiopia

248PhilippinesPhilippinesPhilippinesPhilippines

1455Other 13 HBCsOther 13 HBCsOther 13 HBCsOther 13 HBCs

1823Non-HBCsNon-HBCsNon-HBCsNon-HBCs

Et

India20%

Sheet1

IndiaChinaIndonesiaNigeriaSouth AfricaBangladeshPakistanEthiopiaPhilippinesOther 13 HBCsNon-HBCs

1933131153445045435129230624814551823

Et

*In 2008 WHO report-9.4 million new cases equivalents to 139 cases per 100,000 population of TB globally. 1.98 million were estimated to have occurred in India (0.87 million---infectious cases, fifth of the global burden of TB. About 40% of Indian population is infected with TB bacillus.

Evolution of TB Control in India1950s-60s Important TB research at TRC and NTI1962 National TB Control Program (NTCP)1992 Program Review only 30% of patients diagnosed; of these, only 30% treated successfully1993 RNTCP pilot began1998 RNTCP scale-up 2001 450 million population covered2004 >80% of country covered 2006Entire country covered by RNTCP

*TRC- Tuberculosis research Centre, ChennaiNTI National Tuberculosis Institute, BengalaruStill a third of worlds population has been exposed so called THE CAPTAIN OF ALL MEN OF DEATH(19TH century)

From robbins 9th EditionPathogenesis

PneumoniaGranuloma formation with fibrosisCaseous necrosisTissue becomes dry & amorphous (resembling cheese)Mixture of protein & fat (assimilated very slowly)CalcificationCa++ salts depositedCavity formationCenter liquefies & empties into bronchiTypical Progression of Pulmonary Tuberculosis

From robbins 9th Edition

From robbins 9th EditionFrom robbins 9th Edition

From robbins 9th EditionPrimary Pulmonary Tuberculosis

Red Arrow GW parenchymal focus under the pleura(lower part upper lobe)Blue Arrow Hilar lymph nodes with caseation*

Characteristic Tubercle (Central granular caseation surrounded by epitheloid and MN giant cells) at -Lower MagnificationHigher Magnification

*

C. Tubercular Granuloma in Immunocompromised patient with no central caseationD. Sheets of foamy macrophages packed with mycobacteria.*

From robbins 9th EditionSecondary Pulmonary TBMiliary Tuberculosis

Upper parts of both lungs are riddled with GW areas of caseation and multiple areas of softening and cavitation.Cut surface show numerous GW to GY tubercles.

*

*

HaematologyComplete Blood Count is usually Normal.Usually moderate Normochromic or slightly hypochromic anemia.Anemia may be there due to the chronic debiliting natureof the disease. Megaloblastic Anemia (Macrocytes in blood) in cases of abdominal TB with malabsorption.Thrombocytosis may be seen.

ESR & CRP are Raised.

Isoniazid may cause sideroblastic anemia showing hypochromic microcytes to normochromic macrocytes, often with few stippled red cells.Thrombocytosis due to increase no of small megakaryocytes in the marrow.ESR depends more on amount of fibrinogen than the amount of globulins present in the plasma.*

Tubercular LymphadenitisNeedle aspiration(FNAC) is a good test for TB lymphadenitis in HIV-infected persons Can be done the same day in the health facility.Has a low rate of adverse effects.Has a high yield for diagnosing TB.

TB is the most common cause of adenopathy among HIV-infected patients in sub-Saharan Africa. When biopsy is necessary, excisional biopsy is preferred because incisional biopsies in the setting of TB carry a high risk for poor wound healing and/or chronic fistula formation

TB Lymph NodeH&EMGG Stain

Epithelioidhistiocytes(Epithelioid cells) are activatedmacrophagesresemblingepithelial cells: elongated, with finely granular, pale eosinophilic (pink) cytoplasm and central, ovoid nucleus (oval or elongate), which is less dense than that of alymphocyte. They have indistinct shape contour, often appear to merge into one another and can form aggregates known as giant cells.*

TB Meningitis

Subacute or chronicHeadache, fever, neck stiffness, decreasing mental statusLumbar puncture essential for diagnosisCSF

clear or slightly turbid, forms fibrin coagulum on standing. Raised white cell count (100-1000 lymp/ul)Elevated protein (>45mg/dl), Reduced glucose (

Traditional Methods for Diagnosis of Tuberculosis

PresumptiveDefinitive1. Clinical2. RadiologicalIsolation & Identification3. AFB Microscopyof M. Tuberculosis4. Tuberculin Test5. Pathological

Tuberculosis bacilli is a great IMITATOR & may simulate many other diseases like sarcoidosis, pneumoconiosis, neoplasms, lung abscess and fungal infection.*

Mantoux Tuberculin Skin Test (TST)

0.1 ml of tuberculin purified protein derivative (-PPD) into inner surface of forearm intradermally. Read between 48-72 hrs.

A positive tuberculin skin test result is supportive evidence in the diagnosis of TB in areas of low prevalence (or no vaccination); however, a negative tuberculin skin test result may occur in approximately one third of patients.

Only the induration which is hard, dense raised formation is measured. Erythema area is not measured.*

PPD Tuberculosis Skin Test Criteria

A Negative Test Result could result from the following:

(1) Anergy secondary to immunosuppression or malnourishment; (2) Recent infection; (3) Circulating mononuclear cells suppressing the specifically sensitized circulating T-lymphocytes in the peripheral blood; or (4) Sequestration of purified protein derivative specific reactive T-lymphocyte.

However, results of a tuberculin skin test is repeated 6 to 8 weeks later would usually be positive. False positive in Atypical mycobacterial infection and previous BCG vaccination.

Disseminated TB have negative mantoux b/c of release of large amount of tuberculoproteins from endogenous lesions maskinh Hypersensitivity rxn.Anergy d/t sarcoidosis, viral, hodgkins ds, fulminant tb*

Role of RadiographyChest X-Ray (CXR) can support a diagnosis of PTB

Not used routinely for follow-upPTB can exist with normal CXRMust be interpreted with other information

History and examSputum smear resultsAlso useful in diagnosing other types of TB, especially in bones, joints, and spine

Radiography has more than 90% sensitivity but only 65-70% specificity for detecting PTB. A chest x-ray is an important tool in supporting the diagnosis of PTB in symptomatic individuals whose sputum smears are negative for AFB but it is not possible to diagnose PB using chest x-rays only. Therefore, always request sputum smear examination for all TB suspects.

Chest X-Ray of Patient with Active Pulmonary Tuberculosis

Roentgen discovered Xray in 1895 (Nobel prize in 1901)Radiographic appearances suggestive of active TB: Shadows in one or both upper zones. Cavities in one or both upper zones. Miliary pattern. Persistent shadows after pneumonia treatment. Pleural effusion. Intrathoracic adenopathy. Pericardial effusionA combination of any of the above, especially in HIV-infected patientsEarlier by fluoroscopy without film. Later with Xray films(around 1935s)*

CT by Godrey in 1972(Nobel prize in 1979)Later HRCT developed*

Sputum Examination

CASE FINDING TOOLS

Sputum examination -:- sputum smear examination by direct microscopy is the method of choice.

Collection of sputum

Day 1 Sample 1 - Patient provide an on the spot sampleDay 2 Sample 2 - Patient bring early morning sample.

Because increment yields from sputum specimens are small, WHO recommends examining 2 smears(workload less, less time diagnosis, drop out less)A Minimum 5.0 ml of Sputum Improves the Sensitivity of Acid-fast Smear for Mycobacterium tuberculosis*

ZN Technique 5min heated stain till cooling. Wash with clean water.5min for acid alcohol..1-2min for methylene blue..Processing of sputum use of NaOH 40g/L then centrifugation(18-23% more sensitive).*

Slide reporting The number of bacilli seen in a smear reflects disease severity & patient infectivity.

The table shows the standard method of reporting using 100X magnification (WHO).

Number of bacilliResult reportedNO AFB per 100 oil immersion field01-9 AFB per 100 oil immersion fieldScanty10-99 AFB per 100 oil immersion field+ (1+)1-10 AFB per oil immersion field+ + (2+) >10 AFB per oil immersion field + + + (3+)

Scanty may be due to contamination from tap water/deionized water.Never write negative.. Report as NO AFB SEEN*

Smear and CultureDirect examination by Zeihl-Neelsen staining requires bacillary densities of 5000-10,000/mL

Culture requires a minimum of 10 to 100 viable bacilli.

Cant reliably distinguish MTB from NTM*

Lipid-Rich Cell Wall of MycobacteriumCMN Group: Unusual cell wall lipids (mycolic acids,etc.)

Red,straight slightly curved rods occurring singly or in small groups.. may appear beaded..*

Mycobacterium Tuberculosis Stained with Fluorescent DyeFrom Carl Zeiss microimaging GmbH (FluoLED)Yellow bacilli with green background.

Fluorescence microscopy is 10% more sensitive than conventional microscopy.Used to determine viability of M.Tb (auramine O stained) in follow up sputum specimens to treatment failure.LED(light-emitting diode) light source used. Cheaper, last longer.Attractive alternative to mercury vapour lamps. Hazards of dye toxicity.*

Mycobacterial CultureReasons to request mycobacterial culture:Patient previously on anti-TB treatment (Relapse, Defaulter)Still smear-positive after intensive phase of treatment or after finishing treatmentSymptomatic and at high-risk of MDR-TBTo test fluids potentially infected with M. tuberculosisInvestigation of patients who develop active PTB during or after IPT.TB in health workers

Culture Based MethodsLiquid Culture (e.g., automated mycobacteria growth indicator tube) Faster and more sensitive than solid media)Microscopic Observation Drug Susceptibility Testing (DST) Yields fasterb culture and DST results than do liquid or solid media and is inexpensive(Requires Skilled Technician to interpret)Thin Layer Agar Methodology (same as above)Calorimetric DST methods using redox tetrazolium slats, or a nitrate reductase assay Lower cost, less time (Potential Biohazard)

LJ egg medium protein enriched media with optimum temp of 35-37c.LJ is an egg based media with addition of salts, 5% glyecerol and Malachite green.*

Eight Week Growth of Mycobacterium tuberculosis on Lowenstein-Jensen Agar

With the Lowenstein-Jensen (L-J) method, a positive result (growth of mycobacteria) is usually apparent after three weeks. If there is no growth by 8 weeks, the result is negative. Approximately 4 weeks from receipt of specimen to culture resultsAn additional 3-4 weeks for susceptibility resultsTherefore minimum of 7-8 weeks for DST results (Botswana National Tuberculosis Programme Manual, page 33-34)*

Raised, dry cream (aka buff,rough and tough) coloured colonies. Visible colonies are usually produced weeks after incubation but culture should be incubated for upto 6weeks before being discarded.Nitrate reduction and niacin production are definitive for M.tb*

DST performed on all culturesTests for isoniazid, rifampicin, ethambutol, and streptomycinIf found to be multi-drug resistant, then send for additional testing for susceptibility to second-line medicines

TB Drug Susceptibility Testing (DST)

DST is a routine performed on all positive culturesMDR = specimen shown to be resistant to isoniazid AND rifampicin +/- any other drugs

BACTEC 460 TB System (radiometric)Developed in 1969 by Deland and Wagner.

Principle

BACTEC 12B vial, utilize 14C labeled substrate (Palmitic acid).On inoculation, mycobacteria, grow and release 14CO2.The BACTEC instrument measures quantitatively the radioactivity on a scale ranging from 0-999, as GI (Growth Indicator).The daiy increase in GI is proportional to growth in the medium.DST (Drug Susceptibility Test) When ATT is introduced in the medium, reduced production of 14CO2 & decrease in GI.

Specimens are cultured in Mycobacteria Growth Indicator tube (MGIT) with liquid media (Middle brook7H9 broth base) containing C14 labelled palmitic acid, OADC enrichment and PANTA antibiotic mixture.*

Specially designed to accommodate Mycobacteria Growth Indicator Tube and incubate them at 37c.Scans tubes every 60min for increased fluorescence.CONTINUOUS MONITORING BLOOD CULTURING INSTRUMENTS.MGIT 960 is a nonradimeteric system.*

New Approach in Diagnosis of TB

Replication of M. TuberculosisAntigen Detection Tests LAM ELISA Urinary Antigen Test(ELISA BASED TEST, detect LAM, Antigen 85 LipoArabinoMannon) still developingSputum Antigen Test2. Microscopic Visualization of bacteria LED MicroscopyBleach Microscopy3. Culture based Detection Tests Microscopic observation drug susceptibility assays (MODS)Thin Layer AgarPhage based testsCalorimetric media

Antigen detection tests are almost absolute now*

Replication of M. Tuberculosis4. Nucleic Acid Amplification Tests (NAATs)- LAMPGeneXpert MTBTransrenal DNA detectionGenotype MTBDRPlus(High Specificity and Positive Predictive Value)5. Volatile Organic Compounds (VOC) detection-E-noseBiosensors(Emitted from the infected cells & released in exhaled breath through nanomaterial biosensors or Gas Chromatography)

CBNAATs used in MYH.LAMP Loop Mediated Isothermal Amplification of DNA(Phenotypic)Fast Plaque TB principle mycobacteriophage based assay. Detects only live bacteria.*

New Approach in Diagnosis of TB

Immune Response to M. TuberculosisI. Cellular Immune Response -INF-Y release assays(IGRA)Quanti-FELON TB goldT-SPOT TB Rd ESAT-6 skin testII. Humoral Immnune Response Antibody Detection TestsSerological tests

Immunochromatographic tests (Serological test) are absolute now.*

Polymerase Chain Reaction

Polymerase chain reaction (PCR) is based on amplification of mycobacterial DNA fragments.

It can detect as few as 10 mycobacteria.

Advantages of PCR include rapid diagnosis, improved specificity and sensitivity, and no requirement of intact immunity.

Molecular Beacon Assay(at MDL)Target: DNARealtime PCRPCR to amplify target sequencesAt the same time, Molecular beacon probes are used to detect INH and RIF resistance mutations.2 MBs for INH (targeting katG & inhA)3 MBs for RIF (targeting core of rpoB)

*Molecular beacon test goes beyond what MTD & Amplicor can offer. It provides Identification of MTB and drug susceptibility of INH and RIF.There is tremendous impact on TB patient management and control.

I will discuss this in detail on the 2nd part of my talk.

Real-Time PCR2 componentsPCR to amplify target sequences.A system to monitor PCR product.Fluorophore-labeled probesAn optical device to detect fluorescenceSoftware to record dataNo post-PCR manipulationsFast when PCR is done, results are ready for interpretation.No amplicon contaminations

iCycler IQ5

*Bought a backup iCycler, newer version, named iQ5.

What is a Molecular Beacon?

Loop (15-30 nt)

Stem (5-7 nt)

FluorophoreQuencherHair-pin structure

*MB is an oligonucleotide probe with a hair-pin or loop-and-stem structure. When the MB is at rest, the fluorophore is at the close proximity of the quencher, therefore, it is not emitting fluorescence.When the MB is in action, that is, when the target is 100% complementary to MB loop SQ, the MB will undergo a conformational change, from hairpin to linear structure, and hybridizes to its target. The conformational change forces the fluorophore away from the quencher and therefore, fluorescence will be emitted.This structure creates a competition for annealing between the arms and between the MB and the target SQ. The MB loses its competition when mismatches exist between its loop and the target. This unique property renders MB a great discriminary power to detect a single-nucleotide mismatch.

MolecularBeacon (off)Hybrid (Molecular Beacon - On)Detection of Mutations with a Molecular Beacon(Loop portion containing wildtype SQ) Mutant SequenceWildtype SequenceAmpliconHeatLight+Courtesy of Dr. ProbertLoopQuencherFluorophoreFluorophore

*

An Example of a Good MB

No mutations, Susceptible

Mutant,Resistant

RFUThreshold

Causes of false-positive results include DNA contamination or presence of nonviable organisms

The disadvantages of PCR include high cost, risk of contamination, and the technology involved in the procedure does not permit routine diagnostic use at present.

*LimitationsLimited genes & sites are targeted.

Some mutations are not detected.Emerging resistance in mixed populations may not be detected.Some mutations do not confer resistance.

Rare occurring, but lead to wrong interpretation.Silent mutation in rpoB: codon 514.Not a silent mutation but only cause little change in MIC.Available for INH and RIF only.New MBs for other drugs not developed yet.Phenotypic drug susceptibility testing is still needed.

*

GeneXpert Automated System at MYH, Indore Resp. Medicine (TCD) Department and TB Hospital

CBNAAT Report

Line Probe AssaysTarget: DNA Traditional PCR (not realtime)Amplify target sequences. Reverse hybridizationAmplicons hybridize to probes immobilized on membrane (strip).Colorimetric detection of captured amplicons on strip.Observation of bands. One probe for one band.

*

*Line ProbesHybridization and colorimetric detection

Amplicons bind to probesColor reaction to form bands

Identify the bacilli while simultaneously identify drug resistamt strains by detecting the most common Single Nucleotide Polymorphisms(SNPs) associated with resistance.*

*Conjugate ctrlrpoB wild-type, 5 segments4 rpoB mutationsrpoB universal ctrlMTBDR by HAIN Lifescience LiPA RIF.TB by INNOGENETICSkatG universal ctrlkatG wild-type2 katG mutationsUniversal ctrlMTBC516526531315marker lineMTBCMore probes are added in MTBDRsl to detected 2nd-line drug R-mutations.

*

*Line Probes FeaturesMany controls; more objectiveMTBDRsl (HAIN) added embB, gyrA, rrs (screen for XDR).Exact mutations are available for most prevalent mutations only.Some mutations are detected by lacking bands in wild-type sequences.Emerging resistance in mixed populations may not be detected.Phenotypic drug susceptibility testing is still needed.

*

Loop Mediated Isothermal Amplification(LAMP)of DNASmall Heating DeviceRuns at High Temperature(avoids nonspecific amplifications)Multiple primers sets (increased specificity and speed)Direct from Sputum.Closed System (No risk of contamination)Minimal instrumentationFast (Less than 2hrs total)Visual Detection(no instrumentation- Mg2P2O7 ppt - white)Test Under Development still.

Cytokine AssaysT- cell Interferon-Gamma Release Assay (IGRA)INF- y produced by T-lymphocytes, is capable of activating macrophages, increasing their bactericidal capacity against M tuberculosis and is involved in granuloma formation.

Elevated concentrations of INF-y in TB is related to increased production at the disease site by effectors T cells.

The sensitivity of an elevated level varies from 78 to 100% and specificity from 95 to 100%

*

IGRA is useful in targeted strategy for latent TB infection(LTBI) detection in low TB incidence settingsMore specific than Tuberculin Skin TestCant distinguish active from treated TB or LTBI.False positive results in-Hematologic malignancies Empyema.

Note :- Immunosuppressed patients (HIV or after renal transplant) had INF- Y levels similar to immunocompetent individuals retaining its efficacy as a diagnostic test

Methods for detection of IFN-yTwo new blood tests T-SPOT.TB [Oxford Immunec] directly count the no of IFN-y secreting T cells. QuantiFERON-TB Gold [Cellestis Limited] measures the concentration of IFNy secretion. Both tests based on detection of IFN-y in blood have been found to be more accurate than the tuberculin skin test in the diagnosis of latent TB infection. Future research should focus on the potential efficacy of quantification of specifically activated lymphocytes in body fluid and blood using IFN-Y release assay in the diagnosis of TB.

Marketed as TB Gold.. Low specificity of IGRA and consequent unnecessary t/t. WHO discouraged this test in developing countries.*

IGRA discontinued after 2012 as it cant differentiate Active from latent tb.*

Immunodiagnosis

Serological tests are simple to perform & can be developed into a rapid method for wide screening.

Immuno- chromatographic card test Sensitivity 80 % & specificity 88 %.Principle - the test employs antihuman Ig-G, A, M antibody rabbit dye conjugate highly purified antigen A60 from Mycobacterium bovis stain BCG (cell culture), fixed in the test line, & anti-rabbit antibodies in the control line. As the sample flows through the absorbent pad, human immunoglobulins are bound by the antihuman Ig- dye conjugates to form an immunocomplex. This binds to the A60 proteins in the test line & produce a red violet test line, if the anti- Tb antibodies were present in the sample. In control line excess conjugate react with the anti rabbit antibodies forming second red violet line to demonstrate the correct function of the reagent.

Absolute now*

Cytokines: Significantly higher levels of IL-6 have been demonstrated in TB (A Potent Biomarker/Biosignature).

Furthermore, the serum/pleural fluid IL-6 ratio was significantly higher in TB.

IL-1b and Tumor necrosis factor(TNFa), produced predominantly by mononuclear phagocytes, have been shown to be present in TB.

Levels of soluble IL-2 receptors, IL-18, immunosuppressive acidic protein, and IL-12p40 are all significantly elevated in TB

LAM in cell wall of M.Tb induce IL-6.IL-6 is the most potent stimulator for hepatic synthesis of CRP which also has thrombopoietic activity.*

Adenosine DeAminase (ADA) in Pleural FluidADA levels in Pleural fluid are measured by colorimetric method.Increased ADA levels (>36IU/L) observed in Tubercular Pleural Effusion. Quick test.Adjunct test to help rule in or rule out tuberculosis in pleural fluid.

ADA is an enzyme involved in purine metabolism. Asso with greater lymphocyte proliferation.>100IU/L is exclusively seen in tubercular pleural effusion.ADA needed for breakdown of adenosine from food and for the turnover of nucleic acids in tissues.*

Tulip Diagnostic ADA kit in MYH*

LysozymeLysozyme, a bacteriolytic enzyme, have been found to be higher in patients with TB.

However, a fluid to serum lysozyme ratio > 1.2 has been found to be a better tool for the diagnosis of TB.

IHC with an antibody to MPT64, a secreted antigen specific to theM. tuberculosiscomplex, is a specific and sensitive technique for diagnosis of EPTB.*

ConclusionNewer Technologies offer a significant time savings. However these tests have limitations 1. Costly.2. Complex & Cumbersome.3. Only smear positive (50% of culture positives)4. Recommended only in special cases.5. Add-on tests.Culture is still the Gold Standard.

Asrecommended by CDC/WHO* Whenever possible, use liquid culture & DST.* Rapid testing and reporting essential for TB control.

*

Progress in TB Diagnosis

PastPresentKoch discovered tubercle bacillus 133 yrs backNo major discovery Except TB Genome, IS6110, BACTEC 460 (liquid media)TB diagnosed by symptoms - prehistoricStill the same practice in many High Bruden Countries (HBCs)Tuberculin test - > 100yrsStill Commonly usedEgg based media almost 100yrsStill most commonly usedAFB Smear for diagnosis 133 yrs backStill the major diagnostic tool in many countriesRadiological DiagnosisStill Important (X-ray, CT Scan)

We are still in need of a costeffective, handy and accurate test for the diagnosis of latent tb cases.GeneXpert gave a hope.. Awaiting for even better technologies..*

TB CowBoysThank You!

*

Earlier known as Consumption (severe wt loss)/ phthisis pulmonaris and the white plaque( extreme pallor in infected) .Potts disease (Egyptian mummies spinal tb) by archaeologist.*Mycobacterium tubercule bacilli demonstrated by Robert Koch(coke) in 1882. (Kochs bacilli)Nobel prize 1905.Every third person is infected.*Generation timeis simply the time it takes for one cell to become two.M. Tb is slow growing, a nonchromogen, doesnt grow at 25c or in PNB medium.**Follows iceberg phemomenon.. As only tip of iceberg is visible.. Part below the iceberg indicates a lot of dormant infection in individuals, requiring urgent need of screening clinical findings and investigations for finding the dormant infections*In 2008 WHO report-9.4 million new cases equivalents to 139 cases per 100,000 population of TB globally. 1.98 million were estimated to have occurred in India (0.87 million---infectious cases, fifth of the global burden of TB. About 40% of Indian population is infected with TB bacillus. *TRC- Tuberculosis research Centre, ChennaiNTI National Tuberculosis Institute, BengalaruStill a third of worlds population has been exposed so called THE CAPTAIN OF ALL MEN OF DEATH(19TH century)Red Arrow GW parenchymal focus under the pleura(lower part upper lobe)Blue Arrow Hilar lymph nodes with caseation*Characteristic Tubercle (Central granular caseation surrounded by epitheloid and MN giant cells) at -Lower MagnificationHigher Magnification

*C. Tubercular Granuloma in Immunocompromised patient with no central caseationD. Sheets of foamy macrophages packed with mycobacteria.*Upper parts of both lungs are riddled with GW areas of caseation and multiple areas of softening and cavitation.Cut surface show numerous GW to GY tubercles.

*

*Isoniazid may cause sideroblastic anemia showing hypochromic microcytes to normochromic macrocytes, often with few stippled red cells.Thrombocytosis due to increase no of small megakaryocytes in the marrow.ESR depends more on amount of fibrinogen than the amount of globulins present in the plasma.*TB is the most common cause of adenopathy among HIV-infected patients in sub-Saharan Africa. When biopsy is necessary, excisional biopsy is preferred because incisional biopsies in the setting of TB carry a high risk for poor wound healing and/or chronic fistula formation Epithelioidhistiocytes(Epithelioid cells) are activatedmacrophagesresemblingepithelial cells: elongated, with finely granular, pale eosinophilic (pink) cytoplasm and central, ovoid nucleus (oval or elongate), which is less dense than that of alymphocyte. They have indistinct shape contour, often appear to merge into one another and can form aggregates known as giant cells.*The test for cryptococcal meningitis is Indian Ink stainPleural or pericardial fluids are not very sensitive samples for the detection of M. tuberculosis.Tuberculosis bacilli is a great IMITATOR & may simulate many other diseases like sarcoidosis, pneumoconiosis, neoplasms, lung abscess and fungal infection.*Only the induration which is hard, dense raised formation is measured. Erythema area is not measured.*Disseminated TB have negative mantoux b/c of release of large amount of tuberculoproteins from endogenous lesions maskinh Hypersensitivity rxn.Anergy d/t sarcoidosis, viral, hodgkins ds, fulminant tb*Radiography has more than 90% sensitivity but only 65-70% specificity for detecting PTB. A chest x-ray is an important tool in supporting the diagnosis of PTB in symptomatic individuals whose sputum smears are negative for AFB but it is not possible to diagnose PB using chest x-rays only. Therefore, always request sputum smear examination for all TB suspects. Roentgen discovered Xray in 1895 (Nobel prize in 1901)Radiographic appearances suggestive of active TB: Shadows in one or both upper zones. Cavities in one or both upper zones. Miliary pattern. Persistent shadows after pneumonia treatment. Pleural effusion. Intrathoracic adenopathy. Pericardial effusionA combination of any of the above, especially in HIV-infected patientsEarlier by fluoroscopy without film. Later with Xray films(around 1935s)*CT by Godrey in 1972(Nobel prize in 1979)Later HRCT developed*Because increment yields from sputum specimens are small, WHO recommends examining 2 smears(workload less, less time diagnosis, drop out less)A Minimum 5.0 ml of Sputum Improves the Sensitivity of Acid-fast Smear for Mycobacterium tuberculosis*ZN Technique 5min heated stain till cooling. Wash with clean water.5min for acid alcohol..1-2min for methylene blue..Processing of sputum use of NaOH 40g/L then centrifugation(18-23% more sensitive).*Scanty may be due to contamination from tap water/deionized water.Never write negative.. Report as NO AFB SEEN*Cant reliably distinguish MTB from NTM*Red,straight slightly curved rods occurring singly or in small groups.. may appear beaded..*Fluorescence microscopy is 10% more sensitive than conventional microscopy.Used to determine viability of M.Tb (auramine O stained) in follow up sputum specimens to treatment failure.LED(light-emitting diode) light source used. Cheaper, last longer.Attractive alternative to mercury vapour lamps. Hazards of dye toxicity.*

LJ egg medium protein enriched media with optimum temp of 35-37c.LJ is an egg based media with addition of salts, 5% glyecerol and Malachite green.*With the Lowenstein-Jensen (L-J) method, a positive result (growth of mycobacteria) is usually apparent after three weeks. If there is no growth by 8 weeks, the result is negative. Approximately 4 weeks from receipt of specimen to culture resultsAn additional 3-4 weeks for susceptibility resultsTherefore minimum of 7-8 weeks for DST results (Botswana National Tuberculosis Programme Manual, page 33-34)*Raised, dry cream (aka buff,rough and tough) coloured colonies. Visible colonies are usually produced weeks after incubation but culture should be incubated for upto 6weeks before being discarded.Nitrate reduction and niacin production are definitive for M.tb*DST is a routine performed on all positive culturesMDR = specimen shown to be resistant to isoniazid AND rifampicin +/- any other drugsSpecimens are cultured in Mycobacteria Growth Indicator tube (MGIT) with liquid media (Middle brook7H9 broth base) containing C14 labelled palmitic acid, OADC enrichment and PANTA antibiotic mixture.*Specially designed to accommodate Mycobacteria Growth Indicator Tube and incubate them at 37c.Scans tubes every 60min for increased fluorescence.CONTINUOUS MONITORING BLOOD CULTURING INSTRUMENTS.MGIT 960 is a nonradimeteric system.*Antigen detection tests are almost absolute now*CBNAATs used in MYH.LAMP Loop Mediated Isothermal Amplification of DNA(Phenotypic)Fast Plaque TB principle mycobacteriophage based assay. Detects only live bacteria.*Immunochromatographic tests (Serological test) are absolute now.**Molecular beacon test goes beyond what MTD & Amplicor can offer. It provides Identification of MTB and drug susceptibility of INH and RIF.There is tremendous impact on TB patient management and control.

I will discuss this in detail on the 2nd part of my talk.*Bought a backup iCycler, newer version, named iQ5.*MB is an oligonucleotide probe with a hair-pin or loop-and-stem structure. When the MB is at rest, the fluorophore is at the close proximity of the quencher, therefore, it is not emitting fluorescence.When the MB is in action, that is, when the target is 100% complementary to MB loop SQ, the MB will undergo a conformational change, from hairpin to linear structure, and hybridizes to its target. The conformational change forces the fluorophore away from the quencher and therefore, fluorescence will be emitted.This structure creates a competition for annealing between the arms and between the MB and the target SQ. The MB loses its competition when mismatches exist between its loop and the target. This unique property renders MB a great discriminary power to detect a single-nucleotide mismatch. *

*

*Identify the bacilli while simultaneously identify drug resistamt strains by detecting the most common Single Nucleotide Polymorphisms(SNPs) associated with resistance.**

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*Marketed as TB Gold.. Low specificity of IGRA and consequent unnecessary t/t. WHO discouraged this test in developing countries.*IGRA discontinued after 2012 as it cant differentiate Active from latent tb.*Absolute now*LAM in cell wall of M.Tb induce IL-6.IL-6 is the most potent stimulator for hepatic synthesis of CRP which also has thrombopoietic activity.*ADA is an enzyme involved in purine metabolism. Asso with greater lymphocyte proliferation.>100IU/L is exclusively seen in tubercular pleural effusion.ADA needed for breakdown of adenosine from food and for the turnover of nucleic acids in tissues.*Tulip Diagnostic ADA kit in MYH*IHC with an antibody to MPT64, a secreted antigen specific to theM. tuberculosiscomplex, is a specific and sensitive technique for diagnosis of EPTB.*

*We are still in need of a costeffective, handy and accurate test for the diagnosis of latent tb cases.GeneXpert gave a hope.. Awaiting for even better technologies..**