Diagnostic Tests for TB A Comprehensive Overview website: www.drsarma.in YouTube: drsarmaji channel Diagnostic Tests for TB A Comprehensive Overview Prof. Dr. Sarma Rachakonda M.D., M.Sc., (Canada), FCGP, FIMSA, FRCP (Glasgow), Consultant Physician and Cardio-metabolic Specialist Visiting Professor of Internal Medicine, SBMC, FLL

DST means Drug Sensitivity Test MDR, XDR and TDR TB MDR TB : Multi drug Resistant TB Rifampicin (RIF) and INH resistance XDR TB : Extensive Drug Resistant TB INH and RIF (MDR-TB) and Amikacin or Kanamycin or Capreomycin and Ofloxacin, Moxifloxacin (Fluroquinolones) TDR TB : Totally Drug Resistant TB Resistant to almost all known anti TB drugs DST means Drug Sensitivity Test

Sensitivity and Specificity GOLD STANDARD SnNOUT (Minimum FN) Sensitivity and Specificity CAD by CAG No CAD ECG +VE True Positives a False Positives b ECG – VE False Negative c True Negatives d Total CAD a + c Total No CAD b + d Sensitivity is True positives a Total CAD a + c TEST Specificity is True Negatives d Total No CAD b + d SpPIN (Minimum FP)

Sensitivity and Specificity GOLD STANDARD SnNOUT (Rules out 70%) Sensitivity and Specificity CAD by CAG No CAD ECG + VE True Positives 70 False Positives 120 ECG – VE False Negative 30 True Negatives 180 Total CAD 100 Total No CAD 300 Sensitivity is True positives 70 Total CAD 100 TEST Specificity is True Negatives 180 Total No CAD 300 SpPIN (Confirms 60%)

PPV and NPV Test True positives 70 Total Positives 190 GOLD STANDARD PPV is 37%) PPV and NPV Test CAD by CAG No CAD ECG + VE True Positives 70 False Positives 120 Total +ves 190 ECG – VE False Negative 30 True Negatives 180 Total -ves 210 Total CAD 100 300 Grand Total 400 Positive Predictive Value True positives 70 Total Positives 190 Negative Predictive Value True Negatives 180 Total Negatives 210 NPV is 86%)

Key Concept Sensitivity is the ability of the test to rule out disease confidently when the test result is negative. Specificity is the ability of the test to confirm disease confidently when the test result is positive. Positive Predict Value (PPV): useful in high prevalence situations Negative Predictive Value (NPV): useful in low prevalence situations Sensitivity and Specificity are unaffected by prevalence

Under Diagnosis - 50%

Co Infection HIV BOTH TB 3.5m 1.7m 2.5m

The Burden of Illness 10 million new cases every year globally 3 million deaths annually worldwide Leading cause of death due to infectious disease 25% of all avoidable deaths are due to TB 95% of TB cases and 98% of deaths are in developing countries India contributes 2 million cases every year 0.5 million die of TB annually – 1 death / min 75% of cases are in productive age groups Increasing HIV infection increases TB burden.

New Problems – New Needs More than 10 m cases of TB yearly - world wide India ranks 1st among the top 22 TB countries 5 lakh cases of MDR TB per year (WHO) Accurate and early diagnosis is most important for effective case management and prevention of transmission of MTC We do not have an effective vaccine to prevent Case finding and case holding are key issues MDR, XDR and TDR cases are due to improper Rx. Anonymous or atypical mycobacteria MOTT

We are always different Tuberculosis is endemic in our country Ours is high prevalence, high incidence scenario Poverty, over crowding, lack of education, ½ Rx. Large population and wide area – large burden BCG vaccination is common – confounds skin test MOTT – Anonymous mycobacteria are ubiquitous Subclinical MTB infections are very common New HIV burden throws a new serious challenge MDR TB, XDR TB and now TDR TB – big challenge Non compliance, self medication, quackery We very well know about our Govt. programme

Genes and Drug Resistance MDR TB Rifampicin rpoB 95% INH KatG 70% inhA 30% XDR TB FQ- gyrA AG/CP -16S rRNA (rrs) embB Mutations in the MTB Genome

Clinical Conundrum Fever, cachexia, cough, sputum, chest pain, hemoptysis, fatigue, night sweats, raised ESR are notoriously present in many similar disorders Clinical signs of consolidation, cavitation, fluid, thickened pleura, neck glands can occur in a variety of conditions. Old treated TB is big confounder, DST is not done Partially treated cases pose resistance problems COPD, emphysema mimic and mask TB signs Early lesions often are unsuspected until X-ray The sensitivity and specificity are rather low. Extra pulmonary poses even greater confusion

X-ray and Imaging Radiological features when clear cut are highly specific. Often early lesions may be missed. X-ray shadows can’t tell activity of disease Old shadows are superimposed on fresh ones. Effusions may be due to many causes HRCT is expensive and again cannot speak of the activity of disease. Treatment decisions on imaging alone will be risky as TB Rx is prolonged and potentially toxic Over all - X-ray has high sensitivity, mod. Specificity We can’t treat shadows. What about extra pulmonary ?

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST – The Need for Fast track Indirect Methods Interferon Gamma - IGRA TBFeron / Quanti-FERON T Spot TB Test Adenosine Deaminase (ADA) Serological Tests for TB Tuberculin Test – Skin Tests MTB and Its Products Humoral & Cellular Response

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear - Sp. Microscopy BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST for MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

AFB Smear Examination 120 years old technique, relatively simple Special techniques to improve the yield ZN method and Fluorescent microscopy Results will be reported with in hours Most cost effective method – RNTCP Requires good training & observer dependent Requires 5 x 103 bacilli per ml of sputum Proper collection of sputum is essential Three specimens are needed for Dx. Sensitivity is 30 to 60%, Specificity is high - 97%

AFB Smear Microscopy

Lab. Diagnosis of Tuberculosis Direct Methods MTB culture – Solid / Liquid BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST for MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

Cultures for MTB Corner stone of definitive diagnosis. Gold standard. LJ medium or Middle Brook 7H10 & 11 Solid media Kirchner’s or Middle Brook 7H9 broth – Liquid media Slow growth – Mean time of 4 to 6 weeks (2-3 wks) DST requires another 4 weeks; Contaminants problem Combination of solid & liquid media is better Micro colony detection, Sept Check AFB, MODS Proper collection of sputum is essential. High infrastructure cost, Not available readily. Many factors decide the yield of positive culture Highly specific 99.5% but moderately sensitive - 70%.

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST for MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

BACTEC 460 Uses 14C labeled palmitic acid in the medium Based on metabolism of MTB – not on visible growth If the medium is metabolized - 14CO2 is released BACTEC system radiometric measurement gives GI Same BACTEC can be used for DST Significantly faster – 87% +ves in 7 d, 96% in 14 d DST can be completed in 8 days This proves cost effective in high prevalence areas

MIGT BACTEC 960 Uses 14C labeled palmitic acid in the medium Based on metabolism of MTB – not on visible growth Mycobacteria Growth Indicator Tube (MIGT) Observed every 60 min for increase in fluorescence AFB metabolic utilization of O 2 in the fluorescent dye Intensification of fluorescence in the tube Rapid, accurate and cost effective method for high volume labs 960 tubes can be computer monitored simultaneously Can be used for DST also; Rapid in 4-6 days results

Other Culture based Tests MB / BacT System Non radiometric continuous monitoring system with computerized data base management. The system is based on colorimetric detection of CO2 ESP Culture System Fully automated continuous monitoring of pressure changes with in the head space above the broth culture medium in a sealed bottle. Gas production or gas consumption by bacterial growth. Results in about 2 weeks

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST for MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

PCR for TB Sequences of bacterial DNA will be amplified 10-1000 bacilli are sufficient for detection Rapid and results are available in a day Target IS6110, 65 kDa, 65 SrNA MTB Specific It is present up to 20 times in the MTB genome It can detected both in blood and in sputum Sensitivity 84%, Specificity 99%, PPV 94.2% NAAT – Nucleic Acid Amplification Test and TMA, SDA, NASBA, b-DNA, LiPA – other tests

Molecular Dx of MTB MD of MTB Mycoreal RT PCR Mycoresist MDR TB GeneXpert MTB RIF Myco3Plex Multiplex

TB Molecular Diagnosis “Mycoreal” - A rapid real time PCR test for MTB Utilizes UTP/UDP system to avoid contamination Sensitivity 99%, Specificity 99.5% Detects all members of MTC group Can be used in pulmonary and extra pulmonary Sputum, Blood, Tissues - all can be tested No “ post PCR processing”. So no contamination Detects as low as 2 fg of MTB DNA Negative, positive and no template controls

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST for MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

GeneXpert/MTB-RIF It is two-in-one: Detects presence of MTB by Real Time PCR + identifies if it is resistant to RIF Developed by Cepheid – Endorsed by WHO, TB Culture take 3 to 6 weeks, DST further 3 to 4 wks Rapid 100’, very simple, minimal training, field use Completely closed, No man errors, No contamination RIF resistance is a surrogate of MDR TB Sensitivity 91% detection in S-C+ / 100% in S+C+ Specificity very high 99.2%, 98% for RIF resistance Govt. of India has started in 4 places, Available in labs

On Gene Xpert 22nd October 2011

GeneXpert/MTB-RIF

Cepheid - GeneXpert

GeneXpert Cartridge

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF LiPA - MDR & XDR test DST for MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

Rapid Genotypic MDR & XDR TB Test This is a LiPA endorsed by WHO and FIND Detects mutations in rpoB , Kat G and inhA Rapid detection of RIF & INH Resistance – MDR inhA inclusion detects even low levels of INH R Sensitivity 93.6% RIF, 92.6% INH, 88.9% for MDR Specificity is 100% for all types of patients PPV is 100%, NPV is 90.3% Smear +ve or culture +ve specimens In smear –ve cases bacillary load will be low to detect mutations for the drug resistance.

MDR TB - Dx. Time Line 1st Line DST 1st Line LiPA Microscopy One Day Liquid Culture 2 to 3 weeks 1st Line DST 1 to 3 weeks MDR TB - Dx. Time Line Microscopy One Day RT PCR 1st Line LiPA 3 to 6 weeks 3 days

XDR TB - Dx. Time Line 4 to 9 weeks 4 days Microscopy One Day Liquid Culture 2 to 3 weeks 1st Line DST 1 to 3 weeks 2nd Line DST XDR TB - Dx. Time Line Microscopy One Day RT PCR 1st Line LiPA 2nd Line LiPA 4 to 9 weeks 4 days

Rapid Genotypic 2nd line Drug Resistance This is also a LiPA – rapid test for XDR Detects mutations in gyrA, 16S rRNA, embB Sputum or Culture specimens of MDR TB only are tested for second line drug resistance India, China, SA and Russia, BD – together contribute for largest number of XDR TB. Culture and DST is not routinely done in our country. We miss a lot of MDR and XDR TB We realize only when the patient does not respond after 6 months of treatment.

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF New Hope – The TB LAMP DST for MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST – Need For Fast Track Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

DST – Need for Fast Track Routine DST is now essential in view of MDR, XDR TB burden. Also TDR TB will be most challenging DST based on solid media cultures take 3 to 4 weeks for 1st line and a further 3 to 4 weeks for 2nd line drug sensitivity RT PCR combined with Line Probe Assay (LiPA) will shorten this time most efficiently to 3 to 4 days If we use GeneXpert – MDR TB can be diagnosed in 100 minutes, even before the smear results come. The world is on the fast track but our TB Dx is on 120 years old ultra slow track. How do we win TB?

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

Interferon Gamma Release Assay (IGRA) MTB genome has regions of difference (RD) These RDs encode potential antigens for Dx. RD1 is responsible for secretion of ESAT6 ESAT6 (6kDa) is specific antigen and a strong inducer of IFG by T cells of the TB patient. IFG is a cytokine secreted by sensitized T cells This ESAT6 is not recognized by BCG or NTM IFG levels increase with treatment – prognostic It is a measure of CMI in TB patients Sensitivity is 82 to 90%, Specificity is 96 to 99%

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron / Quanti-FERON T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

TBFeron /Quanti-FERON TB Gold These are IGRA – use three proteins specific for MTB – ESAT6 (early Secretory Antigen TB), CFP (Colony Forming Protein) and TB7.7 – All are proteins from different RDs This combination makes the test very specific as these are absent in BCG and NTM Rapid test – 1 day, available – moderately priced Does not distinguish between Latent Infection (LTBI) and active disease. It is an ELISA based test. Specificity 96 to 99%, Sensitivity 82 to 90%

The IGRA Test Steps

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

T Spot TB Test It is one of the latest tests – based on IGRA An FDA approved in vitro test based on ELISPOT Uses two separate panels of MTB complex – the ESAT6 and CFP10 – not present in BCG, NTM Detects both LTBI and Active TB (all forms) Useful in all ages, ethnicity, immunocompromised More sensitive compared to ELISA-TBFeron Sensitivity 95% and Specificity 97% Rapid test – in one day result is available

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase -ADA Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

Adenosine Deaminase (ADA) Useful to test body fluids like plural, peritoneal, pericardial and cerebrospinal fluid. Commonly used for Dx. of TB pleural effusion Increased T lymphocytes and their increased activity increases ADA in the exudate In low prevalence areas – more false positives and low specificity. Combined with lymphocyte count it may be useful as a screening test. Not a replacement for culture or biopsy. Simple, cheap test to R/o TB in exudate effusions.

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test - PPD MTB and Its Products Humoral & Cellular Response

Serological Tests for TB Rarely useful as screening tests. Industry bias Low turn around time, Not available in all labs Very low sensitivity in smear negative cases and HIV positive cases Not useful in our country of high endemicity Ubiquitous nature of MOTT Sero conversion would have already occurred High cost, require trained staff, costly equipment Can’t separate MTB and NTM Strongly recommended by WHO – Not to be used

Lab. Diagnosis of Tuberculosis Direct Methods AFB Smear and MTB culture BACTEC 460 - MGIT 960 Molecular Dx MTB - PCR Gene Xpert - MTB-RIF Rapid Geno MDR & XDR test DST MOTT - rapid growers Indirect Methods Interferon Gamma - IGRA TBFeron T Spot TB Test Adenosine Deaminase Serological Tests for TB Tuberculin Test – Skin Tests MTB and Its Products Humoral & Cellular Response

Do not Use Manteaux Test Useful in non endemic countries with low TB rates Almost all adults in our population are manteaux positive because they are either BCG vaccinated or are exposed MTB or NTM in the air and soil. The protein (PPD) used in Manteaux test is non specific and is shared by BCG, MTB and NTM A positive Test in Indian adults has no meaning. In very young children high positivity may mean recent infection. Infection is not synonymous with disease. Sensitivity is also low. Specificity is nearing zero

Limitations of Manteaux Test False negatives In severe disease – Miliary TB In HIV infected In Sarcoidosis Technical factors Application, Reading, Improper storage of PPD Biological factors Poor nutrition, Infection Immunosuppressive drugs Malignancy, Age, Stress False positives (Major Issue) Infection with NTM - non tuberculous mycobacteria BCG vaccination Booster effect or retest Natural infection with MTB in endemic counties and acquired CMI (herd immunity

TB MPB 64 Patch Test MPB 64 is a specific MTB antigen Patch test becomes positive after 3-4 days of application and lasts for a week Specificity of 100% and sensitivity of 98.1% Evaluated only in Philippines and needs to be reproduced in other settings. ESAT 6 (Early Secretory Antigen for TB) and CFP 10 (Colony Forming Protein) are specific proteins of MTB and are being tried in skin tests.

New Dx Tests for TB – Bench, Bedside and Beyond – by Susan E Dorman

Take Home Points We need to move fast from our ESR and Mx. test Clinical feature are good screening tool for tests Radiology has a good role but understand its limits Costly imaging like HRCT, MRI add nothing for Dx. Accurate Diagnosis, Complete Rx are the answers MDR, XDR and now TDR TB are great challenges DST has to be done routinely – at least on diagnosis RT PCR, Gene Xpert, IGRA, LiPA – a sea change Use ADA to exclude. Serology should not be used. Above all, remember our burden & poverty. Stop TB.